CN117143299A - Preparation method and application of functionalized magnetic microsphere - Google Patents

Preparation method and application of functionalized magnetic microsphere Download PDF

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CN117143299A
CN117143299A CN202311115992.8A CN202311115992A CN117143299A CN 117143299 A CN117143299 A CN 117143299A CN 202311115992 A CN202311115992 A CN 202311115992A CN 117143299 A CN117143299 A CN 117143299A
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functionalized magnetic
oil
polymerizable monomer
sio
magnetic microspheres
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陈志坚
刘峰
林志铿
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Xiamen Wiz Biotech Co ltd
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    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads

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Abstract

The application relates to the technical field of aqueous adhesives, and in particular provides a preparation method of a functional magnetic microsphere, which is characterized by comprising the following steps: s1, dispersing tetraethoxysilane, a silane coupling agent containing carbon-carbon double bonds, a catalyst and water in an organic solvent, and processing to obtain SiO 2 A dispersion; s2, fe 3 O 4 The nano magnetic particles are dispersed inAdding water into oil-soluble polymerizable monomer containing free radical initiator and emulsifier for emulsification to obtain hybrid emulsion, heating to polymerization reaction temperature, reacting for 0.5-2 hr, and dropwise adding SiO within 5-60 min 2 And (3) continuously reacting the dispersion liquid for 1-5 hours after dripping, filtering, cleaning and drying to obtain the product. The magnetic microsphere has the structure that polymer wraps Fe 3 O 4 The surfaces of the nanometer magnetic particles and the polymer are coated with nanometer silicon dioxide particles, so that the surfaces of the magnetic microspheres are rich in silicon hydroxyl groups.

Description

Preparation method and application of functionalized magnetic microsphere
Technical Field
The application relates to the technical field of functional microspheres, in particular to a preparation method and application of a functional magnetic microsphere.
Background
By Fe 3 O 4 The representative nano magnetic particles have wide application in the fields of biological medicine, detection and the like. At Fe 3 O 4 The surface of the nano magnetic particle is coated with silicon dioxide, so that the surface of the magnetic particle contains silicon hydroxyl, and the nano magnetic particle can be applied to the extraction and purification of nucleic acid. The prior art discloses various compositions of Fe 3 O 4 The method for coating the surface of the nano magnetic particle with silicon dioxide, but the obtained particle is nano-sized, and has higher separation difficulty and poorer extraction effect when being applied to the extraction and purification of nucleic acid. To solve the problem, the prior art discloses that Fe is used first 3 O 4 The polymer is coated on the surfaces of the nano magnetic particles, and then the method of coating the nano silicon dioxide particles is continued, so that the micron particles can be obtained. The applicant believes that there is a continuing need for improvements in the art, such as increasing the binding of nanosilica particles to polymers.
Disclosure of Invention
In order to solve the technical problems, the application provides a preparation method and application of a functionalized magnetic microsphere.
The application adopts the following technical scheme:
a method for preparing functionalized magnetic microspheres, comprising:
s1, dispersing tetraethoxysilane, a silane coupling agent containing carbon-carbon double bonds, a catalyst and water in an organic solvent, and processing to obtain SiO 2 A dispersion;
s2, fe 3 O 4 Dispersing the nano magnetic particles in oil-soluble polymerizable monomer containing free radical initiator and emulsifier, adding water for emulsification to obtain hybrid emulsion, heating to polymerization reaction temperature, reacting for 0.5-2 hrAnd (3) dropwise adding the SiO obtained in the step S1 within 5-60 min 2 And (3) continuously reacting the dispersion liquid for 1-5 hours after dripping, filtering, cleaning and drying to obtain the product.
Preferably, the structural general formula of the silane coupling agent containing carbon-carbon double bonds in the step S1 is R 1 CH＝CR 2 R 3 Si(OR 4 ) 3 Wherein R is 1 Selected from H or methyl, R 2 Selected from H or methyl, R 3 Is absent or selected from C1-C6 alkylene or C2-C6 substituted alkylene, R 4 Selected from C1-C4 alkyl or C2-C4 acyloxy.
Preferably, the molar ratio of the tetraethoxysilane to the silane coupling agent containing carbon-carbon double bonds in the step S1 is 1-50:1.
Preferably, the treatment in step S1 is ultrasonic for 3-30min or stirring at 200-1000rpm for 10-180min.
Preferably, the radical initiator in step S2 is selected from oil-soluble azo radical initiators and/or organic peroxide radical initiators.
Preferably, the oil-soluble polymerizable monomer in step S2 is selected from monofunctional polymerizable monomers or a combination of monofunctional polymerizable monomers and polyfunctional polymerizable monomers in a weight ratio of 5 to 200:1.
Preferably, the oil-soluble polymerizable monomer in step S2 is at least one selected from the group consisting of an acrylic monomer, a styrene monomer, and an acrylamide monomer.
Preferably, the Fe in step S2 3 O 4 The weight ratio of the nano magnetic particles to the oil-soluble polymerizable monomer is 0.01-0.3:1.
Preferably, in step S2, the oil-soluble polymerizable monomer and the SiO 2 The weight ratio of the dispersion liquid is 1:0.1-2.
The functionalized magnetic microsphere obtained by the preparation method of the functionalized magnetic microsphere according to any one of the embodiments, which is applied to nucleic acid extraction and purification or applied to a detection kit.
The principle of the preparation method of the functionalized magnetic microsphere is as follows: fe (Fe) 3 O 4 Nanometer magnetismDispersing the particles in an oil-soluble polymerizable monomer, forming O/W emulsion under the action of an emulsifier and water, heating, initiating the oil-soluble polymerizable monomer to start polymerization by a free radical initiator in an oil phase, gradually adding SiO containing nano-scale carbon-carbon double bonds on the surface after the oil-soluble polymerizable monomer starts to polymerize to a part 2 When nano SiO is used as the dispersion liquid of (a) 2 When moving in the emulsion and contacting the micron-sized latex particles undergoing polymerization, the surface carbon-carbon double bonds can participate in the polymerization reaction inside the latex particles, thus, the nano-sized SiO 2 Is firmly coated on the surface of the polymer to form silicon dioxide with silicon hydroxyl groups coated on the surface of the polymer and Fe coated in the polymer 3 O 4 Functionalized magnetic microspheres of nano magnetic particles.
In summary, the application has the following beneficial effects:
1. the application adopts a free radical polymerization method, combines emulsion polymerization and interfacial polymerization of oil phase/water phase to obtain the silicon dioxide with the structure that the polymer surface is coated with silicon hydroxyl and the polymer inside is coated with Fe 3 O 4 The functionalized magnetic microsphere particles of the nano magnetic particles have the advantages that the surface of the magnetic microsphere is wrapped with rich silicon hydroxyl groups, the extraction effect on nucleic acid is good, and the separation is easy.
2. As the particles of the functionalized magnetic microsphere are larger, the particle size can reach several micrometers to tens of micrometers, and the application of a single microsphere can be realized when the functionalized magnetic microsphere is applied to a microfluidic chip.
Drawings
FIG. 1 is a schematic structural diagram of a functionalized magnetic microsphere of the present application;
wherein 1-polymer, 2-silica nanoparticles, 3-Fe 3 O 4 Nano magnetic particles.
Detailed Description
In order to make the objects, technical solutions and advantages of the present application more apparent, the technical solutions of the present application will be described in detail below.
Throughout the specification, unless specifically indicated otherwise, the terms used herein should be understood as meaning as commonly used in the art. Accordingly, 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 application belongs. In case of conflict, the present specification will control.
In one aspect, the application provides a method for preparing a functionalized magnetic microsphere, comprising:
s1, dispersing tetraethoxysilane, a silane coupling agent containing carbon-carbon double bonds, a catalyst and water in an organic solvent, and processing to obtain SiO 2 A dispersion;
wherein the catalyst is alkaline matter, such as ammonia water, diethanolamine, triethanolamine, sodium hydroxide, potassium carbonate, sodium carbonate, potassium hydroxide, etc., and the dosage of the catalyst is to adjust the pH of the reaction system to 9-11; the organic solvent can be water-soluble organic solvent such as acetone, methanol, ethanol, isopropanol, etc., and the weight of the organic solvent can be 50-300 times of that of the ethyl orthosilicate; the weight of the water can be 3-20 times of that of the ethyl orthosilicate;
s2, fe 3 O 4 Dispersing the nano magnetic particles in an oil-soluble polymerizable monomer containing a free radical initiator and an emulsifier, adding water for emulsification to obtain a hybrid emulsion, heating to a polymerization reaction temperature, reacting for 0.5-2 hours, and dropwise adding the SiO obtained in the step S1 within 5-60 minutes 2 And (3) continuously reacting the dispersion liquid for 1-5 hours after dripping, filtering, cleaning and drying to obtain the product.
In the above step S2, in order to increase Fe 3 O 4 Compatibility of the nano magnetic particles and the oil-soluble polymerizable monomer can be used for Fe 3 O 4 The nano-magnetic particles are surface modified, such as with a silane coupling agent, which is well known to those skilled in the art, and may be, for example, methyltrimethoxysilane, vinyltriethoxysilane, propyltrimethoxysilane, vinyltrimethoxysilane, etc. When the silane coupling agent contains a carbon-carbon double bond, the silane coupling agent can be polymerized together with the oil-soluble polymerizable monomer, fe 3 O 4 The dispersion of the nano magnetic particles in the microsphere is very stable. Fe of the present application 3 O 4 The average particle size of the nano magnetic particles may be 10 to 50nm。
The emulsifier is not particularly limited, and may be Span20, span40, span60, span80, tween20, tween40, tween60, tween80, AEO-3, AEO-9, AEO-12, etc., one or a combination of several emulsifiers, and the amount of the emulsifier may be 3 to 10% by weight of the oil-soluble polymerizable monomer.
In a preferred embodiment of the present application, the silane coupling agent containing carbon-carbon double bonds in step S1 has the general structural formula R 1 CH＝CR 2 R 3 Si(OR 4 ) 3 Wherein R is 1 Selected from H or methyl, R 2 Selected from H or methyl, R 3 Is absent or selected from C1-C6 alkylene or C2-C6 substituted alkylene, R 4 Selected from C1-C4 alkyl or C2-C4 acyloxy. For example, the silane coupling agent having a carbon-carbon double bond may be vinyltrimethoxysilane, vinyltriethoxysilane, methacryloxypropyl trimethoxysilane, methacryloxypropyl triethoxysilane, or the like, but is not limited to the above list.
In a preferred embodiment of the present application, the molar ratio of the ethyl orthosilicate to the silane coupling agent containing carbon-carbon double bond in step S1 is 1-50:1. The silicon dioxide nano-particles are prepared by adopting the combination of the tetraethoxysilane and the silane coupling agent containing carbon-carbon double bonds to carry out hydrolytic condensation, so that the surfaces of the silicon dioxide nano-particles are modified with the carbon-carbon double bonds. The weight ratio of the carbon-carbon double bond-containing silane coupling agent in the tetraethoxysilane cannot be too high, so that the formation of the silicon dioxide nano-particles is influenced, the weight ratio of the carbon-carbon double bond-containing silane coupling agent in the tetraethoxysilane cannot be too low, and the carbon-carbon double bond groups on the surfaces of the silicon dioxide nano-particles are insufficient, so that the reaction of the silicon dioxide nano-particles and the latex particles is influenced. Further, the molar ratio of the tetraethoxysilane to the silane coupling agent containing carbon-carbon double bond is 5-30:1, and for example, the molar ratio may be 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, etc.
In a preferred embodiment of the present application, the treatment in step S1 is ultrasonic for 3-30min, the ultrasonic may be 100-1000W, or stirring at 200-1000rpm for 10-180min, or the stirring time may be further 60-180min.
In a preferred embodiment of the present application, the radical initiator in step S2 may be selected from oil-soluble azo-based radical initiators and/or organic peroxide-based radical initiators. The azo radical initiator may be azobisisobutyronitrile AIBN, azobisisoheptonitrile AIVN, dimethyl azobisisobutyrate AIBME, etc., and the organic peroxide radical initiator may be benzoyl peroxide, etc.
In a preferred embodiment of the present application, the oil-soluble polymerizable monomer in step S2 is selected from monofunctional polymerizable monomers or a combination of monofunctional polymerizable monomers and polyfunctional polymerizable monomers in a weight ratio of 5-200:1. In the present application, the functionality in the polymerizable monomer means the number of carbon-carbon double bonds in the molecule thereof, such as 1 in the single-functionality polymerizable monomer molecule, 2 or more in the multi-functionality polymerizable monomer molecule, such as 2 in the di-functionality polymerizable monomer molecule, 3 in the tri-functionality polymerizable monomer molecule, and so on. For example, the monofunctional polymerizable monomer may be methyl acrylate, methyl methacrylate, butyl acrylate, isooctyl acrylate, isobornyl acrylate, butyl methacrylate, styrene, acrylamide, N-dimethylacrylamide, etc., the difunctional polymerizable monomer may be 1, 6-adipic acid diacrylate, 1, 4-succinic acid diacrylate, polyether diacrylate, diethylene glycol diacrylate, triethylene glycol diacrylate, di-1, 3-propanediol diacrylate, tri-1, 3-propanediol diacrylate, divinylbenzene, etc., the trifunctional polymerizable monomer may be pentaerythritol triacrylate, trimethylolpropane triacrylate, etc., and the tetrafunctional polymerizable monomer may be pentaerythritol tetraacrylate, etc. Further, the weight ratio of monofunctional polymerizable monomer to polyfunctional polymerizable monomer may be 10 to 130:1.
In a preferred embodiment of the present application, the oil-soluble polymerizable monomer in step S2 is selected from at least one of an acrylic monomer, a styrene monomer, and an acrylamide monomer. For example, the acrylic monomer may be methyl acrylate, methyl methacrylate, butyl acrylate, isooctyl acrylate, isobornyl acrylate, butyl methacrylate, 1, 6-adipic acid diacrylate, 1, 4-succinic acid diacrylate, polyether diacrylate, diethylene glycol diacrylate, triethylene glycol diacrylate, di-1, 3-propylene glycol diacrylate, tri-1, 3-propylene glycol diacrylate, etc., the styrene monomer may be styrene, divinylbenzene, vinyl toluene, etc., and the acrylamide monomer may be acrylamide, N-dimethylacrylamide, N-methylacrylamide, N-diethylacrylamide, N' -methylenebisacrylamide, etc.
In a preferred embodiment of the present application, fe in step S2 3 O 4 The weight ratio of the nano magnetic particles to the oil-soluble polymerizable monomer is 0.01-0.3:1.Fe (Fe) 3 O 4 The content of the nano magnetic particles is too high, so that the density of the emulsion particles in emulsion polymerization is too high, the emulsion particles are easy to subside, the emulsion is layered, and Fe 3 O 4 The content of the nano magnetic particles is too low, and the magnetic performance of the obtained microsphere is poor. Further, fe 3 O 4 The weight ratio of the nanomagnetic particles to the oil-soluble polymerizable monomer is 0.06-0.25:1, for example, the weight ratio may be 0.06:1, 0.07:1, 0.08:1, 0.09:1, 0.1:1, 0.11:1, 0.12:1, 0.13:1, 0.14:1, 0.15:1, 0.16:1, 0.17:1, 0.18:1, 0.19:1, 0.2:1, 0.21:1, 0.22:1, 0.23:1, 0.24:1, 0.25:1, etc.
In a preferred embodiment of the present application, the oil-soluble polymerizable monomer and SiO in step S2 2 The weight ratio of the dispersion liquid is 1:0.1-2. Further, an oil-soluble polymerizable monomer and SiO 2 The weight ratio of the dispersion is 1:0.3-2, for example, the weight ratio may be 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, etc. In the application, the hybrid emulsion is reversely heated to the polymerization reaction temperatureThe time is 0.5-2 hours, partial polymerization reaction occurs in the hybrid emulsion, and the polymer in the emulsion particles is initially formed and relatively stable, so that the hybrid emulsion can be smoothly matched with SiO 2 The particles collide and react; the SiO is added dropwise when the hybrid emulsion is just heated to the polymerization temperature 2 The dispersion liquid or insufficient polymerization reaction time can lead to unstable emulsion particles, precipitation of oil phase and layering; if the hybrid emulsion has been polymerized for a longer period of time, the polymerization within the latex particles is complete and the SiO 2 It is more difficult for the particles to continue grafting to the polymer surface.
In another aspect, the application provides the use of the functionalized magnetic microsphere obtained by the method for preparing a functionalized magnetic microsphere according to any one of the embodiments above, for nucleic acid extraction and purification, or for a detection kit.
The technical scheme of the application is described in detail below with reference to examples and comparative examples.
Example 1
Dispersing 0.1mol of tetraethoxysilane, 0.005mol of vinyltrimethoxysilane, ammonia water and 100g of water in 2Kg of absolute ethyl alcohol, and carrying out ultrasonic treatment for 20min at the reaction system pH of 9.5 and 500W to obtain SiO 2 A dispersion;
12g of Fe with an average particle diameter of 25nm 3 O 4 The nano magnetic particles are dispersed in 100g of monomer (composed of 99g of styrene and 1g of divinylbenzene) containing 1.5g of AIBN and 5g of emulsifier (composed of 3g of span80 and 2g of Tween 80) by ultrasonic, 170g of water is gradually added for emulsification under the stirring speed of 1200rpm, and the hybrid emulsion is obtained; the temperature of the hybrid emulsion is raised to 72 ℃, the mixture is reacted for 1 hour at constant temperature, and 50g of SiO is added dropwise in 25min 2 And (3) continuously reacting the dispersion liquid at constant temperature for 3 hours after dripping, filtering, cleaning with pure water for 2 times, and drying in a 60 ℃ oven in vacuum overnight to obtain the magnetic microsphere.
Example 2
The differences between example 2 and example 1 are: in example 1, vinyltrimethoxysilane was adjusted from 0.005mol to 0.012mol. The remaining steps remain unchanged.
Example 3
Example 3 differs from example 1 in that: in example 1, vinyltrimethoxysilane was adjusted from 0.005mol to 0.02mol. The remaining steps remain unchanged.
Example 4
The differences between example 4 and example 2 are: in example 2, fe 3 O 4 The nano magnetic particles are adjusted from 12g to 6g. The remaining steps remain unchanged.
Example 5
The differences between example 5 and example 2 are: in example 2, fe 3 O 4 The nano magnetic particles are adjusted from 12g to 25g. The remaining steps remain unchanged.
Example 6
The differences between example 6 and example 2 are: in example 2, siO was added dropwise 2 The dispersion was adjusted from 50g to 15g. The remaining steps remain unchanged.
Example 7
The differences between example 7 and example 2 are: in example 2, siO was added dropwise 2 The dispersion was adjusted from 50g to 100g. The remaining steps remain unchanged.
Example 8
The differences between example 8 and example 2 are: in example 2, siO was added dropwise 2 The dispersion was adjusted from 50g to 200g. The remaining steps remain unchanged.
Example 9
The differences between example 9 and example 2 are: in example 2, the hybrid emulsion was warmed to 72℃and allowed to react at constant temperature for 1 hour, and then allowed to react at constant temperature for 2 hours. The remaining steps remain unchanged.
Example 10
The differences between example 10 and example 2 are: the hybrid emulsion in example 2 was warmed to 72℃and allowed to react at constant temperature for 1 hour, and then allowed to react at constant temperature for 0.5 hour. The remaining steps remain unchanged.
Comparative example 1
The difference between comparative example 1 and example 2 is: in example 2, the hybrid emulsion was heated to 72℃and allowed to react at constant temperature for 1 hour, and 50g of the above SiO was added dropwise over 25 minutes 2 The dispersion is adjusted to the hybridized emulsion, the temperature is raised to 72 ℃, and 50g of SiO is added dropwise within 25min 2 And (3) a dispersion. The remaining steps remain unchanged. During the experiment, it was found that comparative example 1The emulsion showed a distinct oil phase layer and the polymer at the bottom was more agglomerated. Indicating that SiO was added to the emulsion under the condition that the emulsion particles were not stabilized yet 2 The dispersion may cause instability of the emulsion, resulting in precipitation of the oil phase.
Comparative example 2
The difference between comparative example 2 and example 2 is: in example 2, the hybrid emulsion was heated to 72℃and allowed to react at constant temperature for 1 hour, and 50g of the above SiO was added dropwise over 25 minutes 2 The dispersion is adjusted to the hybridized emulsion, the temperature is raised to 72 ℃, the constant temperature reaction is carried out for 2.5 hours, and 50g of SiO is added dropwise within 25min 2 And (3) a dispersion. The remaining steps remain unchanged.
Comparative example 3
The difference between comparative example 2 and example 2 is: in example 2, the hybrid emulsion was heated to 72℃and allowed to react at constant temperature for 1 hour, and 50g of the above SiO was added dropwise over 25 minutes 2 The dispersion is adjusted to the hybridized emulsion, the temperature is raised to 72 ℃, the constant temperature reaction is carried out for 3.5 hours, and 50g of SiO is added dropwise within 25min 2 And (3) a dispersion. The remaining steps remain unchanged.
Example 11
Dispersing 0.1mol of tetraethoxysilane, 0.004mol of methacryloxypropyl trimethoxysilane, ammonia water and 150g of water in 3Kg of absolute ethyl alcohol, stirring the reaction system at pH of 9.5 and 600rpm for 120min to obtain SiO 2 A dispersion;
10g of Fe with an average particle diameter of 45nm 3 O 4 The nano magnetic particles are dispersed in 100g of monomer (composed of 57g of butyl acrylate, 40g of methyl methacrylate and 3g of tripropylene glycol diacrylate) containing 1.2g of AIVN and 6g of emulsifier (composed of 2.8g of span80 and 2.2g of Tween 80) by ultrasonic, 150g of water is gradually added for emulsification under the stirring speed of 1000rpm, and the hybrid emulsion is obtained; the temperature of the hybrid emulsion is raised to 53 ℃, the constant temperature reaction is carried out for 1.5 hours, and 50g of SiO is added dropwise within 25min 2 And (3) continuously reacting the dispersion liquid at constant temperature for 4 hours after dripping, filtering, cleaning with pure water for 2 times, and drying in a 60 ℃ oven in vacuum overnight to obtain the magnetic microsphere.
Comparative example 4
The difference between comparative example 4 and example 11 is: in example 11, methacryloxypropyl trimethoxysilane was replaced with equimolar ethyl orthosilicate. The remaining steps remain unchanged.
Comparative example 5
The difference between comparative example 5 and example 11 is: in example 11, ethyl orthosilicate was adjusted from 0.1mol to 0.08mol, methacryloxypropyl trimethoxysilane was adjusted from 0.004mol to 0.024mol, and the rest of the procedure remained unchanged. As a result of the experiment, it was found that SiO in example 11 2 The dispersion exhibited a clear to translucent blue light, whereas the SiO of comparative example 4 2 The dispersion was transparent and blue light was very slight, indicating that the nanoparticles formed in comparative example 4 were much less than in example 11.
The D50 particle size of the microspheres to be tested was measured using a laser particle sizer and the results are shown in Table 1 below.
TABLE 1
The magnetic microsphere to be tested is applied to the extraction of novel coronavirus nucleic acid by applying test contrast to replace the silicon-based material of the existing centrifugal column. Novel coronavirus samples were taken from pharyngeal swabs. Adding the sample into a commercially available lysate (general RNA extraction kit (centrifugal column type) of Shanghai Yi Shang Ji Biotechnology Co., ltd.), mixing uniformly, adsorbing with a magnetic microsphere to be detected, centrifuging at 8000rpm for half an hour, washing, eluting with an eluent (general RNA extraction kit (centrifugal column type) of Shanghai Yi Shang Ji Biotechnology Co., ltd.), obtaining extracted RNA, detecting the concentration of the extracted RNA, and detecting the absorbance OD260/OD280 value of the extracted RNA at 260nm and 280 nm.
The results are shown in Table 2 below.
TABLE 2
RNA concentration (ng/. Mu.L) OD260/OD280
Example 2 21.73 1.87
Comparative example 2 14.54 1.51
Comparative example 3 13.79 1.48
Example 11 20.85 1.89
Comparative example 4 6.71 1.32
Comparative example 5 8.94 1.47
As is clear from the data in Table 2, when a large amount of nano silica particles cannot be adsorbed on the surface of the magnetic microsphere, the silicon hydroxyl group content on the surface is small, and when the magnetic microsphere is applied to the extraction and purification of nucleic acid, the nucleic acid with high concentration cannot be obtained effectively, and the purity is low.
The present embodiment is only for explanation of the present application and is not to be construed as limiting the present application, and modifications to the present embodiment, which may not creatively contribute to the present application as required by those skilled in the art after reading the present specification, are all protected by patent laws within the scope of claims of the present application.

Claims (10)

1. A method for preparing a functionalized magnetic microsphere, comprising:
s1, dispersing tetraethoxysilane, a silane coupling agent containing carbon-carbon double bonds, a catalyst and water in an organic solvent, and processing to obtain SiO 2 A dispersion;
s2, fe 3 O 4 Dispersing the nano magnetic particles in an oil-soluble polymerizable monomer containing a free radical initiator and an emulsifier, adding water for emulsification to obtain a hybrid emulsion, heating to a polymerization reaction temperature, reacting for 0.5-2 hours, and dropwise adding the SiO obtained in the step S1 within 5-60 minutes 2 And (3) continuously reacting the dispersion liquid for 1-5 hours after dripping, filtering, cleaning and drying to obtain the product.
2. The method for preparing functionalized magnetic microspheres according to claim 1, wherein the silane coupling agent containing carbon-carbon double bonds in step S1 has a general structural formula of R 1 CH=CR 2 R 3 Si(OR 4 ) 3 Wherein R is 1 Selected from H or methyl, R 2 Selected from H or methyl, R 3 Is absent or selected from C1-C6 alkylene or C2-C6 substituted alkylene, R 4 Selected from C1-C4 alkyl or C2-C4 acyloxy.
3. The method for preparing functionalized magnetic microspheres according to claim 1, wherein the molar ratio of the tetraethyl orthosilicate to the silane coupling agent containing carbon-carbon double bonds in step S1 is 1-50:1.
4. The method of preparing functionalized magnetic microspheres according to claim 1, wherein the treatment in step S1 is ultrasonic for 3-30min or stirring at 200-1000rpm for 10-180min.
5. The method of preparing functionalized magnetic microspheres according to claim 1, wherein the radical initiator in step S2 is selected from oil-soluble azo-based radical initiators and/or organic peroxide-based radical initiators.
6. The method of preparing functionalized magnetic microspheres according to claim 1, wherein the oil-soluble polymerizable monomer in step S2 is selected from monofunctional polymerizable monomers or a combination of monofunctional polymerizable monomers and polyfunctional polymerizable monomers in a weight ratio of 5-200:1.
7. The method of preparing functionalized magnetic microspheres according to claim 1, wherein the oil-soluble polymerizable monomer in step S2 is at least one selected from the group consisting of acrylate monomers, styrene monomers, and acrylamide monomers.
8. The method of claim 1, wherein the Fe in step S2 is 3 O 4 The weight ratio of the nano magnetic particles to the oil-soluble polymerizable monomer is 0.01-0.3:1.
9. The method of preparing functionalized magnetic microspheres according to claim 1, wherein the oil-soluble polymerizable monomer and the SiO in step S2 2 The weight ratio of the dispersion liquid is 1:0.1-2.
10. Use of the functionalized magnetic microspheres obtained by the method of preparation of the functionalized magnetic microspheres according to any one of claims 1-9, characterized in that it is applied in nucleic acid extraction and purification or in a detection kit.
CN202311115992.8A 2023-08-31 2023-08-31 Preparation method and application of functionalized magnetic microsphere Pending CN117143299A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117861624A (en) * 2023-12-18 2024-04-12 广州奕昕生物科技有限公司 Preparation method and application of DNA fragment sorting and purifying magnetic beads

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117861624A (en) * 2023-12-18 2024-04-12 广州奕昕生物科技有限公司 Preparation method and application of DNA fragment sorting and purifying magnetic beads

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