CN113354764B - Preparation of layer-assembled multifunctional nano magnetic beads - Google Patents
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- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
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- 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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- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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Abstract
The invention discloses a Fe-containing alloy3O4A preparation method of @ PS (ferroferric oxide @ styrene-functional group copolymer). The monodisperse magnetic bead adopts a multilayer/layer-by-layer packaging method. Compared with a method of coating magnetic beads, the method can avoid PCR fluorescence quenching caused by exposure of the magnetic beads, has controllable particle size and narrow particle size distribution, can uniformly distribute functional groups such as surface carboxyl, amino and the like, and can be widely applied to a plurality of biological fields such as nucleic acid purification, protein purification, affinity chromatography, immunoassay and the like.
Description
The invention is a divisional application of a layer-assembled multifunctional nano magnetic bead preparation (application No. 201910906745.7, application No. 20190924).
Technical Field
The invention belongs to the field of biological materials, and particularly relates to a layer-assembled multifunctional nano magnetic bead and a preparation method thereof.
Background
The gene precise rapid detection technology has wide application prospects in the fields of clinic, POCT (point of care testing), field detection and the like, and how to realize the rapid extraction of the specificity of nucleic acid is a great problem which needs to be solved at present. The existing nucleic acid extraction process is complex in operation and low in specificity, and cannot meet the requirement of a gene accurate and rapid detection technology. Among various extraction technologies, the magnetic bead extraction technology has the widest application range and the widest application prospect.
The conventional magnetic bead preparation method is complex, and the conventional steps are roughly divided into the following three steps: firstly, ferroferric oxide magnetic particles are synthesized, then silicon dioxide with a certain thickness grows outside the ferroferric oxide particles, and finally, the surface of the silicon dioxide is modified to bring various groups required by the next step of nucleic acid extraction. The process has many steps and is troublesome to operate. There are also disadvantages as follows: the wide particle size distribution causes the difficulty in ensuring the stability of the material; partial magnetic bead particles are easy to be incompletely coated, so that fluorescence quenching is caused; the coating and re-modification are easy to cause uneven distribution of surface functional groups.
Disclosure of Invention
The invention aims to provide a layer-assembled multifunctional nano magnetic bead and a preparation method thereof, which are applied to the biological fields of nucleic acid extraction and the like.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a preparation method of a layer-assembled multifunctional nano magnetic bead is characterized by comprising the following steps: the multifunctional nanometer magnetic bead is Fe3O4The (ferroferric oxide) and the styrene-functional group copolymer are used as matrixes to be assembled layer by layer, the morphology is regular spherical, the particle size increases along with the number of layers, the particle size range is 200 nm-2 mu m, and the functional group is carboxyl, hydroxyl or amino.
Specifically, when the styrene-functional group copolymer is a styrene-carboxyl copolymer, the styrene-functional group copolymer is prepared by polymerizing styrene and carboxylic acid containing carboxyl and double bonds as a matrix, wherein the carboxylic acid is one or more of acrylic acid, methacrylic acid, itaconic acid and maleic acid.
Specifically, when the styrene-functional group copolymer is a styrene-hydroxyl copolymer, the styrene-functional group copolymer is prepared by polymerizing styrene and alcohol containing hydroxyl and double bonds as matrixes, wherein the alcohol is hydroxyethyl methacrylate.
Specifically, when the styrene-functional group copolymer is a styrene-amino copolymer, the styrene-functional group copolymer is prepared by polymerizing styrene and amine containing amino and double bonds as matrixes, wherein the amine is allyl amine.
The preparation method of any layer-by-layer assembled multifunctional nano magnetic bead comprises the following steps:
1) preparing lipophilic magnetic beads and dispersing the lipophilic magnetic beads in a styrene solution;
2) assembling layer 1, namely adding styrene serving as an oil-soluble monomer into a reaction system serving as a seed, wherein the reaction system contains water, a surfactant, a polymerization initiator and a curing agent;
3) layer 2, adding the magnetic bead-styrene solution prepared in the step 1) into a reaction system for polymerization and solidification;
4) repeating the step 3) to carry out multilayer assembly;
5) and adding a styrene-functional group copolymer into the last layer to form a magnetic bead with a spherical surface and a functional group.
Specifically, the magnetic beads prepared by a hydrophilic small-size coprecipitation method in the step 1) are modified by lipophilic groups, and the modified small-size magnetic beads are dispersed in styrene by using oleic acid for modification.
Specifically, in the reaction system constructed in the step 2), the surfactant is stearic acid, the polymerization initiator is azobisisobutyronitrile, and the curing agent is n-dodecyl mercaptan.
Specifically, the reaction temperature in the step 3) is 80-100 ℃, and the reaction time is 0.5-1.5 h.
Specifically, the number of layers repeatedly added in the step 4) is 3-10.
Specifically, the mass ratio of the added materials of each layer in the steps 3) to 5) is water: styrene: surfactant (b): polymerization initiator: curing agent 100: 20-35: 1-1.5: 2-5: 1.5 to 2.
The invention also provides a kit applied to nucleic acid purification, protein purification, nucleic acid extraction and amplification and immunoassay, which comprises the layer-by-layer assembled multifunctional nano magnetic beads.
It should be noted that: the multifunctional nano magnetic bead is characterized by adopting a scanning electron microscope, a magnetic hysteresis loop, infrared spectroscopy, thermogravimetric analysis, spectrum detection and the like.
Compared with the prior art, the invention has the advantages that:
1) conventional encapsulation methods with one-time addition of magnetic beads, e.g. SiO2Coating, the prepared magnetic beads have wide particle size distribution; the magnetic beads prepared by the novel layer-by-layer assembly method adopted by the invention have narrow particle size distribution, namely monodispersion;
2) the magnetic beads prepared by the conventional wrapping method of adding the magnetic beads at one time have some Fe3O4Not completely coated; the multiple coating of the magnetic bead preparation method can coat Fe3O4The coating is complete;
3) the preparation method of the magnetic bead combines two steps of surface functional group modification and shell coating into one step, and has the advantages of simple method, easy operation and good reproducibility.
Drawings
FIG. 1 SEM image of magnetic beads obtained in example 2;
FIG. 2 is a diagram of a magnetic bead hysteresis loop obtained in example 2;
FIG. 3 is an infrared spectrum of a layer-by-layer assembled carboxyl magnetic bead obtained in example 2;
FIG. 4 PCR amplification of DNA extracted from magnetic beads as described in example 9.
Detailed Description
The invention is further illustrated by the following examples, without restricting the invention thereto. All changes, modifications and equivalents that do not depart from the spirit of the invention are intended to be included within the scope thereof.
Example 1 preparation of lipophilic magnetic beads
Placing 15g of magnetic beads prepared by coprecipitation in a three-neck flask, adding 1L of water for dispersion, stirring and reacting for half an hour under the protection of nitrogen, adding 1g of oleic acid for continuous reaction, and reacting for 1 h. Magnetic attraction, washing with water and ethanol twice.
EXAMPLE 25 preparation of layer-assembled carboxyl magnetic beads
Adding 5g of itaconic acid into 500ml of water, heating to 85 ℃, stirring and dissolving, dissolving 10g of stearic acid and 8g of n-dodecyl mercaptan into 20g of styrene, adding the styrene into the system, preparing two solutions in another, wherein the solution A is prepared by dissolving 10g of azodiisobutyronitrile in 40g of styrene and equally dividing the solution A into 5 parts, and the solution B is prepared by dissolving 5g of lipophilic Fe in 5 parts3O4Dispersing in 40g of styrene, equally dividing into 3 parts, adding 1 part of solution A into the system, reacting for half an hour until the solution is milky, then adding 1 part of solution A and 1 part of solution B, repeating the operation for 3 times, adding the last 1 part of solution A, reacting for 8 hours, finishing the reaction, and magnetically attracting and washing to obtain the sample. The magnetic bead sample obtained in example 2 is characterized by using a Scanning Electron Microscope (SEM), and specifically, the obtained sample is dispersed in water and coated on a silicon wafer, and as a result, as shown in fig. 1, it can be seen that the morphology of the microcapsule-type phase change energy storage material obtained in example 2 is a sphere with excellent dispersibility and a regular shape, and the particle size distribution is relatively uniform.
EXAMPLE 35 preparation of layer-assembled magnetic hydroxyl beads
Similar to example 2, 6g of hydroxyethyl methacrylate was added to 500ml of water, the mixture was heated to 85 ℃ and stirred to dissolve, 10g of stearic acid and 8.2g of n-dodecylmercaptan were dissolved in 20g of styrene, two solutions were prepared, solution A was prepared by dissolving 40g of styrene in 9.8g of azobisisobutyronitrile and equally divided into 5 parts, solution B was prepared by dissolving 5g of lipophilic Fe3O4Dispersing in 40g of styrene, equally dividing into 3 parts, adding 1 part of solution A into the system, reacting for half an hour until the solution is milky, then adding 1 part of solution A and 1 part of solution B, repeating the operation for 3 times, adding the last 1 part of solution A, reacting for 8 hours, finishing the reaction, and magnetically attracting and washing to obtain the sample.
EXAMPLE 45 preparation of amino magnetic beads for layer Assembly
Similar to example 2, 5.5g of allylamine was added to 500ml of water, heated to 85 ℃ and stirred to dissolve, 10g of stearic acid and 8.2g of n-dodecylmercaptan were dissolved in 20g of styrene, and two solutions were prepared, wherein solution A was prepared by dissolving 40g of styrene in 9.8g of azobisisobutyronitrile and was divided into 5 parts, and solution B was prepared by dissolving 5g of lipophilic Fe3O4Dispersing in 40g of styrene, dividing into 3 parts, adding 1 part of solution A into the system, and reactingAnd (3) adding 1 part of solution A and 1 part of solution B after half an hour till the solution is milky white, repeating the operation for 3 times, adding the last 1 part of solution A, reacting for 8 hours, finishing the reaction, and magnetically attracting and washing to obtain the sample.
Example 5 hysteresis Loop test
The magnetic hysteresis loop tester is used for testing and magnetic analysis of the sample in the embodiment 2, and the tested magnetic hysteresis loop is shown in fig. 2, so that the magnetic performance is better.
Example 6 Infrared Spectroscopy testing
The 3 magnetic bead samples obtained in examples 2 to 4 were subjected to infrared spectroscopic measurement using an infrared spectrometer, and the surface functional groups of the obtained magnetic beads were analyzed. The IR spectrum of the carboxyl magnetic beads of example 2 is shown in FIG. 3.
Example 7 thermogravimetric testing
The carboxyl magnetic beads obtained in example 2 were tested by a thermogravimetric analyzer, and the carboxyl content of the surfaces of the magnetic beads was determined by the characteristics of the carboxyl groups. The measured carboxyl content was 12.5%
Example 8 amino content testing
The amino group content of the beads obtained in example 4 was determined by the reactivity of amino groups with ninhydrin. The amino magnetic beads obtained in example 4 were dispersed in water, ninhydrin solution was added thereto, and after reaction at 110 ℃ for 2 hours, the ultraviolet-visible light spectrum absorption was measured, and the amino content was measured to be 8.9% by conversion of characteristic peaks.
Example 9 nucleic acid extraction
Nucleic acid extraction and PCR amplification experiments Using the magnetic beads prepared in example 2
1. Taking 8 μ L of magnetic beads (the dosage can be optimized), adding 200 μ L of lysine, and mixing well.
2. The nucleic acid sample to be tested (100. mu.L) was added and mixed well.
3. Incubate at room temperature with low speed rotation or gentle shaking for 13min, and magnetically attract for 2min to remove supernatant.
4.250 μ L W1 and W2 were washed once each.
5. Add 20. mu.L EB, incubate at 56 ℃ for 3min with shaking.
6. Magnetic attraction, discarding magnetic beads, keeping supernatant and PCR mixed solution, transferring all the solution to an eight-connecting tube, and performing direct PCR.
The PCR amplification results are shown in FIG. 4. As can be seen from FIG. 4, our beads have high sensitivity and wider linear range.
Claims (2)
1. A preparation method of a layer-assembled multifunctional nano magnetic bead is characterized by comprising the following steps: the method comprises the following steps: adding 6g of hydroxyethyl methacrylate into 500ml of water, heating to 85 ℃, stirring and dissolving, dissolving 10g of stearic acid and 8.2g of n-dodecyl mercaptan in 20g of styrene, adding two solutions into the system, preparing a solution A by dissolving 9.8g of azodiisobutyronitrile in 40g of styrene and equally dividing the solution A into 5 parts, and preparing a solution B by dissolving 5g of lipophilic Fe in solution B3O4Dispersing in 40g of styrene, equally dividing into 3 parts, adding 1 part of solution A into the system, reacting for half an hour until the solution is milky, then adding 1 part of solution A and 1 part of solution B, repeating the operation for 3 times, adding the last 1 part of solution A, reacting for 8 hours, finishing the reaction, and magnetically attracting and washing to obtain a sample;
said lipophilic Fe3O4The preparation method comprises the following steps: placing 15g of magnetic beads prepared by coprecipitation in a three-neck flask, adding 1L of water for dispersion, stirring and reacting for half an hour under the protection of nitrogen, adding 1g of oleic acid for continuous reaction for 1 hour, magnetically attracting, and washing with water and ethanol twice respectively.
2. A kit applied to nucleic acid purification, protein purification, nucleic acid extraction and amplification and immunoassay, comprising the layer-by-layer assembled multifunctional nano magnetic bead prepared by the method of claim 1.
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