CN112121768A - Amino magnetic bead, preparation method and application thereof - Google Patents
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Abstract
The invention discloses an amino magnetic bead, a preparation method and application thereof. The amino magnetic bead comprises silicon dioxide coated ferroferric oxide nanoparticles, amino-rich groups are covalently grafted on the surfaces of the ferroferric oxide nanoparticles, and the density of bonded amino groups on the surfaces of the magnetic nanoparticles is more than 0.18 mu mol/mg. The preparation method comprises the following steps: (1) preparing a silicon dioxide coated ferroferric oxide nano magnetic bead particle dispersion liquid; (2) preparing a precursor dispersion; (3) and performing alkylation reaction, washing and drying to obtain the amino magnetic beads. The amino magnetic beads are used for whole blood DNA extraction, and can extract a large amount of DNA samples for amplification and detection at one time.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an amino magnetic bead, and a preparation method and application thereof.
Background
Genomic DNA carries all the genetic information of life activities and is a biological macromolecule essential for the development and normal operation of organisms. Extracting DNA from whole blood is a simpler method for obtaining biological genome genetic information, and is also an important means and technology for gene correlation research. The quality and yield of DNA extraction directly affects the accuracy of subsequent experiments. The nano magnetic bead is a functional material with excellent performance, which is prepared by a chemical or physical bonding method of a magnetic material and a high polymer material.
The nanometer magnetic bead method belongs to a solid phase extraction method, and the use of magnetic nanometer materials enables the solid phase separation process to be simpler, more convenient and more efficient. The magnetic nano-particles have the excellent characteristics of large specific surface area, high surface activity, good biocompatibility and the like, can fully interact with DNA, and can efficiently carry out solid-liquid separation under the action of an external magnetic field, thereby improving the extraction efficiency of the DNA. Because the DNA nucleic acid skeleton has phosphate groups and is negatively charged, DNA can be effectively adsorbed on the surface of the magnetic bead modified by the positively charged groups without any additional binding agent, and the adsorbed DNA is protected from enzyme cleavage. Based on the advantages, the magnetic bead method DNA extraction kit can be used in various fields such as clinical disease diagnosis, environmental microorganism detection, food safety detection, laboratory research and the like.
The amino magnetic beads are magnetic particles with surfaces modified with amino functional groups and superparamagnetism, are widely applied functional biological magnetic beads and are mainly used for preparing immunomagnetic beads. Under certain conditions, the amino magnetic beads can be covalently coupled with biomolecules such as protein ligands (such as antigens, antibodies and the like) and oligonucleotide probes through the mediation of cross-linking reagents (such as glutaraldehyde and the like), and the amino magnetic beads coupled with the biological ligands are immunomagnetic beads. Amino magnetic beads are widely used in many fields such as separation and purification of proteins, cell separation, immobilization of enzymes, and immunoassay.
The amino groups grafted on the surface of the amino magnetic beads can interact with phosphate groups on DNA molecules through electrostatic interaction to achieve the purpose of adsorption, can be well compatible with DNA downstream analysis conditions under mild adsorption conditions, and reduces purification steps. Therefore, in order to improve the efficiency of DNA capture, the density of amino group modification on the surface of the magnetic nanoparticle should be increased as much as possible.
Disclosure of Invention
Aiming at the defects or improvement requirements of the prior art, the invention provides an amino magnetic bead, a preparation method and application thereof, and aims to improve the affinity of the amino magnetic bead for DNA specificity by further improving the surface bonding amino density of the amino magnetic bead and by the structure of amino-rich groups, thereby simplifying the extraction steps, reducing the extraction difficulty and shortening the extraction time, and thus solving the problems of longer extraction time, fussy steps and low recovery rate in the prior art.
To achieve the above object, according to one aspect of the present invention, there is provided an amino magnetic bead comprising silica-coated ferroferric oxide nanoparticles, the surfaces of which are covalently grafted with amino-rich groups, and the surface bonding amino density of the magnetic nanoparticles is greater than 0.18 μmol/mg.
Preferably, the amino magnetic beads of which the amino-rich groups are linear chains of C3 or more.
Preferably, the amino magnetic bead has an amino-rich group of-CH2NH-C2H5NH-C2H5NH2。
Preferably, the particle size of the ferroferric oxide nano particle of the amino magnetic bead is less than or equal to 30nm, and the particle size of the silicon dioxide coated ferroferric oxide nano particle is 35nm-50 nm.
According to another aspect of the present invention, there is provided a method for preparing amino magnetic beads, comprising the steps of:
the ferroferric oxide nano magnetic bead particles coated by silicon dioxide adopt 3- [2- (2-amino ethyl amino) ethyl amino ] propyl trimethoxy silane as an alkylating reagent for surface modification and covalent grafting of amino-rich groups.
Preferably, the preparation method of the amino magnetic bead comprises the following steps:
(1) dispersing the ferroferric oxide nano magnetic bead particles coated with silicon dioxide in an organic solution to enable the concentration of the ferroferric oxide nano magnetic bead particles coated with the silicon dioxide to be 2-7mg/mL, and obtaining ferroferric oxide nano magnetic bead particle dispersion liquid coated with the silicon dioxide;
(2) adding an alkylating reagent 3- [2- (2-aminoethylamino) ethylamino ] propyl trimethoxy silane and ammonia water into the step (1), so that 4-10mmol of alkylating reagent and 0.15-0.2mL of ammonia water are correspondingly added to each 0.1g of silicon oxide coated ferroferric oxide nano magnetic bead particles, and the dispersion state of the nano particles is maintained to obtain a precursor dispersion liquid;
(3) and (3) reacting the precursor dispersion liquid obtained in the step (2) at room temperature to 100 ℃ for 8-12 h to generate alkylation reaction, and washing and drying to obtain the amino magnetic beads.
Preferably, in the preparation method of the amino magnetic beads, the organic solution in the step (1) is preferably Dimethylformamide (DMF), and/or toluene; the particle size of the ferroferric oxide nano magnetic bead particles coated by the silicon oxide is between 35nm and 50 nm.
Preferably, in the preparation method of the amino magnetic beads, the silica-coated ferroferric oxide nano magnetic bead particles are prepared according to the following method:
dispersing nano ferroferric oxide particles into an alcohol-water solution, adding an ethanol solution of tetraethyl orthosilicate and ammonia water to enable each 0.1g of ferroferric oxide powder to be correspondingly added with 0.4-0.6g of tetraethyl orthosilicate and 1-2.5mL of ammonia water to generate sol-gel reaction, washing and drying to obtain the silica-coated ferroferric oxide nano magnetic bead particles, wherein the particle size of the nano ferroferric oxide particles is less than or equal to 30nm, preferably 20nm, and the volume ratio of the alcohol-water solution ethanol to the water is 3:1-4: 1; the concentration of the ethyl alcohol solution of the tetraethyl silicate is between 0.1 and 0.5 g/mL; the sol-gel reaction is carried out under the reaction conditions of maintaining the dispersion of the nano particles and reacting for 8-12 h at the temperature of 60-100 ℃.
According to another aspect of the invention, the application of the amino magnetic bead is provided, which is applied to whole blood DNA extraction.
Preferably, the application of the amino magnetic bead comprises the following steps:
s1, adding DNA binding solution and the amino magnetic beads provided by the invention into the whole blood sample pretreated by protease, uniformly mixing and reacting for 10-40min, and carrying out solid-liquid separation to obtain the amino magnetic beads adsorbed with DNA; 2mg-3mg of the amino magnetic beads are used for every 200 mu L of whole blood sample;
s2, washing with an ethanol water solution; 1-3 mL of ethanol aqueous solution, the concentration of which is 70-75 v/v%, and the washing times are more than or equal to two times, so as to obtain a magnetic bead-DNA compound; the magnetic bead-DNA complex can be directly used for amplification and sequencing, and can also be eluted in step S3;
and S3, adding an eluent into the magnetic bead-DNA compound, eluting DNA, performing solid-liquid separation, and collecting a liquid phase to obtain an extracted whole blood DNA sample.
In general, compared with the prior art, the above technical solution contemplated by the present invention can achieve the following beneficial effects:
the surface bonding amino density of the amino magnetic bead provided by the invention is further improved, the concentration and purity of the adsorbed DNA are obviously improved, and the recovery rate is good.
The amino magnetic beads provided by the invention are used for extracting DNA from whole blood, and can extract a large amount of DNA samples for amplification and detection at one time.
Drawings
FIG. 1 is a diagram showing agarose gel electrophoresis bands for extracting DNA from whole blood using the magnetic beads of example 1 in examples 5 to 7 of the present invention;
FIG. 2 is an elution-free agarose gel electrophoresis strip of whole blood DNA extracted using the magnetic beads of example 1 in examples 8 to 9 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
The invention provides an amino magnetic bead which comprises silicon dioxide coated tetraoxideThe surface of the iron nano-particle is covalently grafted with an amino-rich group, and the density of the bonded amino groups on the surface of the magnetic nano-particle is more than 0.18 mu mol/mg. The amino-rich group is a linear chain of C3 or more, and is preferably-CH2NH-C2H5NH-C2H5NH2Because the amino adsorbs DNA, if the density of the bonded amino on the surface of the magnetic nano particle is too high, the adsorption effect is possibly improved to a limited extent due to sufficient space, and the steric hindrance of DNA adsorption can be reduced by adopting the straight-chain amino-rich group; -CH2NH-C2H5NH-C2H5NH2The groups and amino groups are concentrated and arranged in a straight chain, so that adsorption/separation with DNA is easier to occur, the groups and amino groups are easier to combine with DNA to form a DNA-magnetic bead complex during adsorption reaction, thus the adsorption rate is higher, magnetic beads are easier to separate from DNA during elution reaction, high recovery rate can be easily obtained overall, and even in the case of eluting DNA, PCR amplification can be directly performed by using the DNA-magnetic bead complex.
The particle size of the ferroferric oxide nano particles is less than or equal to 30nm, and the particle size of the ferroferric oxide nano particles coated by the silicon dioxide is between 35nm and 50 nm. The particle size of the silicon dioxide coated ferroferric oxide nano particles is too small, and due to the fact that steric hindrance and intermolecular force are large, the difficulty of grafting amino-rich groups on the surfaces of the silicon dioxide coated ferroferric oxide nano particles is increased, and the reaction is difficult; too large particle size results in a small specific surface area and insufficient contact with DNA during DNA adsorption, resulting in poor final extraction effect.
The preparation method of the amino magnetic bead provided by the invention comprises the following steps:
carrying out surface modification on ferroferric oxide nano magnetic bead particles coated by silicon dioxide by using 3- [2- (2-aminoethylamino) ethylamino ] propyl trimethoxy silane as an alkylating reagent, and covalently grafting amino-rich groups; the method specifically comprises the following steps:
(1) dispersing the ferroferric oxide nano magnetic bead particles coated with silicon dioxide in an organic solution to enable the concentration of the ferroferric oxide nano magnetic bead particles coated with the silicon dioxide to be 2-7mg/mL, and obtaining ferroferric oxide nano magnetic bead particle dispersion liquid coated with the silicon dioxide; the organic solution is preferably Dimethylformamide (DMF), and/or toluene; the particle size of the ferroferric oxide nano magnetic bead particles coated by the silicon oxide is between 35nm and 50 nm;
the silicon dioxide coated ferroferric oxide nano magnetic bead particles are prepared by the following method:
dispersing nano ferroferric oxide particles into an alcohol-water solution, adding an ethanol solution of tetraethyl orthosilicate and ammonia water to enable each 0.1g of ferroferric oxide powder to be correspondingly added with 0.4-0.6g of tetraethyl orthosilicate and 1-2.5mL of ammonia water to generate sol-gel reaction, washing and drying to obtain the silica-coated ferroferric oxide nano magnetic bead particles, wherein the particle size of the nano ferroferric oxide particles is less than or equal to 30nm, preferably 20nm, and the volume ratio of the alcohol-water solution ethanol to the water is 3:1-4: 1; the concentration of the ethyl alcohol solution of the tetraethyl silicate is between 0.1 and 0.5 g/mL; the sol-gel reaction is carried out under the reaction conditions of maintaining the dispersion of the nano particles and reacting for 8-12 h at the temperature of 60-100 ℃.
(2) Adding an alkylating reagent 3- [2- (2-aminoethylamino) ethylamino ] propyl trimethoxy silane and ammonia water into the step (1), so that 4-10mmol of alkylating reagent and 0.15-0.2mL of ammonia water are correspondingly added to each 0.1g of silicon oxide coated ferroferric oxide nano magnetic bead particles, and the dispersion state of the nano particles is maintained to obtain a precursor dispersion liquid;
(3) and (3) reacting the precursor dispersion liquid obtained in the step (2) at room temperature to 100 ℃ for 8-12 h to generate alkylation reaction, and washing and drying to obtain the amino magnetic beads.
The invention provides an amino magnetic bead applied to whole blood DNA extraction, which specifically comprises the following steps:
s1, adding DNA binding solution and the amino magnetic beads provided by the invention into the whole blood sample pretreated by protease, uniformly mixing and reacting for 10-40min, and carrying out solid-liquid separation to obtain the amino magnetic beads adsorbed with DNA; 2-3mg of the amino magnetic beads are used for every 200 mu L of whole blood sample;
s2, washing with an ethanol water solution; 1-2mL of ethanol aqueous solution, wherein the concentration of the ethanol aqueous solution (the concentration of the ethanol adopted by the invention is calculated by volume ratio) is 70-75%, and the washing times are more than or equal to two times, so that a magnetic bead-DNA compound is obtained; the magnetic bead-DNA complex can be directly used for amplification and sequencing, and can also be eluted in step S3;
s3, adding an eluent into the magnetic bead-DNA compound, eluting DNA, performing solid-liquid separation, and collecting a liquid phase to obtain an extracted whole blood DNA sample; 200 to 1000. mu.l of eluent was used per 2.5mg of amino magnetic beads.
In the general whole blood DNA extraction method, the sample volume after elution of the whole blood sample DNA extraction method of 200 mu L is 50-80 mu L, and the concentration can reach the requirement of PCR reaction. The amino magnetic bead provided by the invention has higher recovery rate, 200-1000 mu L of sample required by PCR reaction is finally eluted from 200 mu L of whole blood sample, the DNA concentration is more than 20 mu g/mL, and the sample can be used for multiple experiments or large amplification.
The following are examples:
example 1
The preparation method of the amino magnetic bead provided by the invention comprises the following steps:
carrying out surface modification on ferroferric oxide nano magnetic bead particles coated by silicon dioxide by using 3- [2- (2-aminoethylamino) ethylamino ] propyl trimethoxy silane as an alkylating reagent, and covalently grafting amino-rich groups; the method specifically comprises the following steps:
(1) dispersing the ferroferric oxide nano magnetic bead particles coated with the silicon dioxide in an organic solution to enable the concentration of the ferroferric oxide nano magnetic bead particles coated with the silicon dioxide to be 5.5mg/mL, and obtaining ferroferric oxide nano magnetic bead particle dispersion liquid coated with the silicon dioxide; the organic solution is Dimethylformamide (DMF); the particle size of the ferroferric oxide nano magnetic bead particles coated by the silicon oxide is between 35nm and 50 nm;
the silicon dioxide coated ferroferric oxide nano magnetic bead particles are prepared by the following method:
dispersing nano ferroferric oxide particles into an alcohol-water solution, adding an ethanol solution of tetraethyl orthosilicate and ammonia water, correspondingly adding 0.5g of tetraethyl orthosilicate and 2mL of ammonia water into every 0.1g of ferroferric oxide powder to perform sol-gel reaction, washing and drying to obtain the silica-coated ferroferric oxide nano magnetic bead particles, wherein the particle size of the nano ferroferric oxide particles is 20nm, and the volume ratio of ethanol to water in the alcohol-water solution is 2.5: 1; the concentration of the ethyl alcohol solution of the tetraethyl silicate is between 0.25 g/mL; the reaction conditions of the sol-gel reaction are that the dispersion of the nano particles is maintained and the reaction is carried out for 10 hours at 80 ℃.
(2) Adding an alkylating reagent 3- [2- (2-aminoethylamino) ethylamino ] propyl trimethoxy silane and ammonia water into the step (1), so that 5.4mmol of alkylating reagent and 0.18mL of ammonia water are correspondingly added to each 0.1g of silicon oxide coated ferroferric oxide nano magnetic bead particles, and the dispersion state of the nano particles is maintained to obtain a precursor dispersion liquid;
(3) and (3) reacting the precursor dispersion liquid obtained in the step (2) at 60 ℃ for 10 hours to generate alkylation reaction, and washing and drying to obtain the amino magnetic beads.
The prepared amino magnetic beads are grafted with-CH 2NH-C2H5NH-C2H5NH2 on the surface, and the surface bonding amino density is 0.1884 mu mol/mg.
Example 2
The preparation method of the amino magnetic bead provided by the invention comprises the following steps:
carrying out surface modification on ferroferric oxide nano magnetic bead particles coated by silicon dioxide by using 3- [2- (2-aminoethylamino) ethylamino ] propyl trimethoxy silane as an alkylating reagent, and covalently grafting amino-rich groups; the method specifically comprises the following steps:
(1) dispersing the ferroferric oxide nano magnetic bead particles coated with silicon dioxide in an organic solution to ensure that the concentration of the ferroferric oxide nano magnetic bead particles coated with silicon dioxide is 6.5mg/mL, and obtaining ferroferric oxide nano magnetic bead particle dispersion liquid coated with silicon dioxide; the organic solution is Dimethylformamide (DMF); the particle size of the ferroferric oxide nano magnetic bead particles coated by the silicon oxide is between 35nm and 50 nm;
the silicon dioxide coated ferroferric oxide nano magnetic bead particles are prepared by the following method:
dispersing nano ferroferric oxide particles into an alcohol-water solution, adding an ethanol solution of tetraethyl orthosilicate and ammonia water, correspondingly adding 0.55g of tetraethyl orthosilicate and 2mL of ammonia water into every 0.1g of ferroferric oxide powder to perform sol-gel reaction, washing and drying to obtain the silica-coated ferroferric oxide nano magnetic bead particles, wherein the particle size of the nano ferroferric oxide particles is 30nm, and the volume ratio of ethanol to water in the alcohol-water solution is 4: 1; the concentration of the ethyl alcohol solution of the tetraethyl silicate is between 0.45 g/mL; the reaction conditions of the sol-gel reaction are that the dispersion of the nano particles is maintained and the reaction is carried out for 10 hours at 80 ℃.
(2) Adding an alkylating reagent 3- [2- (2-aminoethylamino) ethylamino ] propyl trimethoxy silane and ammonia water into the step (1), so that 10mmol of alkylating reagent and 0.2mL of ammonia water are correspondingly added to each 0.1g of silicon oxide coated ferroferric oxide nano magnetic bead particles, and the dispersion state of the nano particles is maintained to obtain a precursor dispersion liquid;
(3) and (3) reacting the precursor dispersion liquid obtained in the step (2) at room temperature for 12 hours to generate alkylation reaction, and washing and drying to obtain the amino magnetic beads.
The prepared amino magnetic bead is grafted with-CH on the surface2NH-C2H5NH-C2H5NH2The density of surface-bonded amino groups was 0.1968. mu. mol/mg.
Example 3
The preparation method of the amino magnetic bead provided by the invention comprises the following steps:
carrying out surface modification on ferroferric oxide nano magnetic bead particles coated by silicon dioxide by using 3- [2- (2-aminoethylamino) ethylamino ] propyl trimethoxy silane as an alkylating reagent, and covalently grafting amino-rich groups; the method specifically comprises the following steps:
(1) dispersing the ferroferric oxide nano magnetic bead particles coated with silicon dioxide in an organic solution to enable the concentration of the ferroferric oxide nano magnetic bead particles coated with the silicon dioxide to be 2.2mg/mL, and obtaining ferroferric oxide nano magnetic bead particle dispersion liquid coated with the silicon dioxide; the organic solution is Dimethylformamide (DMF); the particle size of the ferroferric oxide nano magnetic bead particles coated by the silicon oxide is between 35nm and 50 nm;
the silicon dioxide coated ferroferric oxide nano magnetic bead particles are prepared by the following method:
dispersing nano ferroferric oxide particles into an alcohol-water solution, adding an ethanol solution of tetraethyl orthosilicate and ammonia water, correspondingly adding 0.4 of tetraethyl orthosilicate and 1 of ammonia water into every 0.1g of ferroferric oxide powder to perform sol-gel reaction, washing and drying to obtain the silica-coated ferroferric oxide nano magnetic bead particles, wherein the particle size of the nano ferroferric oxide particles is 20nm, and the volume ratio of the alcohol-water solution ethanol to the water is 3: 1; the concentration of the ethyl alcohol solution of the tetraethyl silicate is between 0.15 g/mL; the reaction conditions of the sol-gel reaction are that the dispersion of the nano particles is maintained and the reaction is carried out for 12 hours at the temperature of 60 ℃.
(2) Adding an alkylating reagent 3- [2- (2-aminoethylamino) ethylamino ] propyl trimethoxy silane and ammonia water into the step (1), so that 4mmol of alkylating reagent and 0.15mL of ammonia water are correspondingly added to each 0.1g of silicon oxide coated ferroferric oxide nano magnetic bead particles, and the dispersion state of the nano particles is maintained to obtain a precursor dispersion liquid;
(3) and (3) reacting the precursor dispersion liquid obtained in the step (2) at 60 ℃ for 12hh to perform alkylation reaction, and washing and drying to obtain the amino magnetic beads.
The prepared amino magnetic bead is grafted with-CH on the surface2NH-C2H5NH-C2H5NH2, surface-bonded amino density 0.1933. mu. mol/mg.
Example 4 Whole blood DNA extraction experiment
Using the amino magnetic beads of examples 1 to 3 and magnetic beads 1, 2 and 3 used for comparison, the surface-bonded amino density is shown in the following table:
magnetic bead 1: APTES bonded silicon oxide coated ferroferric oxide
The synthesis method comprises the following steps: 0.1g of silicon oxide coated ferroferric oxide is dispersed in a mixed solution consisting of 95mL of ethanol and 5mL of ethanol by ultrasonic wave for 30min, 0.65mL of APTES is added, and the mixture is mechanically stirred at room temperature for reaction for 12 h.
Magnetic beads 2: EPTES bonded silicon oxide coated ferroferric oxide
The synthesis method comprises the following steps: 0.1g of silicon oxide coated ferroferric oxide is dispersed in 16mL of DMF by ultrasonic wave for 30min, 0.18mL of ammonia water and 2mL of EPTES are added, and the mixture is mechanically stirred at room temperature for reaction for 12 h.
Magnetic bead 3: the method for synthesizing the EDPS bonded silicon oxide coated ferroferric oxide comprises the following steps: 0.1g of silicon oxide coated ferroferric oxide is dispersed in 16mL of DMF by ultrasonic wave for 30min, 0.18mL of ammonia water and 2mL of EDPS are added, and the mixture is mechanically stirred at room temperature for reaction for 12 h.
The four magnetic beads are used for extracting DNA from whole blood as follows:
1. adding 20 μ L TritonX-100 and 20 μ L0.5 μ g/mL proteinase K into 200 μ L whole blood, standing at 56 deg.C for 10 min;
2. adding 1.76mL of DNA binding solution (pH 3.0,10mM Tris-HCl, 1mM EDTA) into the treated whole blood sample, respectively adding more than 2.5mg of four amino bonded magnetic beads, and shaking for 10 min;
washing with 3.70% ethanol for three times, and separating with magnet;
4. adding 200 μ L of DNA eluent (pH10.0,10mM Tris-HCl, 1mM EDTA), 70 deg.C, 500rpm, 20 min;
5.8000rpm for 3min, and taking the supernatant, and determining the DNA concentration and purity, the results are shown in the following table.
| For short | DNA concentration (μ g/mL) | A260/A280 |
| APTES | 25.1 | 1.42 |
| EPTES | 25.0 | 1.59 |
| EDPS | 32.2 | 2.16 |
| Example 1 | 49.0 | 1.85 |
| Example 2 | 47.9 | 1.80 |
| Example 3 | 47.7 | 1.82 |
The A260/A280 index is used to characterize the purity of DNA, and approaching 1.80 indicates that the sample has a higher purity. The table shows that the concentration and purity of the adsorbed DNA are significantly higher than those of the amino magnetic beads for comparison when the amino magnetic beads prepared in examples 1 to 3 of the present invention are subjected to DNA extraction.
The detection sensitivity to nucleic acid samples is high, the recovery rate of extraction calculated by taking salmon sperm DNA as model DNA can reach more than 92 percent, and the recovery rate is obviously higher than that of magnetic beads prepared by other methods. The method can not only adopt an elution method to recover DNA for downstream analysis; the magnetic bead-DNA compound can also be directly used as a template of PCR reaction for PCR amplification, and good effect can be achieved.
Example 5
1. Adding 20 μ L TritonX-100 and 20 μ L0.5 μ g/mL proteinase K into 200 μ L whole blood, standing at 56 deg.C for 10 min;
2. adding 1.76mL of DNA binding solution (pH 3.0,10mM Tris-HCl, 1mM EDTA) into the treated whole blood sample, adding 2.5mg of the amino-bonded magnetic beads of example 1, and shaking for 10 min;
washing with 3.70% ethanol for three times, and separating with magnet;
4. adding 200 μ LDNA eluate (pH10.0,10mM Tris-HCl, 1mM EDTA), 70 deg.C, 500rpm, 20 min;
5.8000rpm for 3min, and taking the supernatant as a DNA template for PCR and RT-PCR.
PCR primer beta-acting sequence
| Name (R) | Sequence of |
| β-actin-F | GGCATGGGTCAGAAGGATT |
| β-actin-R | CACACGCAGCTCATTGTAGA |
PCR reaction System
| Components | Volume of |
| 2×PCRMix | 12.5μL |
| F/R | Each 1 mu L |
| DNA | 0.5μL |
| Sterilized water for injection | 10.5μL |
| Total volume | 25μL |
PCR reaction procedure
Example 6
1. Adding 20 μ L TritonX-100 and 20 μ L0.5 μ g/mL proteinase K into 200 μ L whole blood, standing at 56 deg.C for 10 min;
2. adding 1.76mL of DNA binding solution (pH 3.0,10mM Tris-HCl, 1mM EDTA) into the treated whole blood sample, adding 2.5mg of the amino-bonded magnetic beads of example 1, and shaking for 10 min;
washing with 3.70% ethanol for three times, and separating with magnet;
4. adding 600 μ LDNA eluate (pH10.0,10mM Tris-HCl, 1mM EDTA), 70 deg.C, 500rpm, 20 min;
5.8000rpm for 3min, and taking the supernatant as a DNA template for PCR and RT-PCR.
The PCR primer sequences, PCR reaction system and PCR reaction procedure were the same as in example 5.
Example 7
1. Adding 20 μ L TritonX-100 and 20 μ L0.5 μ g/mL proteinase K into 200 μ L whole blood, standing at 56 deg.C for 10 min;
2. adding 1.76mL of DNA binding solution (pH 3.0,10mM Tris-HCl, 1mM EDTA) into the treated whole blood sample, adding 2.5mg of the amino-bonded magnetic beads of example 1, and shaking for 10 min;
washing with 3.70% ethanol for three times, and separating with magnet;
4. adding 1ml DNA eluent (pH10.0,10mM Tris-HCl, 1mM EDTA, 70 deg.C, 500rpm, 20 min);
5.8000rpm for 3min, and taking the supernatant as a DNA template for PCR and RT-PCR.
The PCR primer sequences, PCR reaction system and PCR reaction procedure were the same as in example 5.
Example 8
1. Adding 20 μ L TritonX-100 and 20 μ L0.5 μ g/mL proteinase K into 200 μ L whole blood, standing at 56 deg.C for 10 min;
2. adding 1.76mL of DNA binding solution (pH 3.0,10mM Tris-HCl, 1mM EDTA) into the treated whole blood sample, adding 2.5mg of the amino-bonded magnetic beads of example 1, and shaking for 10 min;
washing with 3.70% ethanol for three times, and separating with magnet;
4. after air-drying at room temperature, the magnetic bead-DNA complexes were dispersed in 500. mu.L of TE buffer (pH10.0,10mM Tris-HCl, 1mM EDTA);
5. PCR was performed by directly taking 5. mu.L of the dispersion of the magnetic bead-DNA complex as a DNA template.
PCR primer EGFR sequence
| Name (R) | Sequence of |
| EGFR-F | CAGGAGGTGGCTGGTTATGT |
| EGFR-R | AGCTCCTTCAGTCCGGTTTT |
PCR reaction System
PCR reaction procedure
Example 9
1. Adding 20 μ L TritonX-100 and 20 μ L0.5 μ g/mL proteinase K into 200 μ L whole blood, standing at 56 deg.C for 10 min;
2. adding 1.76mL of DNA binding solution (pH 3.0,10mM Tris-HCl, 1mM EDTA) into the treated whole blood sample, adding 2.5mg of the amino-bonded magnetic beads of example 1, and shaking for 10 min;
washing with 3.70% ethanol for three times, and separating with magnet;
4. after air-drying at room temperature, the magnetic bead-DNA complexes were dispersed in 500. mu.L of TE buffer (pH10.0,10mM Tris-HCl, 1mM EDTA);
5. directly taking 5 mu L of the dispersion liquid of the magnetic bead-DNA compound as a DNA template to carry out PCR and RT-PCR.
PCR primer EGFR sequence
| Name (R) | Sequence of |
| EGFR-F | CAGGAGGTGGCTGGTTATGT |
| EGFR-R | AGCTCCTTCAGTCCGGTTTT |
PCR reaction System
| Components | Volume of |
| 2×PCR Mix | 12.5μL |
| F/R | Each 1 mu L |
| DNA | 10μL |
| Sterilized water for injection | 0.5μL |
| Total volume | 25μL |
PCR reaction procedure
It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (10)
1. The amino magnetic bead is characterized by comprising silicon dioxide coated ferroferric oxide nanoparticles, wherein amino-rich groups are covalently grafted on the surfaces of the ferroferric oxide nanoparticles, and the density of bonded amino groups on the surfaces of the magnetic nanoparticles is more than 0.18 mu mol/mg.
2. The amino magnetic bead according to claim 1, wherein the amino-rich group is a linear chain having at least C3.
3. The amino magnetic bead of claim 2, wherein the amino-rich group is-CH2NH-C2H5NH-C2H5NH2。
4. The amino magnetic bead as claimed in claim 1, wherein the particle size of the ferroferric oxide nanoparticle is less than or equal to 30nm, and the particle size of the silica-coated ferroferric oxide nanoparticle is between 35nm and 50 nm.
5. A method for preparing amino magnetic beads according to any one of claims 1 to 4, comprising the steps of:
the ferroferric oxide nano magnetic bead particles coated by silicon dioxide adopt 3- [2- (2-amino ethyl amino) ethyl amino ] propyl trimethoxy silane as an alkylating reagent for surface modification and covalent grafting of amino-rich groups.
6. The method of claim 5, comprising the steps of:
(1) dispersing the ferroferric oxide nano magnetic bead particles coated with silicon dioxide in an organic solution to enable the concentration of the ferroferric oxide nano magnetic bead particles coated with the silicon dioxide to be 2-7mg/mL, and obtaining ferroferric oxide nano magnetic bead particle dispersion liquid coated with the silicon dioxide;
(2) adding an alkylating reagent 3- [2- (2-aminoethylamino) ethylamino ] propyl trimethoxy silane and ammonia water into the step (1), so that 4-10mmol of alkylating reagent and 0.15-0.2mL of ammonia water are correspondingly added to each 0.1g of silicon oxide coated ferroferric oxide nano magnetic bead particles, and the dispersion state of the nano particles is maintained to obtain a precursor dispersion liquid;
(3) and (3) reacting the precursor dispersion liquid obtained in the step (2) at room temperature to 100 ℃ for 8-12 h to generate alkylation reaction, and washing and drying to obtain the amino magnetic beads.
7. The method for preparing amino magnetic beads according to claim 5, wherein the organic solution in step (1) is preferably Dimethylformamide (DMF), and/or toluene; the particle size of the ferroferric oxide nano magnetic bead particles coated by the silicon oxide is between 35nm and 50 nm.
8. The method for preparing amino magnetic beads according to claim 5, wherein the silica-coated ferroferric oxide nano magnetic bead particles are prepared by the following method:
dispersing nano ferroferric oxide particles into an alcohol-water solution, adding an ethanol solution of tetraethyl orthosilicate and ammonia water to enable each 0.1g of ferroferric oxide powder to be correspondingly added with 0.4-0.6g of tetraethyl orthosilicate and 1-2.5mL of ammonia water to generate sol-gel reaction, and washing and drying to obtain the ferroferric oxide nano magnetic bead particles coated by silicon dioxide; the particle size of the nano ferroferric oxide particles is less than or equal to 30nm, preferably 20nm, and the volume ratio of ethanol to water in the alcohol-water solution is 3:1-4: 1; the concentration of the ethyl alcohol solution of the tetraethyl silicate is between 0.1 and 0.5 g/mL; the sol-gel reaction is carried out under the reaction conditions of maintaining the dispersion of the nano particles and reacting for 8-12 h at the temperature of 60-100 ℃.
9. Use of amino magnetic beads according to any of claims 1 to 4, for whole blood DNA extraction.
10. Use of an amino magnetic bead according to claim 9, comprising the steps of:
s1, adding DNA binding solution and the amino magnetic beads provided by the invention into the whole blood sample pretreated by protease, uniformly mixing and reacting for 10-40min, and carrying out solid-liquid separation to obtain the amino magnetic beads adsorbed with DNA; 2mg-3mg of the amino magnetic beads are used for every 200 mu L of whole blood sample;
s2, washing with an ethanol water solution; 1-3 mL of ethanol aqueous solution, the concentration of which is 70-75 v/v%, and the washing times are more than or equal to two times, so as to obtain a magnetic bead-DNA compound; the magnetic bead-DNA complex can be directly used for amplification and sequencing, and can also be eluted in step S3;
and S3, adding an eluent into the magnetic bead-DNA compound, eluting DNA, performing solid-liquid separation, and collecting a liquid phase to obtain an extracted whole blood DNA sample.
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