CN112427027A - Preparation method and application of aminated magnetic microspheres - Google Patents
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- 239000004005 microsphere Substances 0.000 title claims abstract description 58
- 238000002360 preparation method Methods 0.000 title claims abstract description 10
- 238000006243 chemical reaction Methods 0.000 claims abstract description 35
- 238000001179 sorption measurement Methods 0.000 claims abstract description 28
- 238000000034 method Methods 0.000 claims abstract description 27
- 239000002122 magnetic nanoparticle Substances 0.000 claims abstract description 26
- LSHROXHEILXKHM-UHFFFAOYSA-N n'-[2-[2-[2-(2-aminoethylamino)ethylamino]ethylamino]ethyl]ethane-1,2-diamine Chemical compound NCCNCCNCCNCCNCCN LSHROXHEILXKHM-UHFFFAOYSA-N 0.000 claims abstract description 18
- 238000003795 desorption Methods 0.000 claims abstract description 16
- 239000012265 solid product Substances 0.000 claims abstract description 16
- 102000053602 DNA Human genes 0.000 claims abstract description 14
- 108020004414 DNA Proteins 0.000 claims abstract description 14
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- 238000001035 drying Methods 0.000 claims abstract description 6
- 239000000047 product Substances 0.000 claims abstract description 6
- 238000005576 amination reaction Methods 0.000 claims abstract description 5
- 238000007885 magnetic separation Methods 0.000 claims abstract description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 10
- 239000002105 nanoparticle Substances 0.000 claims description 10
- 230000035484 reaction time Effects 0.000 claims description 7
- 229920006395 saturated elastomer Polymers 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 229910045601 alloy Inorganic materials 0.000 claims description 3
- 239000000956 alloy Substances 0.000 claims description 3
- 229920001690 polydopamine Polymers 0.000 claims description 3
- 229910000859 α-Fe Inorganic materials 0.000 claims description 3
- 238000007781 pre-processing Methods 0.000 claims 1
- 239000000463 material Substances 0.000 abstract description 4
- 230000001909 effect on DNA Effects 0.000 abstract 1
- 230000007613 environmental effect Effects 0.000 abstract 1
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- 108020004707 nucleic acids Proteins 0.000 description 5
- 150000007523 nucleic acids Chemical class 0.000 description 5
- 239000007983 Tris buffer Substances 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 3
- 125000004122 cyclic group Chemical group 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000002414 normal-phase solid-phase extraction Methods 0.000 description 2
- 238000001821 nucleic acid purification Methods 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 238000004064 recycling Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 1
- 238000007399 DNA isolation Methods 0.000 description 1
- -1 Poly (2-hydroxyphenyl amines Chemical class 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
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- 238000000576 coating method Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
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- 239000011521 glass Substances 0.000 description 1
- 230000005484 gravity Effects 0.000 description 1
- 231100000086 high toxicity Toxicity 0.000 description 1
- 229910000765 intermetallic Inorganic materials 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- 239000006249 magnetic particle Substances 0.000 description 1
- 230000005389 magnetism Effects 0.000 description 1
- 238000002205 phenol-chloroform extraction Methods 0.000 description 1
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- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
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- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
- B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
- B01J20/28016—Particle form
- B01J20/28021—Hollow particles, e.g. hollow spheres, microspheres or cenospheres
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Abstract
The invention discloses a preparation method and application of aminated magnetic microspheres, and relates to the technical field of deoxyribonucleic acid (DNA) adsorbing materials, wherein the method comprises the following steps: carrying out amination reaction on the pretreated magnetic nanoparticles and pentaethylenehexamine to obtain magnetic composite microspheres; dispersing the magnetic composite microspheres in an alkaline buffer solution, and carrying out stirring reaction with pentaethylenehexamine at room temperature; performing magnetic separation on a product obtained by stirring reaction under the action of a magnetic field to obtain a solid product; and removing impurities from the solid product and drying to obtain the aminated magnetic microsphere. The aminated magnetic microsphere prepared by the method has good adsorption effect on DNA, high adsorption efficiency, environmental friendliness and economy, can be recycled after desorption, and saves the production cost.
Description
Technical Field
The invention relates to the technical field of deoxyribonucleic acid (DNA) adsorption materials, in particular to a preparation method and an application method of aminated magnetic microspheres.
Background
The extraction of nucleic acid from biological samples is a basic technique in the field of life science research such as molecular biology and clinical medicine. The traditional method for extracting nucleic acid, such as phenol-chloroform extraction method, has the defects of high toxicity of organic solvent, more repetitive labor, difficulty in realizing miniaturization and automation operation and the like. In recent years, the solid phase extraction method of nucleic acid using glass powder, ion exchange resin, silica gel or the like as an adsorbing material has been developed, which can not only avoid the use of highly toxic organic solvents, but also greatly improve the extraction efficiency. Nucleic acid purification microchips based on solid phase extraction techniques have attracted considerable attention. As a novel nucleic acid solid phase extracting agent, the magnetic particles can further simplify and accelerate the separation and purification process of nucleic acid by means of a proper magnetic separation device, and realize the miniaturization, automation and parallelization of nucleic acid purification treatment. However, how to improve the specific binding and recycling ability of magnetic microspheres to DNA still needs to be overcome.
Disclosure of Invention
The invention aims to provide a preparation method and application of aminated magnetic microspheres, so as to overcome the defects that an adsorbing material in the prior art is low in adsorption efficiency and cannot be reused.
The technical scheme of the invention is realized through the following aspects:
in a first aspect, a method for preparing an aminated magnetic microsphere is provided, which comprises the following steps:
s1: pretreating magnetic nanoparticles, and carrying out amination reaction on the pretreated magnetic nanoparticles and pentaethylenehexamine to obtain magnetic composite microspheres;
s2: dispersing the magnetic composite microspheres obtained in the step S1 in an alkaline buffer solution, and carrying out stirring reaction with pentaethylenehexamine at room temperature to obtain a reacted product;
s3: performing magnetic separation on the product obtained by the stirring reaction in the step S2 under the action of a magnetic field to obtain a solid product;
s4: and (4) removing impurities from the solid product obtained in the step (S3), and drying to obtain the aminated magnetic microsphere.
Further, the method for pretreating the magnetic nanoparticles in step S1 includes wrapping the magnetic nanoparticles with polydopamine.
Further, in the step S1, the mass ratio of the pretreated magnetic nanoparticles to the pentaethylenehexamine is 1: (1-5).
Further, in step S1, the magnetic nanoparticles are selected from any one or more of alloy nanoparticles, ferrite nanoparticles, and intermetallic nanoparticles.
Further, in the step S2, the reaction time of the stirring reaction is 6 to 10 hours.
Further, in step S4, the method for removing impurities from the solid product includes:
the solid product is washed at least six times with water and/or ethanol.
In a second aspect, there is provided a method for using aminated magnetic microspheres made according to the method of preparation, said method comprising:
and (3) adsorption reaction: putting the aminated magnetic microspheres into deoxyribonucleic acid (DNA) containing to adsorb under a specific environment until the aminated magnetic microspheres are saturated;
and (3) desorption reaction: placing the aminated magnetic microspheres with saturated adsorption into an alkaline buffer solution for desorption operation;
and reusing the aminated magnetic microspheres subjected to the desorption reaction for adsorption reaction.
Further, the reaction time of the adsorption reaction and the desorption reaction is not less than 60 min.
Further, the desorption reaction is performed under the action of a magnetic field.
The invention has the advantages that: the preparation method and the application of the aminated magnetic microsphere comprise (1) the aminated magnetic microsphere is in a positive charge form in a solution and can generate electrostatic interaction with deoxyribonucleic acid (DNA) to generate an adsorption effect, and the adsorption efficiency is up to 95.57%;
(2) the aminated magnetic microspheres have magnetism, can be quickly separated under the condition of an external magnetic field, realize recycling, and can still keep the adsorption rate of the dye above 90 percent after being repeatedly used for 8 times.
Drawings
FIG. 1 is a schematic diagram of the preparation of the aminated magnetic microsphere of the present invention.
FIG. 2 is a graph showing the adsorption effect of the magnetic nanoparticles (pentaethylenehexamine) according to the present invention.
FIG. 3 is a schematic diagram of the cyclic adsorption of the aminated magnetic microsphere of the present invention.
Detailed Description
In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments.
Fe is selected as the magnetic nanoparticles in the following examples3O4For the alkaline buffer, a Tris buffer solution with a pH of 8.5 is used for the description.
As shown in fig. 1 to 3, a method for preparing an aminated magnetic microsphere includes the following steps:
s1: carrying out amination reaction on the pretreated magnetic nanoparticles and pentaethylenehexamine to obtain magnetic composite microspheres;
s2: dispersing the magnetic composite microspheres in an alkaline buffer solution, and carrying out stirring reaction with pentaethylenehexamine at room temperature;
s3: performing magnetic separation on a product obtained by stirring reaction under the action of a magnetic field to obtain a solid product;
s4: and removing impurities from the solid product and drying to obtain the aminated magnetic microsphere.
In this embodiment, the method for pre-treating the magnetic nanoparticles comprises coating the magnetic nanoparticles with polydopamine.
In this embodiment, the mass ratio of the magnetic nanoparticles to the pentaethylenehexamine when performing the amination reaction to obtain the magnetic composite microsphere is 1: 1-1: 5.
in this embodiment, the magnetic nanoparticles comprise at least one of: alloy nanoparticles, ferrite nanoparticles, and intermetallic compound nanoparticles.
In this example, the reaction time of the stirring reaction was 6 to 10 hours.
In this embodiment, the method for removing impurities from the solid product includes:
the solid product is washed at least six times with water and/or ethanol.
A method of using aminated magnetic microspheres, the method comprising:
and (3) adsorption reaction: putting the aminated magnetic microspheres into deoxyribonucleic acid (DNA) containing to adsorb under a specific environment until the aminated magnetic microspheres are saturated;
and (3) desorption reaction: placing the aminated magnetic microspheres with saturated adsorption into an alkaline buffer solution for desorption operation;
and reusing the aminated magnetic microspheres subjected to the desorption reaction for adsorption reaction.
In this example, the reaction time of both the adsorption reaction and the desorption reaction was not less than 60 min.
In this embodiment, the desorption reaction is performed under the action of a magnetic field.
The invention is further illustrated by the following examples:
example 1
Respectively functionalizing according to the mass ratio of the magnetic nanoparticles to the pentaethylenehexamine of 1:1, 1:2, 1:3, 1:4 and 1:5, ultrasonically dispersing the magnetic nanoparticles in a Tris buffer solution with the pH value of 50 mL8.5, adding a certain amount of pentaethylenehexamine, and stirring at room temperature for reacting for 8 hours. And after the reaction is finished, separating by using an external magnetic field to obtain a solid product, repeatedly washing by using water and ethanol to remove impurities, and drying to obtain the aminated magnetic microsphere.
The prepared aminated magnetic microsphere is added into a 50mg/LDNA solution, and after 60min of adsorption, the adsorption effect is shown in figure 2. As can be seen from FIG. 2, the adsorption capacity of the aminated magnetic microsphere is improved with the increase of the mass ratio, and the adsorption capacity reaches the highest value when the mass ratio reaches 1: 3. The mass ratio continues to increase and the maximum adsorption capacity no longer changes significantly. The optimal mass ratio of magnetic nanoparticles to pentaethylenehexamine is 1:3 as illustrated by fig. 2.
Example 2
Respectively functionalizing according to the mass ratio of the magnetic nanoparticles to the pentaethylenehexamine of 1:3, ultrasonically dispersing the magnetic nanoparticles in a Tris buffer solution with the pH value of 50 mL8.5, adding a certain amount of pentaethylenehexamine, and stirring at room temperature for reacting for 8 hours. And after the reaction is finished, separating by using an external magnetic field to obtain a solid product, repeatedly washing by using water and ethanol to remove impurities, and drying to obtain the aminated magnetic microsphere.
Adding the prepared aminated magnetic microspheres into a 50mg/LDNA solution, adsorbing for 60min, then desorbing, and repeating the step of adsorbing DNA; FIG. 3 is a schematic diagram of the cyclic adsorption of the aminated magnetic microsphere, and it can be seen that the removal rate of the dye is still maintained above 90% after the aminated magnetic microsphere is repeatedly used for 8 times.
In summary, the specific gravity of the magnetic nanoparticles and pentaethylenehexamine is 1:3, the adsorption effect of the aminated magnetic microsphere obtained by the reaction is best.
Reference is made to Pan X, Cheng S, Su T, et al Poly (2-hydroxyphenyl amines) functional magnetic polypopamine nanoparticles for high-efficiency DNA isolation applied Surface Science 2019,498: 143888.
It will be appreciated by those skilled in the art that the invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The embodiments disclosed above are therefore to be considered in all respects as illustrative and not restrictive. All changes which come within the scope of or equivalence to the invention are intended to be embraced therein.
Claims (9)
1. A preparation method of aminated magnetic microsphere is characterized by comprising the following steps:
s1: pretreating magnetic nanoparticles, and carrying out amination reaction on the pretreated magnetic nanoparticles and pentaethylenehexamine to obtain magnetic composite microspheres;
s2: dispersing the magnetic composite microspheres obtained in the step S1 in an alkaline buffer solution, and carrying out stirring reaction with pentaethylenehexamine at room temperature to obtain a reacted product;
s3: performing magnetic separation on the product obtained by the stirring reaction in the step S2 under the action of a magnetic field to obtain a solid product;
s4: and (4) removing impurities from the solid product obtained in the step (S3), and drying to obtain the aminated magnetic microsphere.
2. The method for preparing aminated magnetic microsphere according to claim 1, characterized by: the method for preprocessing the magnetic nanoparticles in step S1 includes wrapping the magnetic nanoparticles with polydopamine.
3. The method for preparing aminated magnetic microsphere according to claim 1, characterized by: in the step S1, the mass ratio of the pretreated magnetic nanoparticles to the pentaethylenehexamine is 1: (1-5).
4. The method for preparing aminated magnetic microsphere according to claim 1, characterized by: in step S1, the magnetic nanoparticles are selected from one or more of alloy nanoparticles, ferrite nanoparticles, and intermetallic nanoparticles.
5. The method for preparing aminated magnetic microsphere according to claim 1, characterized by: in the step S2, the reaction time of the stirring reaction is 6-10 h.
6. The method for preparing aminated magnetic microsphere according to claim 1, characterized by: in step S4, the method for removing impurities from the solid product includes:
the solid product is washed at least six times with water and/or ethanol.
7. Use of aminated magnetic microspheres prepared according to the preparation process of any one of claims 1-6, characterized in that it comprises the following applications:
and (3) adsorption reaction: putting the aminated magnetic microspheres into deoxyribonucleic acid (DNA) containing to adsorb under a specific environment until the aminated magnetic microspheres are saturated;
and (3) desorption reaction: placing the aminated magnetic microspheres with saturated adsorption into an alkaline buffer solution for desorption operation;
and reusing the aminated magnetic microspheres subjected to the desorption reaction for adsorption reaction.
8. The use of the aminated magnetic microsphere according to claim 7, characterized in that: the reaction time of the adsorption reaction is not less than 60min, and the reaction time of the desorption reaction is not less than 60 min.
9. The method for applying the aminated magnetic microsphere of claim 7, characterized in that: the desorption reaction is carried out under the action of a magnetic field.
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2020
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