CN110714044B - Magnetic nanoparticle self-assembly method based on polymerase reaction - Google Patents
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Abstract
The invention provides a magnetic nanoparticle self-assembly method based on polymerase reaction, which relates to the technical field of material chemistry and comprises the following steps: s1: preparing a double-stranded DNA molecule with an amino group by using dCTP modified deoxyribonucleic acid (DNA) with the amino group as a raw material through a polymerase chain reaction; s2: performing surface treatment on the magnetic nanoparticles to prepare carboxylated magnetic nanoparticles; s3: and adding the double-stranded DNA molecule with the amino group into the carboxylated magnetic nanoparticles to obtain a reaction solution, and reacting under the conditions of room temperature and alkalinity to obtain the self-assembled magnetic nanoparticles. The magnetic nanoparticle self-assembly method based on polymerase reaction provided by the invention controls the arrangement of the magnetic nanoparticles by combining the amino groups with the magnetic nanoparticles; the random arrangement of the magnetic nanoparticles can be realized by designing the position of the basic group C in the double-stranded DNA molecule.
Description
Technical Field
The invention relates to the technical field of material chemistry, in particular to a magnetic nanoparticle self-assembly method based on polymerase reaction.
Background
Ordered assembly of nanoparticles is a problem in the field of nanotechnology, where self-assembly of nanoparticles using biomaterials as templates is a very efficient method, while DNA is a very desirable biomaterial for assembly of nanoparticles. Different functional groups are modified on DNA molecules to realize the combination of different nanoparticles and DNA, and then the ordered arrangement of the nanoparticles is realized according to the base complementary pairing principle of the DNA molecules.
The magnetic nanoparticles have four basic effects of common nanoparticles, and also have abnormal magnetic properties, such as superparamagnetism, high coercivity, low Curie temperature, high magnetic susceptibility and the like. The application of magnetic nanoparticles in the fields of targeted drugs, clinical diagnosis, nucleic acid analysis, cell separation, Magnetic Resonance Imaging (MRI) detection, etc. has become a research hotspot.
There have been many international reports on the arrangement of magnetic nanoparticles by DNA, but none of these reports modify amino functional groups at the ends of oligonucleotide molecules, and link nanoparticles to DNA by electrostatic adsorption of amino groups to magnetic nanoparticles. The random arrangement of the nano particles cannot be realized because the nano particles can be modified only at the tail end.
Disclosure of Invention
The invention solves the problem that the random arrangement of the nano particles can not be realized when the nano particles are self-assembled by taking the biological material as a template at present.
In order to solve the above problems, the present invention provides a magnetic nanoparticle self-assembly method based on polymerase reaction, comprising the following steps:
s1: preparing a double-stranded DNA molecule with an amino group by using dCTP modified deoxyribonucleic acid (DNA) with the amino group as a raw material through a polymerase chain reaction;
s2: performing surface treatment on the magnetic nanoparticles to prepare carboxylated magnetic nanoparticles;
s3: and adding the double-stranded DNA molecule with the amino group into the carboxylated magnetic nanoparticles to obtain a reaction solution, and reacting under the conditions of room temperature and alkalinity to obtain the self-assembled magnetic nanoparticles.
Optionally, step S1 includes:
s1-1: respectively measuring the dCTP modified amino group-containing deoxyribonucleic acid, a buffer solution, a primer, polymerase and a DNA template, and placing the dCTP modified amino group-containing deoxyribonucleic acid, the buffer solution, the primer, the polymerase and the DNA template into a centrifugal tube to obtain a PCR reaction system;
s1-2: amplifying the PCR reaction system by a PCR instrument through the following procedures: when the temperature of a hot cover of the PCR instrument reaches 98 ℃, placing the PCR reaction system in the PCR instrument, and operating according to the following annealing conditions: denaturation at 95 ℃ for 30s, annealing at 56 ℃ for 30s, and extension at 72 ℃ for 30s, circulating for 40 cycles, purifying the product by a PCR product column type recovery box, and storing at 4 ℃ to obtain the double-stranded DNA molecule with amino groups.
Alternatively, the dCTP amino group-modified deoxyribonucleic acid, the buffer, the primer, the polymerase, and the DNA template in step S1-1 are present in a volume ratio of 2: 10: 1: 2: 4.
optionally, step S2 includes: and mixing the magnetic nanoparticle solution, the mixed solution of carbodiimide and N-hydroxy thiosuccinimide and the boric acid solution, and reacting at room temperature to obtain the carboxylated magnetic nanoparticles.
Optionally, the concentration of the magnetic nanoparticle solution is 5mg/ml, and the addition amount is 10 μ l; the adding amount of the mixed solution of the carbodiimide and the N-hydroxy thiosuccinimide is 5 mul; the amount of the boric acid solution added was 5. mu.l.
Alternatively, the reaction time in step S2 is 20 min.
Optionally, the magnetic nanoparticles comprise ferroferric oxide nanoparticles.
Optionally, in step S3, sodium borate is included in the reaction solution.
Alternatively, the reaction time in step S3 is 2 h.
Compared with the prior art, the magnetic nanoparticle self-assembly method based on polymerase reaction provided by the invention has the following advantages:
the magnetic nanoparticle self-assembly method based on polymerase reaction provided by the invention introduces amino groups onto base C in double-stranded DNA molecules, and controls the arrangement of magnetic nanoparticles by combining the amino groups with the magnetic nanoparticles; the random arrangement of the magnetic nanoparticles can be realized by designing the position of the basic group C in the double-stranded DNA molecule.
Drawings
FIG. 1 is an agarose gel electrophoresis of a product of the invention;
FIG. 2 is a transmission electron micrograph of a product according to the present invention.
Detailed Description
In order to solve the problem that the random arrangement of the nanoparticles cannot be realized when the nanoparticles are self-assembled by taking a biological material as a template at present, the invention provides a magnetic nanoparticle self-assembly method based on polymerase reaction, which comprises the following steps:
s1: preparing a double-stranded DNA molecule with an amino group by using dCTP modified deoxyribonucleic acid (DNA) with the amino group as a raw material through a polymerase chain reaction;
s2: performing surface treatment on the magnetic nanoparticles to prepare carboxylated magnetic nanoparticles;
s3: adding double-stranded DNA molecules with amino groups into the carboxylated magnetic nanoparticles to obtain reaction liquid, and reacting under the conditions of room temperature and alkalinity to obtain the self-assembled magnetic nanoparticles.
The method for preparing the deoxyribonucleic acid modified by dCTP with the amino group by using dCTP as a raw material specifically means that the deoxyribonucleic acid is used as a raw material, wherein the dCTP is modified with the amino group; wherein dCTP modified amino group deoxyribonucleic acid can be purchased directly; introducing an amino group into a double-stranded DNA molecule by taking dCTP modified deoxyribonucleic acid with the amino group as a raw material to perform polymerase chain reaction; and since the amino group is introduced through the base C on dCTP, the double-stranded DNA molecule is prepared such that the amino group is located at the position of the base C in the double-stranded DNA molecule, i.e., the amino group is not located only at the end of the double-stranded DNA molecule but corresponds to the position of the base C in the double-stranded DNA molecule; when the carboxylated magnetic nanoparticles react with double-stranded DNA molecules with amino groups, the magnetic nanoparticles are combined with the amino groups to realize the self-assembly of the magnetic nanoparticles; since the position of the amino group corresponds to the position of the base C, the arrangement position of the magnetic nanoparticles on the double-stranded DNA molecule also corresponds to the position of the base C. It can be seen that the position of the base C in the double-stranded DNA molecule, that is, the position of the base C in dCTP as a raw material, can be designed to control the arrangement position of the magnetic nanoparticles.
The magnetic nanoparticle self-assembly method based on polymerase reaction provided by the invention introduces amino groups onto base C in double-stranded DNA molecules, and controls the arrangement of magnetic nanoparticles by combining the amino groups with the magnetic nanoparticles; the random arrangement of the magnetic nanoparticles can be realized by designing the position of the basic group C in the double-stranded DNA molecule.
Wherein step S1 specifically includes:
s1-1: respectively measuring dCTP modified amino group-containing deoxyribonucleic acid, a buffer solution, a primer, polymerase and a DNA template, and placing the dCTP modified amino group-containing deoxyribonucleic acid, the buffer solution, the primer, the polymerase and the DNA template in a centrifugal tube to obtain a PCR reaction system;
s1-2: the PCR reaction system was amplified by a PCR instrument by the following procedure: when the temperature of a hot cover of the PCR instrument reaches 98 ℃, placing the PCR reaction system in the PCR instrument, and operating according to the following annealing conditions: denaturation at 95 ℃ for 30s, annealing at 56 ℃ for 30s, and extension at 72 ℃ for 30s, circulating for 40 cycles, purifying the product by a PCR product column type recovery box, and storing at 4 ℃ to obtain the double-stranded DNA molecule with amino groups.
In step S1-1, the volume ratio of the amino group-modified dCTP to the deoxyribonucleic acid, buffer, primer, polymerase and DNA template is 2: 10: 1: 2: 4.
wherein, the buffer solution, the primer, the polymerase and the DNA template in the step S1-1 are all related substances commonly used in PCR reaction, and are not described herein again.
During PCR amplification, bases A-T, C-G are paired to amplify a DNA double-stranded molecule with a plurality of amino sites, and the amino position can be controlled by designing the position of the base C.
Step S2 includes: mixing the magnetic nanoparticle solution, a mixed solution of carbodiimide (EDC) and N-hydroxythiosuccinimide (sulfo-NHS) and a boric acid solution, and reacting at room temperature to obtain the carboxylated magnetic nanoparticles.
The magnetic nanoparticles are subjected to surface treatment through the reaction, carboxyl groups are activated, sulfonate is generated, and the carboxylated magnetic nanoparticles are obtained.
Preferably, the concentration of the magnetic nanoparticle solution in the step is 5mg/ml, and the addition amount is 10 mul; the adding amount of the mixed solution of carbodiimide and N-hydroxy thiosuccinimide is 5 mul; the addition amount of the boric acid solution is 5 mul; the reaction time in step S2 was 20 min; the magnetic nanoparticles herein include ferroferric oxide nanoparticles.
Adding double-stranded DNA molecules with amino groups into the carboxylated magnetic nanoparticles to obtain reaction liquid, and reacting under the conditions of room temperature and alkalinity to obtain the self-assembled magnetic nanoparticles.
Further, double-stranded DNA molecules with amino groups are added into the carboxylated magnetic nanoparticles for reaction, so that the carboxylated magnetic nanoparticles are combined with the amino groups in the double-stranded DNA molecules with the amino groups, the magnetic nanoparticles are introduced through the amino groups in the double-stranded DNA molecules, the magnetic nanoparticles are sequenced according to the positions of the amino groups, the position control of the magnetic nanoparticles is further realized, and the self-assembled magnetic nanoparticles are obtained.
The reaction is carried out under the alkalescent condition, and the PH of the reaction solution is adjusted by adding sodium borate; the reaction time in step S3 was 2 h.
The principle of the magnetic nanoparticles reacting with the PCR product to realize self-assembly is shown as the following formula:
in order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below.
Example one
S1-1: respectively measuring 2 mul of dCTP modified deoxyribonucleic acid with amino groups, 10 mul of tris-HCl buffer solution with the concentration of 0.05mol/L, 1 mul of primer, 2 mul of polymerase and 4 mul of DNA template, placing the obtained mixture in a centrifugal tube of 0.6ml, and adding water until the total volume is 100 mul; evenly distributing 100 mul of sample into 2 centrifugal tubes of 0.2ml to obtain a PCR reaction system;
in this step, the primers comprise:
an upstream primer: 5'-GACTGCCACTTCCTCGGATT-3'
A downstream primer: 5'-TCACAGTCACAGGCACAGGA-3', respectively;
s1-2: the PCR reaction system was amplified by a PCR instrument by the following procedure: when the temperature of a hot cover of the PCR instrument reaches 98 ℃, the PCR reaction system is placed in the PCR instrument and operated according to the following annealing conditions: denaturation at 95 ℃ for 30s, annealing at 56 ℃ for 30s, and extension at 72 ℃ for 30s, circulating for 40 cycles, purifying the product by a PCR product column type recovery box, and storing at 4 ℃ to obtain the double-stranded DNA molecule with amino groups.
S2: adding 10 mu l of ferroferric oxide nanoparticles with the concentration of 5mg/ml, 5 mu l of mixed solution of carbodiimide (EDC) and N-hydroxy thiosuccinimide (sulfo-NHS) and 5 mu l of boric acid solution into a centrifugal tube with the concentration of 0.6ml for mixing, and reacting for 20min at room temperature to obtain carboxylated magnetic nanoparticles;
s3: adding double-stranded DNA molecules with amino groups into the carboxylated magnetic nanoparticles, adding a sodium borate solution to adjust the pH value to 7-8.2 to obtain a reaction solution, reacting for 2 hours at room temperature, placing the reaction solution on a magnetic frame, and separating the magnetic nanoparticles from the reaction solution to obtain the self-assembled magnetic nanoparticles.
The product obtained in step S3 was detected, and as shown in fig. 1, according to the agarose gel electrophoresis pattern of the product, the amplification was successfully performed by PCR reaction in this example, and a double-stranded DNA molecule was prepared.
Further, the transmission electron microscopy of the product is performed, as shown in fig. 2, it can be seen from the transmission electron microscopy of the product that the magnetic nanoparticles are self-assembled in the product prepared in this embodiment.
According to the detection result, the self-assembled magnetic nanoparticles are successfully prepared by the method provided by the implementation, and the ordering of the magnetic nanoparticles is randomly controllable.
Example two
S1-1: respectively measuring 4 mul of dCTP modified deoxyribonucleic acid with amino groups, 20 mul of tris-HCl buffer solution with the concentration of 0.05mol/L, 2 mul of primer, 4 mul of polymerase and 8 mul of DNA template, placing the obtained mixture in a centrifugal tube of 0.6ml, and adding water until the total volume is 100 mul; evenly distributing 100 mul of sample into 2 centrifugal tubes of 0.2ml to obtain a PCR reaction system;
in this step, the primers comprise:
an upstream primer: 5'-GACTGCCACTTCCTCGGATT-3'
A downstream primer: 5'-TCACAGTCACAGGCACAGGA-3', respectively;
s1-2: the PCR reaction system was amplified by a PCR instrument by the following procedure: when the temperature of a hot cover of the PCR instrument reaches 98 ℃, the PCR reaction system is placed in the PCR instrument and operated according to the following annealing conditions: denaturation at 95 ℃ for 30s, annealing at 56 ℃ for 30s, and extension at 72 ℃ for 30s, circulating for 40 cycles, purifying the product by a PCR product column type recovery box, and storing at 4 ℃ to obtain the double-stranded DNA molecule with amino groups.
S2: adding 10 mu l of ferroferric oxide nanoparticles with the concentration of 5mg/ml, 5 mu l of mixed solution of carbodiimide (EDC) and N-hydroxy thiosuccinimide (sulfo-NHS) and 5 mu l of boric acid solution into a centrifugal tube with the concentration of 0.6ml for mixing, and reacting for 20min at room temperature to obtain carboxylated magnetic nanoparticles;
s3: adding double-stranded DNA molecules with amino groups into the carboxylated magnetic nanoparticles, adding a sodium borate solution to adjust the pH value to be alkalescent 7-8.2 to obtain a reaction solution, reacting for 2 hours at room temperature, placing the reaction solution on a magnetic frame, and separating the magnetic nanoparticles from the reaction solution to obtain the self-assembled magnetic nanoparticles.
And detecting the product obtained in the step S3, wherein the specific detection process refers to relevant contents in the first embodiment, and details are not repeated herein.
According to the detection result, the self-assembled magnetic nanoparticles are successfully prepared by the method provided by the implementation, and the ordering of the magnetic nanoparticles is randomly controllable.
Although the present disclosure has been described above, the scope of the present disclosure is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present disclosure, and such changes and modifications will fall within the scope of the present invention.
Claims (9)
1. A magnetic nanoparticle self-assembly method based on polymerase reaction is characterized by comprising the following steps:
s1: preparing a double-stranded DNA molecule with an amino group by using dCTP modified deoxyribonucleic acid (DNA) with the amino group as a raw material through a polymerase chain reaction;
s2: performing surface treatment on the magnetic nanoparticles to prepare carboxylated magnetic nanoparticles;
s3: adding the double-stranded DNA molecules with amino groups into the carboxylated magnetic nanoparticles to obtain a reaction solution, and reacting at room temperature under an alkaline condition to obtain self-assembled magnetic nanoparticles;
the amino group is introduced via base C on dCTP.
2. The polymerase reaction-based magnetic nanoparticle self-assembly method of claim 1, wherein step S1 comprises:
s1-1: respectively measuring the dCTP modified amino group-containing deoxyribonucleic acid, a buffer solution, a primer, polymerase and a DNA template, and placing the dCTP modified amino group-containing deoxyribonucleic acid, the buffer solution, the primer, the polymerase and the DNA template into a centrifugal tube to obtain a PCR reaction system;
s1-2: amplifying the PCR reaction system by a PCR instrument through the following procedures: when the temperature of a hot cover of the PCR instrument reaches 98 ℃, placing the PCR reaction system in the PCR instrument, and operating according to the following annealing conditions: denaturation at 95 ℃ for 30s, annealing at 56 ℃ for 30s, and extension at 72 ℃ for 30s, circulating for 40 cycles, purifying the product by a PCR product column type recovery box, and storing at 4 ℃ to obtain the double-stranded DNA molecule with amino groups.
3. The method for self-assembly of magnetic nanoparticles based on polymerase reaction according to claim 2, wherein the volume ratio of dCTP-modified amino-group deoxyribonucleic acid, the buffer, the primer, the polymerase and the DNA template in step S1-1 is 2: 10: 1: 2: 4.
4. the polymerase reaction-based magnetic nanoparticle self-assembly method of claim 1, wherein step S2 comprises: and mixing the magnetic nanoparticle solution, the mixed solution of carbodiimide and N-hydroxy thiosuccinimide and the boric acid solution, and reacting at room temperature to obtain the carboxylated magnetic nanoparticles.
5. The method for self-assembling magnetic nanoparticles based on polymerase reaction of claim 4, wherein the concentration of the magnetic nanoparticle solution is 5mg/ml, and the addition amount is 10 μ l; the adding amount of the mixed solution of the carbodiimide and the N-hydroxy thiosuccinimide is 5 mul; the amount of the boric acid solution added was 5. mu.l.
6. The polymerase reaction-based magnetic nanoparticle self-assembly method of claim 4, wherein the reaction time in step S2 is 20 min.
7. The polymerase reaction-based magnetic nanoparticle self-assembly method of any one of claims 1 to 6, wherein the magnetic nanoparticles comprise ferroferric oxide nanoparticles.
8. The polymerase reaction-based magnetic nanoparticle self-assembly method of claim 1, wherein the reaction solution in step S3 comprises sodium borate.
9. The polymerase reaction-based magnetic nanoparticle self-assembly method of claim 1, wherein the reaction time in step S3 is 2 h.
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