WO2018131826A1 - Method and composition for isolating plasmid dna using amine-coated magnetic nanoparticle - Google Patents

Abstract

The present invention provides an amine-coated magnetic nanoparticle for removing non-soluble aggregates to isolate plasmid DNA from cells and a method for isolating plasmid DNA using the same. An amine-coated magnetic nanoparticle of the present invention has the advantage of being interchangeable with a buffer used in the conventional procedure of plasmid DNA isolation and can be added to a buffer for use in plasmid DNA isolation to remove non-soluble aggregates with the aid of a magnetic field rather than by use of a conventional centrifugation or filtration process, thereby further simplifying a plasmid DNA isolation procedure and improving efficiency.

Description

Plasmid DNA Purification Method and Composition Using Amine-Coated Magnetic Nanoparticles

This patent application claims priority to Korean Patent Application No. 10-2017-0007041 filed with the Korean Intellectual Property Office on January 16, 2017, the disclosure of which is incorporated herein by reference.

The present invention relates to amine-coated magnetic nanoparticles for plasmid DNA separation, and more specifically, to insoluble aggregates such as protein-denatured aggregates, bacterial genomic DNA and cell debris produced during nucleic acid purification. Rather than removing by using a magnetic field using a water-soluble amine-coated magnetic nanoparticles (hydrophilic amine-coated magnetic nanoparticles), the plasmid DNA as a nucleic acid binding magnetic particles to the nucleic acid purification using a magnetic particle separation device The present invention relates to a composition and a method thereof.

Plasmid DNA is cloned independently in the cell and possesses a specific antibiotic resistance gene, which can be selected after introduction into a host, thereby inducing cloning, library construction and screening, protein expression, genetic modification and manipulation, and specific gene expression. It is widely used in various fields such as the production of genetically modified organisms (GMO).

For this reason, high purity and high yield plasmid DNA separation and purification methods have been developed, and nucleic acid purification equipment using a high throughput automation system (HTS) has been developed.

Recently, as a technique for separating and purifying nucleic acids or proteins from biological samples using magnetic particles, and efficiently performing concentration and detection of viral samples using magnetic particles bound to specific antibodies, Roche (Roche) has been developed. HTS nucleic acid purification devices using magnetic particles have also been developed by global bio companies such as Qiagen and Thermomoscientific.

Bacterial plasmid DNA purification generally involves four steps: (a) cell suspension; (b) cell lysis; (c) neutralization and binding; And (d) washing and elution.

In the process of purifying plasmid DNA using the HTS device, in order to increase the efficiency and economic efficiency, the most necessary solution is denatured protein aggregates, cell debris and bacterial genomic DNA generated during the neutralization step after cell lysis. Removal of insoluble aggregates, such as bacterial genomic DNA or chromosomal DNA. Generally. As a method for removing insoluble aggregates, a method using a filter (glass fiber filter, nylon filter, silica, etc.) or a method using a high-speed centrifugation method is used to remove the insoluble aggregates, thus simplifying the conventional plasmid DNA purification process. And methods for solving the process are needed to improve the efficiency.

Various techniques have been developed to solve the problem of insoluble aggregates during plasmid DNA purification. Korean Patent No. 10-1193765 (Ultra High Speed Nucleic Acid Purification Method) discloses a non-powerless nucleic acid purification method using a paper chromatography method, but has a limitation of sample volume, automation, and mass production.

US Patent Publication No. 5665554 (Magnetic bead precipitation method) discloses a method of removing aggregates using ferrite magnetic particles (FMP), but FMP itself is not effective in removing aggregates, and the surface is easily oxidized in the air, so storage stability This has a falling problem.

Throughout this specification, many papers and patent documents are referenced and their citations are indicated. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

We efficiently remove insoluble aggregates such as protein denatured aggregates, genomic DNA and cell debris generated during the purification of plasmid DNA, while providing a material compatible with existing commercially available plasmid DNA purification buffers. Efforts have been made to develop. As a result, when the amine-coated magnetic nanoparticles functionalized with the amine group were added to the plasmid DNA purification buffer, the present invention was completed by confirming that the insoluble aggregates could be effectively removed to separate the plasmid DNA.

Accordingly, it is an object of the present invention to provide amine-coated magnetic nanoparticles.

Another object of the present invention to provide a composition for separating plasmid DNA in cells.

Another object of the present invention is to provide a method for removing insoluble aggregates.

Another object of the present invention is to provide a method for separating plasmid DNA in cells.

Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

According to an aspect of the present invention, the present invention provides an amine-coated magnetic nanoparticle for removing an insoluble aggregate for intracellular plasmid DNA separation, wherein the amine-coated magnetic nanoparticle is a functionalized amine group on the surface of the magnetic nanoparticle. An amine-coated magnetic nanoparticle is provided.

The inventors have developed a material compatible with existing commercially available plasmid DNA purification buffers while efficiently removing insoluble aggregates such as protein denatured aggregates, genomic DNA and cell debris generated during the purification of plasmid DNA. I tried to As a result, it was confirmed that when the amine-coated magnetic nanoparticles functionalized with the amine group were added to the plasmid DNA purification buffer, the insoluble aggregates could be effectively removed to separate the plasmid DNA.

Unlike conventional methods for removing insoluble aggregates by filtration using centrifugation or filters for intracellular plasmid DNA separation, the present invention adds the amine-coated magnetic nanoparticles of the present invention to a buffer for plasmid DNA separation. The main feature is the removal of the insoluble aggregates through a magnetic field.

As used herein, the term "magnetic" refers to the magnetic properties represented by a material. All materials interact with the magnetic field to generate an attractive or repulsive force. That is, when a magnetic field is applied to a material, it is magnetized, and the magnetic material is classified into a ferromagnetic material, a paramagnetic material, a diamagnetic material, a ferrimagnetic material, and the like.

As used herein, the term “nanoparticle” refers to a structure or material having a size in nanometers (nm). The nanometer size is a micron meter ( ^10-6 ) scaled down to a thousandth of a millimeter. When the size of the material is reduced to nanometers, it exhibits a variety of unique physical, chemical, mechanical and electronic properties.

The nanoparticles generally have an average size of 1 to 1000 nm, for example 1 to 10 nm, 10 to 50 nm, 50 to 100 nm, 100 to 250 nm, 250 to 500 nm, 500 to 750 nm, or 50 To 1000 nm.

According to one embodiment of the present invention, the nanoparticles of the present invention may be prepared from 50 to 500 nm, 100 to 400 nm, 100 to 300 nm or 100 to 200 nm. When the size of the nanoparticles is 500 nm or more, the settling speed is increased, causing inconvenience in use, and dispersing agents such as glucose, sucrose, glycerolol, polyethyleneamine, and bitine should be used to prevent sedimentation. The dispersant is not necessary at the nanoparticle size of 250 nm or less, and the magnetic property is weak at 50 nm or less, which leads to a long separation time in the magnetic field. not.

As used herein, the term “magnetic nanoparticle” refers to a nanometer-sized structure or material that is magnetic.

The magnetic nanoparticles may be prepared by solution synthesis, co-precipitation, sol-gel method, high energy pulverization, hydrothermal synthesis, microemulsion synthesis, pyrolysis or sonic chemical synthesis, but is not limited thereto.

The magnetic nanoparticles may be selected from one or two or more selected from the group consisting of iron, cobalt, nickel, oxides thereof and alloys thereof, but is not limited thereto.

According to one embodiment of the invention, the magnetic nanoparticles of the present invention are magnetic nanoparticles made of iron.

As used herein, the term “amine-coated magnetic nanoparticle” refers to magnetic nanoparticles in which an amine group is functionalized on a surface. Functionalization of the amine group is amine additives such as aminopropyltriethoxysilane (APTES), polylysine, glycidoxypropyltrimethoxysilane (GPTS), triethoxy The treatment may be carried out by treating one or two or more selected from the group consisting of silane unethcanoic acid (TETU) and 4-trimethoxysilyl benzaldehyde.

As used herein, the term 'functionalization' means modifying the surface to impart physical, chemical or biological properties to the surface of the material.

As used herein, the term 'functionalization of an amine group' means to impart an amine group by functionalizing the surface of the magnetic nanoparticles with a compound having an amine group.

The amine-coated magnetic nanoparticles may be prepared to functionalize an amine group by treating an amine additive on the surface of the magnetic nanoparticles, or finally to functionalize the amine group on the surface of the magnetic nanoparticles by treating the final coating with the amine additive on the magnetic nanoparticles. have.

According to one embodiment of the invention, the amine-coated magnetic nanoparticles of the invention are prepared by functionalizing amine groups on silica coated magnetic nanoparticles.

As used herein, the term “insoluble aggregate” refers to protein aggregates, cell debris, genomic DNA, and the like, produced during the isolation of plasmid DNA. The insoluble aggregate is a concept encompassing an inclusion body.

Removal of insoluble aggregates using the amine-coated magnetic nanoparticles of the invention can be performed on any cell comprising plasmid DNA known in the art.

According to one embodiment of the invention, said cells are bacteria or yeast.

The amine-coated magnetic nanoparticles of the present invention can be treated in a buffer for plasmid DNA isolation to remove insoluble aggregates.

The method for separating the plasmid DNA can be divided into the following four steps. Preparing a cell suspension; Lysis, protein aggregation and DNA denaturation of cells 2; Neutralizing cellular DNA; And obtaining plasmid DNA 4.

The surfactant contained in the buffer of step 2 removes the phospholipids and proteins contained in the cell membranes, and induces the release of cellular components by bursting the cell membranes. NaOH also denatures plasmid DNA, genomic DNA, and proteins. Potassium acetate contained in the buffer of step 3 regenerates the denatured plasmid DNA into double strands, and more complex genomic DNA is not double stranded and forms insoluble DNA complex with the protein.

Buffer 1 used in step 1 may include 1 to 50 mM of Tris-Cl, 1 to 10 mM of ethylenediaminetetraacetic acid (EDTA) and 0.01 to 0.2 mg / ml of RNaseA. The concentration of Tris-Cl is 10 to 50 mM, 20 to 50 mM, 30 to 50 mM or 40 to 50 mM, and the concentration of EDTA is 2 to 10 mM, 4 to 10 mM, 6 to 10 mM or 8 to 8 10 mM and the concentration of RNaseA may be 0.03 to 0.17 mg / ml, 0.06 to 0.14 mg / ml, or 0.09 to 0.11 mg / ml.

Buffer 2 used in step 2 may include 0.05 to 0.2 N NaOH and 0.5 to 5% of sodium dodecyl sulfate (SDS). The concentration of NaOH is 0.08 to 0.2 N, 0.12 to 0.2 N or 0.16 to 0.2 N, the concentration of the SDS may be 0.5 to 4.5%, 1.5 to 4.0% or 1.5 to 3.5%.

Buffer 3 used in step 3 may include guanidine hydrochloride 2 to 5 M, potassium acetate 0.1 to 1.0 mM, acetic acid 0.5 to 3 M. The concentration of guanidine hydrochloride is 2.5 to 4.5 M, 3.0 to 4.5 M or 3.5 to 4.5 M, and the concentration of potassium acetate is 0.2 to 0.9 mM, 0.3 to 0.8 mM, 0.4 to 0.8 mM, 0.5 to 0.8 mM or 0.6 to 0.8 mM, the concentration of acetic acid may be 1.0 to 3.0 M, 1.5 to 3.0 M or 2.0 to 3.0 M.

Compounds included in the buffers 1 to 3 may be replaced with other compounds having the same function, and may be included in the buffer at an appropriate concentration in some cases.

As demonstrated in the examples below, the optimal conditions for the removal of insoluble aggregates or the intracellular plasmid DNA separation method using the amine-coated nanoparticles of the present invention are shown in Buffer 1 (50 mM Tris-Cl (pH 8.0), 10). mM EDTA, 0.1 mg / ml RNase A), Buffer 2 (0.2N NaOH, 2% SDS) and Buffer 3 (4 M guanidine, 750 mM potassium acetate, 2.5 M acetic acid, 60 mg / ml amine-coated magnetic nanoparticles) )to be.

When the cell to be separated from the plasmid DNA is yeast, the buffer of step 2 may further include a yeast cell wall lysing agent such as a zymolyase to decompose the yeast cell wall.

The amine-coated magnetic nanoparticles of the present invention are compatible with the buffer used in steps 1 to 3 above. That is, the amine-coated magnetic nanoparticles of the present invention may be added to the buffer, and after step 3, the insoluble aggregate may be removed using a magnetic field.

According to one embodiment of the invention, the amine-coated magnetic nanoparticles may be treated with a cell suspension, cell lysate or a reactant in which insoluble aggregates are formed by neutralizing the cell lysate.

According to one embodiment of the present invention, the amine-coated magnetic nanoparticles may be added at a concentration of 1 to 100 mg / ml. According to another embodiment of the invention, the amine-coated magnetic nanoparticles are 10 to 90 mg / ml, 20 to 80 mg / ml, 30 to 70 mg / ml, 40 to 70 mg / ml or 50 to 70 mg / May be added in ml.

According to another aspect of the present invention, the present invention provides a composition for separating plasmid DNA in cells comprising the amine-coated magnetic nanoparticles.

According to one embodiment of the invention, the intracellular plasmid DNA separation composition is a buffer for plasmid DNA separation of the amine-coated magnetic nanoparticles of the present invention.

Since the composition for isolating intracellular plasmid DNA of the present invention includes the amine-coated magnetic nanoparticles, the common content between the two is omitted in order to avoid excessive complexity of the present specification.

According to another aspect of the invention, the invention provides a method for removing an insoluble aggregate comprising the following steps:

(a) treating amine-coated magnetic nanoparticles and insoluble aggregates by treating amine-coated magnetic nanoparticles with neutralized suspensions, lysates or lysates of cells containing plasmid DNA to form insoluble aggregates. Inducing adsorption; And

(b) using magnetic fields to remove adsorbed insoluble aggregates.

According to another aspect of the invention, the invention provides a method for separating plasmid DNA in cells comprising the following steps:

(a) treating the cell suspension comprising plasmid DNA or lysate thereof with amine-coated magnetic nanoparticles to induce adsorption of insoluble aggregates;

(b) using a magnetic field to remove the adsorbed insoluble aggregates;

(c) treating silica-coated magnetic nanoparticles to induce adsorption of plasmid DNA; And

(d) separating the adsorbed plasmid DNA using a magnetic field.

The use of the separation of plasmid DNA of the silica-coated magnetic nanoparticles is known in the art.

Since the method for removing the insoluble aggregates of the present invention and the method for separating plasmid DNA in cells are almost identical to those described for the amine-coated magnetic nanoparticles, the contents in common between the two are described in order to avoid excessive complexity of the present specification. Omit.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides an amine-coated magnetic nanoparticle for removing insoluble aggregates for plasmid DNA separation in cells and a plasmid DNA separation method using the same.

(b) The amine-coated magnetic nanoparticles of the present invention have the advantage of being compatible with buffers used in conventional plasmid DNA separation procedures.

(c) The amine-coated magnetic nanoparticles of the present invention are added in the buffer of the plasmid DNA separation process to further simplify the plasmid DNA separation process by removing the insoluble aggregates using a magnetic field rather than the conventional centrifugation or filtration process. Improve efficiency

Figure 1a is an electrophoresis picture of the results of plasmid DNA purification using two water-soluble magnetic particles.

Figure 1b is a schematic diagram showing the plasmid purification process according to the present invention.

Figure 2 is an electrophoresis picture of the purification results using a water-soluble amine magnetic material prepared by using two different amine coating method.

Figure 3a is an electrophoresis picture of restriction enzyme treatment and PCR for plasmid DNA purification results according to the use of chloroform.

Figure 3b shows the result of restriction enzyme treatment of plasmid DNA purified according to the use of chloroform of Figure 3a.

Figure 4 is an electrophoresis picture of the results of small and large scale purification using the method and composition presented in the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example 1. Purification of Plasmid DNA Isolation Using Two Water-Soluble Magnetic Materials

In order to extract plasmid DNA from bacteria, two plasmid DNAs were introduced as follows.

PUC19-B1 plasmid DNA in which 2.5 kb of bacterial DNA was introduced into the multicloning site (MCS) in the pUC19 vector (2.9 kb), which is a high copy number plasmid, is host cell Escherichia coli. (DH5a) strains were transformed and tested. For low copy number plasmids, nucleic acid purification was performed using E. coli DH5a strain into which a pET28a (5.3 kb) vector was introduced. DH5a reloaded with pUC19 vector was in ampicillin-containing medium, and DH5a with pET28a vector was incubated at 37, 250 rpm in kanamycine-containing medium for 16 hours, followed by miniprep. Experiments were taken with 0.5-2 ml and plasmid DNA purification was performed from 50-200 ml of cultured cells for mass purification (Midi / Maxi prep).

Specifically, 2 ml of transformed Escherichia coli cultured for 16 hours were harvested and dispersed in 250 µl buffer 1 (50 mM Tris-Cl (pH 8.0), 10 mM EDTA, 0.1 mg / ml RNase A). After dispersion, 250 μl buffer solution 2 (0.2N NaOH, 2% SDS) and 200 μl chloroform were added and allowed to stand for 1 minute to dissolve, followed by buffer 3 containing amine-coated magnetic nanoparticles (4 M guanidine hydrochloride, 750 mM potassium acetate, 2.5 M acetic acid, 60 mg / ml lysine coated magnetic nanoparticles or APTES coated magnetic nanoparticles) was shaken up and down 5-6 times after the addition of 350 μl. The reaction solution was left in a magnetic particle separation stand for 1 minute to separate insoluble aggregates into amine-coated magnetic particles, and the remaining supernatant (except chloroform layer) was recovered and transferred to a new tube. 100 μl of silica-coated magnetic nanoparticles (30 mg / ml) was added to the supernatant, combined with plasmid DNA, and then left for 1 minute in a magnetic particle separation stand to recover the remaining solution. The plasmid DNA collected on the silica-coated magnetic nanoparticles was washed in the same manner by adding 800 µl of 80% ethanol and then left at 55 for 10 minutes to dry until residual ethanol was removed. After drying, plasmid DNA was eluted from silica-coated magnetic nanoparticles by adding 100-200 μl TE buffer solution (10 mM Tris-Cl (pH 8.0), 1 mM EDTA) or sterile water and placed on a magnetic particle separation stand. Was attached to recover only the eluted plasmid DNA.

The result of purification of the recovered DNA was confirmed by confirming DNA yield and purity using a spectrophotometer (Table 1), and electrophoresed on a 1% agarose gel (Fig. 1).

-	Concentration (ng / ul)	O.D (260/280)	O.D (260/230)	Total amount (ug)
pUC-B1	103.5	2.06	1.93	10.35
pET28a	85.4	2.09	1.85	8.54

Example 2. Nucleic Acid Purification Results Using Two Amine-Coated Magnetic Nanoparticles

Silica-coated magnetic particles were prepared using Stober method (Stober W, Fink A. Controlled growth of monodisperse silica spheres in the micron size range.J Colloid Interface Sci. 1968; 28: 62-9.). The surface of Fe ₃ O ₄ was coated with silica (SiO ₂ ) to produce hydrolysis.

An amine-coated magnetic nanoparticle was prepared by coating an amine group on the surface of the silica coated magnetic particle. There are various methods of coating the surface with amines, but representative methods include L-lysine (amino acid) and coating method using APTES ((3-Aminopropyl) triethoxysilane). The removal efficiency of the insoluble aggregates of the magnetic nanoparticles prepared using these two amine coating methods and the purity of nucleic acid purification were compared. Surface modification of amines using lysine was carried out using Zehra et al. The method proposed by the group (l-lysine coated magnetite nanoparticles: synthesis, structural and conductivity characterization. J alloys Compd 484: 371-376) was performed. The surface modification method using APTES was performed by Cao et. al. It was performed using the method proposed by the group (Fabrication of Cyclodextrin Functionalized Superparamagnetic Fe3O4 / Amino-Silane Core-Shell Nanoparticles via Layer-by-Layer Method.Applied Surface Science, 255, 7974-7980.). In detail, the amine coating method using L-lysine disperses 100 g of silica-coated magnetic particles in sterile water to add L-lysine to a final concentration of 34 mM, and then adds ammonia to a final concentration of 2.8%. The reaction was produced at room temperature for 24 hours. The amine coating method using APTES was produced by dispersing 100 g of silica-coated magnetic particles in 80% ethanol, adding 1.5% APTES to the final mixture, adding ammonia to a final concentration of 0.56%, and reacting at room temperature for 24 hours.

After performing nucleic acid purification using the two water-soluble amine-coated magnetic particles in the same manner as in Example 1, purity and electrophoresis results were confirmed, but there was no significant difference (Table 2 and FIG. 2).

-	Concentration (ng / ul)	O.D (260/280)	O.D (260/230)	Total amount (ug)
Lysine coating	116.9	2.08	1.82	11.69
APTES coating	110.0	2.06	1.80	11.00

Example 3 Insoluble Aggregate Removal Efficiency According to Input Step of Amine Coating Magnetic Particles in Plasmid DNA Purification Step

In order to determine the removal efficiency of insoluble aggregates when used in other buffers, the following experiment was performed. (a) When the amine-coated magnetic particles were diluted in a neutralizing solution (buffer 3) and used as in Example 1, (b) When the magnetic particles were used in the dissolving solution (buffer 2), (c) Dispersion solution (buffer 1) ) Was used to compare the use of magnetic particles. In order to measure the removal efficiency of the insoluble aggregates, the purification purity of the plasmid DNA was measured after removal by centrifugation and removal using the amine-coated magnetic particles included in the buffer. As a result of plasmid purification by adding the water-soluble amine-coated magnetic particles as described above, the yield was similar as shown in Table 3, but it was best to remove the insoluble aggregate by adding the amine magnetic particles to the neutralizing solution and the dissolving solution.

-	Centrifugation		Buffer 1	Buffer 2	Buffer 3
Total DNA yield (ug)	10.35	10.54	11.35	11.61
O.D. 260/280 nm	2.01	2.06	2.03	2.06
O.D. 260/230 nm	1.83	1.62	1.77	1.80

Example 4. Comparison of Purity of Nucleic Acid Purification with or without Chloroform

When chloroform was not used in plasmid purification, the yield and purity of the nucleic acid purification using a spectrophotometer were similar, but the yield was similar when the 260/230 ratio was treated with chloroform (Table 4).

-	Concentration (ng / ul)	O.D. (260/280)	O.D. (260/230)	Gross
Chloroform Treatment ×	111.5	2.03	1.60	11.15
Chloroform treatment	114.5	2.05	1.84	11.45

Electrophoresis and PCR results and restriction enzyme treatment results did not show a significant difference as shown in Figure 3a and 3b.

Example 5 Purification Process in Mass Culture

In Example 1, the plasmid was purified from the cell culture medium in a small amount of culture solution (0.5-2 ml). Plasmid DNA purification was performed by the method described in Example 1 after collecting cells from 50-200 ml of bulk culture using the compositions and methods disclosed herein. Specifically, cells were collected from 50-100 ml culture medium containing the high cloning plasmid pUC19-B1 vector, and then 2.5 ml suspension buffer, 2.5 ml lysis buffer, 3 ml chloroform and 3.5 ml amine. Neutralization buffer containing a coating magnetic material (neutralization buffer + amine coated magnetic nanoparticle) was treated in the same manner as in Example 1 and purified plasmid DNA (Fig. 4).

As shown in Table 5, there was no significant difference in purification efficiency and purity in small and large-scale cultured cells.

-	Concentration (ng / ul)	O.D. (260/280)	O.D. (260/230)	Gross
pUC19 (2 mL culture)	98.7	2.01	1.85	9.87
	106.9	2.00	1.88	10.69
pUC19 (50 ml culture)	108.4	2.03	1.92	216.8
	105.3	2.05	1.85	210.6

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that such a specific technology is only a preferred embodiment, and the scope of the present invention is not limited thereto. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims (8)

1. An amine-coated magnetic nanoparticle for removing insoluble aggregates for intracellular plasmid DNA separation, wherein the amine-coated magnetic nanoparticle has an amine group functionalized on the surface of the magnetic nanoparticle.
2. The amine-coated magnetic nanoparticle of claim 1, wherein the cell is a bacterium or yeast.
3. 2. The amine-coated magnetic nanoparticle of claim 1 wherein the insoluble aggregate is protein aggregate, cell debris or genomic DNA.
4. 2. The amine-coated magnetic nanoparticle of claim 1, wherein the magnetic nanoparticles are silica coated magnetic nanoparticles.
5. 2. The amine-coated magnetic nanoparticle of claim 1, wherein the amine-coated magnetic nanoparticle is treated with a cell suspension, cell lysate or reactant in which insoluble aggregates are formed by neutralizing the cell lysate.
6. An intracellular plasmid DNA separation composition comprising the amine-coated magnetic nanoparticle of any one of claims 1 to 5.
7. Methods for removing insoluble aggregates include the following steps:

   (a) treating the amine-coated magnetic nanoparticles of any one of claims 1 to 5 with a suspension, lysate of the cells or a reactant in which an insoluble aggregate is formed by neutralizing the lysate. Inducing adsorption of nanoparticles and insoluble aggregates; And

   (b) using magnetic fields to remove adsorbed insoluble aggregates.
8. Intracellular plasmid DNA isolation method comprising the following steps:

   (a) treating the cell suspension comprising plasmid DNA or a lysate thereof with the amine-coated magnetic nanoparticle of any one of claims 1 to 5 to induce adsorption of insoluble aggregates;

   (b) using a magnetic field to remove the adsorbed insoluble aggregates;

   (c) treating silica-coated magnetic nanoparticles to induce adsorption of plasmid DNA; And

   (d) separating the adsorbed plasmid DNA using a magnetic field.

Images