KR101800004B1 - Graphene oxide modified magnetic bead, process for preparing the same and process for nucleic acid extraction using the same - Google Patents

Abstract

The present invention provides magnetic beads modified with graphene oxide, a production method of the magnetic beads, and a separation method for nucleic acids using the same. The magnetic beads modified with oxidized graphene of the present invention can be usefully used for a simple and convenient method for purifying single stranded DNA or RNA in biotechnology where extraction of small molecule RNA such as miRNA is essential.

Description

TECHNICAL FIELD [0001] The present invention relates to magnetic beads modified with graphene oxide, a method for producing the same, and a method for separating nucleic acids using the graphene oxide modified magnetic bead,

The present invention relates to magnetic beads modified with graphene oxide, and more particularly to magnetic beads modified with graphene oxide, a method for producing the same, and a method for separating nucleic acids using the same.

In the field of biotechnology, most of the samples to be tested or analyzed are biological samples. Biological samples contain large amounts of cellular or other impurities such as proteins and carbohydrates, and these complex substances act as an obstacle to test reactions or analytical techniques. That is, a series of techniques such as detection, analysis, nucleotide sequence analysis, amplification, hybridization, and cDNA synthesis, which are mainly performed in the field of biotechnology, can not be performed due to the uncleaned sample, . Therefore, the extraction of nucleic acid (NA) can be performed by many molecular biology techniques such as polymerase chain reaction (PCR), complementary DNA (cDNA) synthesis, sequencing, gene cloning, .

Accordingly, various methods for extracting NA from biological samples have been developed and are generally classified into fluid-phase and solid-phase categories. However, fluid-phase processes have the problem of using phenols and / or chloroform compounds that are generally complex, costly, time-consuming, and especially toxic. In addition, due to the contamination of NA by phenol, there is a possibility to influence subsequent steps such as PCR and restriction enzyme digestion (RED). Therefore, there is a need for the development of simple, rapid, economical, and biocompatible NA extraction methods in a large number of biological research areas, such as mass genetic screening and testing.

In this regard, selective separation methods using solid phase supports including silica, glass fibers and nanoparticles have been reported. Recently, various nanomaterials such as carbon nanotubes and magnetic nanoparticles have been utilized for the separation of biomolecules such as proteins and NA. Particularly, the separation of NA by graphene oxide (GO) sheet has been reported, which suggests that a GO having a large surface area has a problem such as pp stacking, van der Waals force, It is based on the principle that single-stranded DNA can be strongly adsorbed through hydrogen bonding. However, in the double-stranded DNA, only the negatively charged group of phosphate is exposed without exposing the DNA base, so that the adsorption of double-stranded DNA to GO is possible only in the presence of a high concentration of salt. However, this method involves time consuming and complicated processes due to the use of centrifugation.

Magnetic separation is a new technology used in the chemical or biological field. The use of this technology in biological sciences was limited until the 1970s. The concept of using magnetic separation techniques to purify physiologically active substances (nucleic acids, proteins, etc.), cells, and organelles has received renewed interest in the last few decades. Accordingly, magnetic particles having improved characteristics have been newly developed and applied to a part where a complicated separation process was required in the field of biotechnology.

Magnetic-related technology has the advantage of non-contact with biomolecules or cells during the separation process, which has been of interest in bio-applications for decades. The size of magnetic nanoparticles / microparticles is an important factor in successful magnetic separation techniques, and the size of magnetic nano / microparticles is often less than 10 μm for accurate and selective interaction with biological targets (objects). In order to prevent aggregation caused by hydrophobic or magnetic attraction and to easily fix a specific ligand such as an antibody, an aptamer and DNA to a biological structure to be studied, magnetic particles Should be modified with biocompatible inorganic or organic materials.

In addition, magnetic microparticles are increasingly utilized in the biological field in recent years because they are easily massed, time-efficient, cost-effective, and have the advantage of easily separating biological materials using an external magnetic field gradient. In particular, magnetic microspheres are being used extensively in the field of biomedicine as solid supports for protein purification, targeted drug delivery, cell separation, medical diagnosis, immunoassay, DNA sequencing and cell analysis.

A variety of materials have been used as matrices containing superparamagnetic nanoparticles in the form of microbeads comprising mesoporous silica, polysaccharides, poly (ethylene glycols) and many other synthetic polymers. The microbeads thus obtained have scalability and superparamagnetic properties. Nucleic acid can be separated directly from non-purified sample materials such as blood, tissue grinds, culture medium, water, etc. by magnetic techniques. The particles are used in a batch process which is virtually unlimited in terms of sample volume. Since the magnetic properties of solid materials can be controlled, solid materials with magnetic properties can be removed relatively easily and selectively even in viscous sample suspensions. Indeed, magnetic separation is the only possible method for the recovery of small particles (about 0.05-1 μm in diameter), even in the presence of similar sized biological debris and other adherent materials.

Moreover, the efficiency of magnetic separation is particularly suitable for mass-scale purification. In addition, this promising separation technique will contribute to the foundation of a variety of automated low- to high-throughput processes that enable time and cost savings. The centrifugation step can also be avoided and the risk of cross-contamination when using traditional methods can be avoided. Various types of magnetic particles are commercially available for nucleic acid purification and magnetic separators that operate in a manual and automatic mode are available.

The efficiency of the magnetic separation strategy is determined by the coating on the magnetic material. Previous studies have reported that silica-coated or amine-modified magnets isolate low concentrations of DNA from solution. Recently, a new charge-reversible surface chemistry has been developed for magnetic nanoparticles, and it has been reported that DNA can be recovered from a single bacterial cell. However, the separation of DNA, particularly RNA, by GO-modified magnetic beads has not been reported yet.

Korean Patent Publication No. 10-2014-0106268 (2014.09.03.) Korean Patent Publication No. 10-2015-0128612 (Nov. Korean Patent Publication No. 10-2011-0026972 (March 16, 2011)

The present inventors have studied a method for separating RNA from a biological sample more effectively, and have found that when a magnetic bead modified with an oxidized graphene is bound to a single-stranded nucleic acid (particularly RNA) and a dissociation of nucleic acid by urea It is possible to extract, isolate and quantitatively analyze single-stranded nucleic acids, and to extract and isolate small-scale RNA molecules from a sample containing nucleic acid more efficiently than a conventional RNA extraction method.

Accordingly, it is an object of the present invention to provide magnetic beads modified with graphene oxide, a method for producing the same, and a method for separating nucleic acids using the same.

According to one aspect of the present invention, magnetic grains modified with graphene oxide are provided.

In one embodiment, the beads may be 0.001 - 10,000 μm in diameter, may have a molecular weight of 10 ³ - 10 ¹² , and may have a magnetization value of 0.1 - 1,000 emu / g.

In one embodiment, the magnetic beads may be magnetic beads having a silica coating layer modified with graphene oxide, and the silica coating layer may have a thickness of 10 ^-4 - 10 ⁴ μm.

In one embodiment, the graphene oxide may have a thickness of 10 ^-4 - 10 ⁴ μm.

According to another aspect of the invention, there is provided a process for preparing a porous polystyrene-divinylbenzene bead comprising: (a) sulfonating a porous polystyrene-divinylbenzene bead; (b) allowing the sulfonated beads obtained in step (a) to adsorb one or more selected from the group consisting of ferrous salts, ferric salts, and mixtures thereof; And (c) reacting the magnetic beads obtained in step (b) with oxidized graphene.

In one embodiment, between step (b) and step (c), the step of (b-1) further comprises the step of aminating and silica coating the magnetized beads obtained in step (b).

According to still another aspect of the present invention, there is provided a method for producing a magnetic bead comprising: (i) mixing and incubating magnetic beads having a silica coating layer modified with a sample containing a nucleic acid and an oxidized graphene; (Ii) separating the nucleic acid-bead complex from the mixture of step (i) into a magnet; And (iii) adding urea to the nucleic acid-bead complex of step (ii) to dissociate the nucleic acid and the bead.

In one embodiment, the magnetic beads of step (i) may be mixed at a concentration of 10 ^-3 - 10 ³ mg / ml and the single stranded nucleic acid is selected from the group consisting of 5S ribosomal RNA, 18S ribosomal RNA and 28S ribosomal RNA It may be at least one selected.

According to the present invention, extraction and separation of a single-stranded nucleic acid can be achieved by simple test procedures such as binding of a magnetic bead modified with an oxidized graphene to single-stranded nucleic acid (in particular, RNA) and dissociation of nucleic acid by urea, (5S, 18S and 28S ribosomal RNA), particularly 5S ribosomal RNA, from a nucleic acid-containing sample, for example, a cell crushing sample, in comparison with a conventional RNA extraction method. Can be separated.

Thus, the magnetic beads modified with the oxidized graphene of the present invention can be usefully used as a simple and convenient method for purifying single stranded DNA or RNA in biotechnology where extraction of small molecule RNA such as miRNA is essential.

FIG. 1 is a schematic view showing a process for manufacturing magnetic grains having oxidized graphene grains.
2 is a SEM photograph of different magnifications of (A) magnetic beads, (B) aminated and silica coated magnetic beads and (C) oxidative graphene modified magnetic beads (silica coated magnetic beads are 50 mg) .
3 is a TEM photograph of different magnifications of (A) magnetic beads, (B) aminated and silica coated magnetic beads and (C) oxidized graphene modified magnetic beads (silica coated magnetic beads are 50 mg) .
4 is a cross-sectional TEM image of (A) SEM and (B) of (i) aminated and silica coated magnetic beads and (ii) oxidized graphene modified magnetic beads, (C) attenuated total reflectance Fourier transform infrared spectrum of GO, aminated and silica coated magnetic beads and oxidized graphene modified magnetic beads, and (D) The supermagnetic hysteresis loop of the bead.
5 is EDX data of (A) polystyrene, (B) magnetic beads and (C) silica coated magnetic beads.
6 is an attenuated total reflection Fourier transform infrared spectrum of (A) graphene oxide, (B) aminated and silica coated magnetic beads and (C) oxidized graphene modified magnetic beads.
7 is a SEM photograph of oxidized graphene-modified magnetic beads modified with 50 mg of (A), 100 mg of (B), 150 mg of (C), 200 mg of (D) The fused and silica coated magnetic beads are 50 mg).
8 is a TEM photograph of graphene-modified magnetic beads modified with 0 mg of (A), 50 mg of (B), 150 mg of (C), and 250 mg of GO (aminated and silica- Bead is 50 mg).
Figure 9 shows (A) RNA extraction by magnetic material, which comprises (i) a representative RNA extraction procedure, (ii) 15% of RNA extracted by aminated and silica coated magnetic beads and GO- (Iii) Results of radioactivity measurement of RNA extracted by aminated and silica-coated magnetic beads and GO-modified magnetic beads (the radioactivity of the gel-like RNA was exposed to a phosphorimager screen, (B) total RNA extracted from GO-modified magnetic beads from cells at different concentrations of 1 - 10 mg / mL [the amount of RNA in each fraction was calculated from the RNA of each lane UV fluorescence intensity was compared with the control group and the control group represents the RNA extracted using conventional methods of guanidinium thiocyanate-phenol-chloroform extraction (TRIZOL) .

The present invention provides magnetic beads modified with graphene oxide.

In one embodiment, the beads may have a diameter of 0.001 - 10,000 μm, preferably 0.01 - 1,000 μm, more preferably 0.1 - 100 μm, most preferably 1 - 10 μm.

In one embodiment, the beads may have a molecular weight of from 10 ³ to 10 ¹² , preferably from 10 ³ to 10 ⁹ , more preferably from 10 ³ to 10 ⁶ , and most preferably from 10 ³ to 10 ⁵ .

In one embodiment, the material of the beads may be a commonly used resin polymer, such as polystyrene-divinylbenzene (PS-DVB), polystyrene (PS), divinylbenzene (DVB) But are not limited to, poly (methyl methacrylate), polyacetylene, poly (p-phenylene), polypyrrole, polyaniline, but are not limited to, polyisothianaphtene, polyacetylene, polythiophene, and mixtures thereof.

In one embodiment, the beads can be sulfonated for magnetization. The magnetized beads may have a diameter of 0.1 to 100 μm, preferably 0.5 to 50 μm, more preferably 1 to 10 μm. The magnetization value of the magnetized beads may be from 0.1 to 1,000 emu / g, preferably from 1 to 100 emu / g, more preferably from 5 to 10 emu / g.

In one embodiment, the bead is not limited in its shape and may be any shape, such as, for example, spherical, rod-shaped, wire-shaped, planar, amorphous.

In one embodiment, the beads may or may not include a silica coating layer, and it is possible to more easily introduce the graphene oxide when the silica coating layer is included. The magnetic beads not including the silica coating layer can be obtained by nonspecific binding, hydrogen bonding, covalent bonding (when a substance such as aminylsilane is reacted at the same time), and the like. By this reaction, As shown in Fig.

In the case of including a silica coating layer, for example, a spherical magnetic bead may be a core-shell structure in which a silica coating layer surrounds. In one embodiment, the silica coating layer may have a thickness of 10 ^-4 - 10 ⁴ μm, preferably 10 ^-3 - 10 ² μm, more preferably 10 ^-2 - 1 μm.

In one embodiment, the graphene oxide may have a thickness of 10 ^-4 - 10 ⁴ μm, preferably 10 ^-3 - 10 ² μm, more preferably 10 ^-2 - 1 μm.

(A) sulfonating the porous polystyrene-divinylbenzene beads; (b) allowing the sulfonated beads obtained in step (a) to adsorb one or more selected from the group consisting of ferrous salts, ferric salts, and mixtures thereof; And (c) reacting the magnetic beads obtained in step (b) with oxidized graphene.

Step (a) is a step of sulfonating the porous beads. Sulfonation can be performed by a conventional sulfonation method. For example, monodisperse polystyrene-divinylbenzene beads are added to acetic acid, sulfuric acid is added thereto at room temperature, and the reaction is carried out by raising the temperature , And then lowering the temperature to terminate the reaction.

Step (b) is a step of activating the sulfonated bead. The magnetisation can be carried out by conventional methods of magnetization, for example by dispersing the porous beads in the water with the sulfonated macromer and then adding a mixture of ferrous salts and ferric salts And then adsorbing the adsorbent.

Step (c) is a step of modifying the magnetized bead with oxidized graphene. The graphene graphene can be modified by a conventional oxidative graphene modification method. For example, a stabilizer is added to an ultrasonic treated graphene oxide solution, a coupling agent is added to activate the carboxyl group , Followed by addition of aminated and silica-coated magnetic beads.

In one embodiment, between step (b) and step (c), the step of (b-1) further comprises the step of aminating and silica coating the magnetized beads obtained in step (b). In this step, step (b-1) is a step of amidizing and silica coating the magnetized beads. The amination and silica coating can be carried out by conventional amination and silica coating methods, for example, by adding (3-aminopyrrole) triethoxysilane [APTS], ammonium hydroxide solution, tetraethyl And then the mixture is washed and then the resulting beads are dispersed in anhydrous ethanol and APTS and ammonium hydroxide are added and reacted.

In one embodiment, the graphene oxide of step (c) is present in an amount of from 10 ^-6 to 10 ⁴ parts by weight, preferably from 10 ^-3 to 10 ³ parts by weight, based on 1 part by weight of the aminated and silica coated magnetic beads, preferably 1 - ² parts by weight to 10 parts, and most preferably 3 may be the reaction of a mixture of 10 wt parts.

The present invention also relates to a method for producing a magnetic bead comprising: (i) mixing a magnetic bead having a silica coating layer modified with a sample containing a nucleic acid and an oxidized graphene and incubating; (Ii) separating the nucleic acid-bead complex from the mixture of step (i) into a magnet; And (iii) adding urea to the nucleic acid-bead complex of step (ii) to dissociate the nucleic acid and the bead.

In one embodiment, the magnetic beads of step (i) have a concentration of 10 ^-3 to 10 ³ mg / ml, preferably 10 ^-2 to 10 ² mg / ml, more preferably 1 to 10 mg / ml, Preferably 5 - 10 mg / ml.

In one embodiment, the single-stranded nucleic acid may be RNA or DNA, preferably one or more selected from the group consisting of 5S ribosomal RNA, 18S ribosomal RNA, and 28S ribosomal RNA.

In one embodiment, the isolated nucleic acid obtained in step (iii) can be quantified to obtain the concentration or amount of single-stranded nucleic acid in a sample containing the nucleic acid.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the following examples are intended to illustrate the present invention, and the scope of the present invention is not limited thereto.

<Examples>

1. Reagents

Styrene, 2,2'-azobis-isobutyronitrile (AIBN), dibenzoyl peroxide (BPO), dibutyl phthalate (DBP) , Fe ₃ Cl ₃揃 6H ₂ O, FeCl ₂揃 4H ₂ O, sodium dodecyl sulfate (SDS), sulfuric acid, acetic acid, polyvinylpyrrolidone-40, PVP- 40,000), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), N-Hydroxysuccinimide (NHS ), Tetraethylorthosilicate (TEOS), ammonium hydroxide (NH ₄ OH), and (3-aminopropyl) triethoxysilane (APTS) were purchased from Sigma-Aldrich , MO, USA). Graphene oxide (GO) was purchased from Graphene Supermarket, NY, USA.

2. Method

Silica-coated microbeads were prepared according to the prior art (Pham, X.-H. et al., Journal of nanomaterials 2016 , 2016 , 9.). The detailed procedure is as follows.

2.1 Silica Coated Magnetic Bead  Produce

(1) Polystyrene- Divinylbenzene ( PS-DVB) Bead  Produce

Monodisperse macroporous PS-DVB (PSD-DVB) was prepared by a seeded polymerization method. Monodispersed polystyrene (PS) seeds (4 μm) were prepared using the dispersion polymerization method. The dispersion medium was ethanol / 2-methoxyethanol (3: 2) (90 mL) containing 1 g of PVP-40 as a steric stabilizer. AIBN (150 mg) was dissolved in the styrene (15 mL) from which the inhibitor had been removed and then added to the prepared dispersion medium. After ultrasonic treatment for 10 minutes, dispersion polymerization was carried out in a cylindrical reaction chamber while stirring at 120 ° C for 20 hours at 70 ° C. The suspension was centrifuged and the precipitate was washed vigorously with distilled water, followed by repeated centrifugation and vortexing. Finally, the PS seeds were washed with ethanol and vacuum dried overnight (4 urn, 8.3 g) on a PS seed.

The swelling and polymerization process was carried out in a reflux condenser in an oil bath equipped with a glass reactor overhead stirrer and thermostat. Two-step seed polymerization was used to prepare poly (styrene-co-DVB), PS-DVB] beads with monodispersed macromolecular poly (styrene-co-DVB). PS seeds (4 μm, 700 mg) were dispersed in DBP (0.7 mL) emulsified aqueous medium (100 mL) containing 0.25% (w / w) SDS. The obtained dispersion medium was stirred at room temperature (400 rpm) for 20 hours to swell the PS seed together with DBP. A mixture of DVB (2.3 mL) styrene (4.6 mL) in which BPO (240 mg) was dissolved was emulsified for 1 minute in a 0.25% (w / w) SDS aqueous medium (100 mL) using a homogenizer. The emulsified monomer solution was added to the stirred DBP-swollen PS seed dispersion medium. Monomer swelling proceeded at room temperature for 20 h with continuous stirring (400 rpm). After the swelling process, 10% (w / v) PVA aqueous solution in deionized water (10 mL) was added to the dispersion medium and nitrogen gas was purged with nitrogen gas for 30 minutes. Seed polymerization was carried out at 70 DEG C for 20 h while continuously stirring (200 rpm) to obtain monodispersed PS-DVB beads. The polymer beads were washed extensively with lukewarm deionized water (50 &lt; 0 &gt; C) by repeated washing and centrifugation. The beads were then pooled and washed vigorously with ethanol and THF to remove DBP and linear polymer. Finally, the beads were vacuum dried at 30 DEG C for 24 h to obtain macroporous PS-DVB beads (7.5 [mu] m, 2.5 g).

(2) Macroporous  PS-DVB Bead Sulfonation

The sulfonation of PS-DVB beads was performed according to the method reported in the prior art (Chambers, TK et al., J. Chromatogr . A 1998 , 797 , 139-147). Briefly, monodispersed PS-DVB beads (1 g) were added to 5 mL of acetic acid in an ice bath. Sulfuric acid (50 mL) was slowly added to the beads at room temperature, the temperature was raised to 90 &lt; 0 &gt; C and the resin mixture was stirred for 30 minutes to 2 hours. The dispersion was poured into ice water (400 mL) to terminate the reaction and centrifuged to collect the sulfonated PS-DVB beads. The resulting beads were washed vigorously with deionized water by repeated centrifugation-decantation. The sulfonated microspheres were then washed three times with ethanol and dried under vacuum (1.1 g).

(3) Sulfonated Macroporous  PS-DVB Bead Self-assimilation

Sulfonated macroporous PS-DVB beads (500 mg) were dispersed in deionized water (10 mL) at room temperature with mechanical stirring (200 rpm) and purged with nitrogen. A mixture of FeCl ₃ .6H ₂ O (618 mg, 2.26 mmol) and FeCl ₂ .4H ₂ O (257 mg, 1.28 mmol) in deionized water (10 mL) was added to the dispersion and adsorbed. After 2 hours, 28% ammonium hydroxide (50 mL) was added to the bead suspension for 40 minutes while stirring was continued. The magnetized microspheres were separated from the mixture by centrifugation, washed with 25% TFA and then washed vigorously with deionized water and ethanol. Finally, the magnetized microspheres were dried under vacuum (magnetized microspheres, 7.5 [mu] m, 653 mg).

(4) Amination  And silica-coated magnetic Bead  Produce

A solution of APTS [(3-aminopropyl) triethoxysilane] (1% (v / v), 100 mL) was added to 100 mg of sulfonated PS-DVB magnetic beads, Min. Then, ammonium hydroxide (28%, 2 mL) was added to the bead dispersion and mixed at room temperature for 20 minutes. TEOS (Tetraethylorthosilicate, 2 mL) was added to the dispersion and vigorously shaken at room temperature for 12 hours. The resulting beads (silica-coated magnetic beads) were collected with magnets and washed five times with ethanol. The silica-coated magnetic beads (100 mg) were dispersed in absolute ethanol (50 mL). APTS (0.5 mL) and NH ₄ OH (0.5 mL) were added to the resulting silica-coated magnetic bead solution and the dispersed solution was spinned at 180 rpm for 6 hours to surface with amine groups. The aminated and silica-coated magnetic beads were collected with magnets and washed several times with ethanol.

2.2 GO-Modified Magnetic Bead  Produce

(1) Silica Shell  Coated magnetic Bead  GO formula

Oxide graphene (300 mg) was dispersed in distilled water and ultrasonicated at 100 m / min in an ice bath for 9 hours (pulse off for 10 seconds and pulse off for 20 seconds). Thereafter, the graphene oxide solution was filtered with a 0.45 μm membrane to remove large graphene oxide. PVP-40 (300 mg) was added as a stabilizer to the oxidized graphene solution (150 mg). EDC (200 mg) and NHS (200 mg) were added to the oxidized graphene to activate the carboxyl group for 1 hour. The aminated and silica coated magnetic beads (50 mg) were then sprinkled on the activated oxidative graphene and spun overnight. The GO-modified magnetic beads were collected in magnets and washed several times with water and ethanol.

(2) Silica Shell  Absent magnetism Bead  GO formula

Oxide graphene (300 mg) was dispersed in distilled water and ultrasonicated at 100 m / min in an ice bath for 9 hours (pulse off for 10 seconds and pulse off for 20 seconds). Thereafter, the graphene oxide solution was filtered with a 0.45 μm membrane to remove large graphene oxide. A solution of APTS [(3-aminopropyl) triethoxysilane] (1% (v / v), 100 mL) and the above prepared graphene oxide were dissolved in 100 mg of sulfonated PS-DVB magnetic beads And mixed at room temperature overnight. The GO-modified magnetic beads were collected in magnets and washed several times with water and ethanol.

2.3 Cell Culture and GO-Modified Magnetic On the bead  Cellular RNA fraction

A549, a non-small cell lung cancer cell (NSCLC), was cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin at 37 ° C and 5% CO _{2 .} To extract total RNA, 1 × 10 ⁵ cells were harvested and sonicated (Vibra-cell, Sonics & Materials Inc., Newtown, CT; pulse on for 5 s and off for 10 s, 3 times at 20% . RNA was isolated from cellular debris by extraction with acid-phenol: chloroform (pH 4.5, Ambion, Austin, TX, USA) and various amounts of GO-modified magnetic beads were added to the RNA. After incubation at room temperature for 15 minutes, the RNA-GO-modified magnetic bead conjugate was separated from unbound RNA (supernatant 1, Sup. 1) for 30 seconds with magnet. Thereafter, 20 μL of 8M urea was added to the pellet (GO-modified magnetic beads) and further incubated at room temperature for 10 minutes. Unbound RNA (Supernatant 2, Sup. 2) was then separated from the GO-modified magnetic beads (pellet) and each fraction was analyzed by 15% denaturing urea-PAGE and stained with ethidium bromide RNA was visualized.

For the extraction of miRNA (microRNA, microRNA), RNA oligonucleotide (miRNA21, 5'-UAG CUU AUC AGA CUG AUG UUG A-3 ') was chemically synthesized (ST Pharm Co. Ltd., Seoul, Korea ), HPLC and polyacrylamide gel electrophoresis (PAGE). 5'the synthesized RNA with ^{[γ- 32 P] ATP (250} μCi, PerkinElmer, Waltham, MA, USA) by T4 polynucleotide kinase (T4 polynucleotide kinase, New England Biolabs , Beverly, MA, USA) terminal - Lt; / RTI &gt;5'-end-labeled miRNA21 (5 nM) was incubated with varying amounts of GO-modified magnetic beads at room temperature for 15 minutes. miRNA21-GO-modified magnetic bead complexes were separated from unbound RNA (supernatant, Sup.) by magnet for 30 seconds. Each fraction was analyzed with 15% denatured urea-PAGE. The radioactivity of the gel-phase RNA was measured with a Cyclone PhosphorImager (PerkinElmer) by exposure to a phosphorimager screen.

3. Results

3.1 GO-Modified Magnetic Bead  Character analysis

The production process of GO-modified magnetic beads is shown in Fig. That is, a method of magnetizing a uniform porous microbead, coating of a silica layer thereafter, and conjugation of GO was applied. As shown in Fig. 1, the monodisperse super magnet microbeads have a spherical core-shell structure surrounded by a silica layer on the magnetized core PS-DVB beads. Monodispersed polystyrene-divinylbenzene (PS-DVB) beads (7.5 μm, cross-linked with 33% DVB) were prepared using the seed polymerization method reported in the previous literature. The PS seed was prepared in the range of 4.0 to 4.2 μm (61% solids yield) and the molecular weight was 29,000 (measured by GPC analysis).

Final PS-DVB beads were prepared with polymerization of seed (4 μm, 2.5 g, 47% solids yield) using DBP and activated swelling agent as porogens. PS-DVB microbeads are not suitable for adsorbing ferrous and ferric salts in an aqueous solution by themselves. Thus, sulfonate groups were introduced on PS-DVB microbeads to allow PS-DVB microbeads to be miscible in aqueous solutions, and iron salts were introduced into the microbeads due to the influence of the sulfonate groups, . According to a recent report, sulfonated PS-DVB was easily prepared by loading -SO ₃ H ranging from 0.27 to 2.63 mmol / g using sulfonic acid and acetic acid. After activating the sulfonated PS-DVB microbeads with a mixture of ferrous ions and ferric ions (2: 1 molar ratio) in excess basic solution of ammonium hydroxide, Since it turned into light, a precipitate of magnetite was observed in the solution as well as in the bead.

The saturation magnetization value of the obtained beads was 7.6 emu / g, which is relatively lower than that of commercial magnetite (~70 emu / g). However, the magnetized beads exhibit strong magnetic properties and can be sufficiently separated from the solvent within 4 seconds by a 4,000 gauss magnet. The magnetized sulfonated PS-DVB microbeads contain a large number of independent tens of nanometers iron oxide grains, encapsulated on or in the polymer matrix, which can lead to their superparamagnetic behavior (Figs. 2A and 3A).

Magnetic beads were coated with silica shells using APTS and tetraethoxyorthosilicate (TEOS) to facilitate chemical modification of their surfaces with water and using various forms of functional silane compounds. The aminosilane compound APTS tends to promote adhesion of the silane layer on the negatively charged sulfonated beads, thereby facilitating additional base-catalyzed condensation of TEOS from the surface . It was confirmed that this pretreatment significantly increased the silica coating efficiency as compared with the non-pretreatment and (3-mercaptopropyl) triethoxysilane solution of (3-mercaptopropyl) triethoxysilane.

The surface morphology of the beads varied markedly after the sol-gel process (Figs. 2B and 3B), thereby indicating the formation of silica grains on the surface. Cross section analysis showed that the silica shell encapsulated core magnetic beads (Fig. 4A, (i)). Silica coated magnetic beads were of uniform size and could be obtained in grams.

To confirm the presence of a silica coating on magnetic beads, EDX analysis was performed on sulfonated PS-DVB, magnetic PS-DVB and silica coated magnetic PS-DVB beads and the results are shown in FIG. The sulfonated PS-DVB beads contained C, O and S components and the atomic compositions were 86.5%, 9.1% and 4.4%, respectively. After deposition of the magnetic nanoparticles in the sulfonated PS-DVB beads, the O component increased to 20.3%, while the C component decreased to 67.2%. In particular, it was confirmed that the magnetic nanoparticles were successfully deposited on the sulfonated PS-DVB beads with 5.7% of the Fe component present. After the silica shell was coated on the magnetic PS-DVB beads, the silica-coated magnetic PS-DVB beads were C (50.3%), O (33.3%), S (5.9% %). The O-content of the silica-coated magnetic beads increased by 13% compared to the magnetic PS-DVB beads, which is about twice the Si content, suggesting the presence of SiO ₂ on the surface of the magnetic PS-DVB beads. Thus, the EDX data were consistent with the TEM photograph and demonstrated that the silica shell was successfully deposited on the magnetic PS-DVB bead surface.

Next, the surface of the silica-coated magnetic beads was functionalized with an amine group by incubation with APTS in the presence of ammonium. The surface of the aminated and silica coated magnetic beads is shown in Figure 4A- (i). The surfaces of aminated and silica coated magnetic beads are surrounded by small magnetic particles and silica particles. The cross-sectional TEM image of FIG. 4B- (i) shows that the thickness of the magnetic and silica particles is about 20-30 nm.

Meanwhile, the carboxyl group of GO was activated by EDC / NHS. The aminated and silica coated magnetic beads were then spotted onto the activated GO. The presence of GO on the surface of the aminated and silica-coated magnetic particles was confirmed by Fig. 4A- (ii). Unlike aminated and silica coated magnetic beads, the surface of the GO-modified magnetic beads became rough, presumably due to the dissolution of the silica in the aqueous solution (FIG. 2). In particular, the GO modified magnetic beads were covered with partially wrinkled material, suggesting that the GO was successfully covered on the surface of the aminated and silica coated magnetic beads. In fact, the cross-sectional TEM image in Figure 4B- (ii) shows the presence of GO on the surface of the aminated and silica coated magnetic beads. The thickness of GO was about 10 nm. However, as shown in the inset of FIG. 4B- (ii), the GO thickness was non-uniform on the surface of the GO-modified magnetic beads.

To confirm conjugation of GO to aminated and silica coated magnetic beads, the functional groups of the prepared material were analyzed by attenuation total Fourier transform infrared spectroscopy (ATR-FTIR). The spectra of GO, aminated and silica coated magnetic beads and GO modified magnetic beads are shown in Figures 4C and 6. It was found that various oxygen functional groups were present in the GO. The broad band at 3000-3700 cm ^{- 1} and the high absorbance intensity are due to the stretching of the hydroxyl group (-OH). The presence of sp ² -hybridized CC was confirmed by a clear band of 2920 and 2850 cm ^-1 . The unconjugated carbonyl group (COOH) of GO appeared at 1720 (stretching of C = O) and 1360 cm ^-1 (stretching of carboxyCO). The aromatic ring of GO CC appeared at 1620 cm ^-1 . The band at 1217 and 1040-1060 cm ^-1 suggests the presence of GO epoxy (C-OH) and alkoxy (CO), respectively. The spectra of aminated and silica coated magnetic beads were obtained at 3000-3700, 2920, 2850, 1625, 1600, 1560, 1510, 1490, 1450, 1410, 1327, 1150, 1120 cm ^-1 , Suggesting the presence of APTS on the surface of the bead. The modes of vibration near 1625 and 1600 cm ^-1 indicate the presence of imine groups produced by oxidation of the amine bicarbonate. In addition, amines on the surface of silica-coated magnetic beads can react with CO ₂ in the air to form bicarbonate forms, resulting in vibration modes at 1560, 1490 and 1327 cm ^-1 . The asymmetric and symmetric modes of CH ₃ group from the ethoxy moiety of APTS appeared near 1450 and 1410 cm ^-1, respectively, suggesting the presence of ethoxy groups in the APTS. The presence of the mode at 1510 cm ^-1 implies that almost all of the amino groups in the APTS film are linked to the surface silanol through electrostatic interactions and / or hydrogen bonding. Finally, bands at 1120, 1010, and 960 cm ^-1 suggest the presence of SiO ₂ . After the GO was conjugated to the surface of the aminated and silica coated magnetic beads, the band of the GO-modified magnetic beads was similar to that of the aminated and silica coated magnetic beads, as shown in Figure 4C- ( i)), because GO was partially covered on the surface of the magnetic beads. The band at 1600-1650 cm &lt;&quot; ¹ &gt; was stronger and wider, suggesting the formation of amide I (Fig. 4C- (ii)). The vibrations of amide II were in the range of 1513 and 1525 cm ^-1 . In addition, two new bands were observed at 1030 and 1050 cm ^-1 , suggesting the presence of epoxy (C-OH) and alkoxy (CO) in the GO. It was therefore confirmed that GO was successfully conjugated to aminated and silica coated magnetic beads.

Next, the concentration of GO conjugated with aminated and silica coated magnetic beads was optimized in Figures 7 and 8. At low concentrations, the GO was hardly conjugated to magnetic beads, but at higher concentrations (> 150 mg) it became apparent that the GO began modifying the surface of the aminated and silica coated magnetic beads. Therefore, further studies were performed at GO 150 mg.

GO The magnetic beads were analyzed for their magnetization, and the results are shown in FIGS. 4D and 8. The saturation magnetization of the aminated and silica coated magnetic beads was 5.0 emu / g, which is lower than the magnetic bead magnetization (7.6 emu / g) due to the silica coating on the surface of the magnetic beads. However, when GO was conjugated to aminated and silica-coated magnetic beads, the saturation magnetization level was slightly increased to 5.2 emu / g, which is due to the dissolution of the silica layer on the surface of the magnetic beads in the aqueous solution as described above It is.

3.2 GO-Modified Magnetic Bead  RNA extraction using

To extract RNA from A549 non-small cell lung cancer cells (NSCLC) cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin at 37 ° C and 5% CO ₂ , The modified magnetic beads were used. RNA was incubated for 15 minutes to be adsorbed on the surfaces of aminated and silica coated magnetic beads and GO-modified magnetic beads. RNA-GO-modified magnetic bead complexes were isolated from unbound RNA (supernatant 1, Sup. 1) using a magnet for 30 seconds and urea was added to the GO-modified magnetic beads for RNA dissociation. After further incubation, unbound RNA (supernatant 2, Sup. 2) was separated from GO-modified magnetic beads (pellet), each fraction was analyzed with 15% denatured urea-PAGE and the RNA was extracted with ethidium bromide And visualized by staining. The RNA extraction results are shown in Fig. 9A.

When aminated and silica-coated magnetic beads were used as adsorbents, most of the RNA remained in the supernatant (supernatant 1, Sup. 1) and RNA showed little RNA (Fig. 9A- (i)). By contrast, when GO-modified magnetic beads were used to extract RNA from the broth, 5S, 18S and 28S ribosomal RNA bands were evident, indicating that RNA was extracted from the solution. However, the band was relatively inexplicable. In order to confirm the adsorption performance of the substance of the present invention, 5'-terminally labeled miRNA 21 with [γ- ³² P] ATP was used. The radioactivity of RNA extracted by aminated and silica coated magnetic beads and GO-modified magnetic beads was measured by cyclophorphage imager exposure to a phosphorimager screen. As shown in Fig. 9A- (ii), both aminated and silica-coated magnetic beads and GO-modified magnetic beads were found to be able to extract RNA from the solution. In particular, the amount of RNA extracted by GO-modified magnetic beads was greater than that by aminated and silica-coated magnetic beads. Specifically, the miRNA band increased with increasing GO-modified magnetic bead concentration, suggesting that the amount of miRNA extracted from solution increased as the GO-modified magnetic bead concentration used increased.

In addition, GO-modified magnetic beads were applied to total RNA extraction from cells using an easy test procedure, such as binding of RNA to GO-modified magnetic beads and dissociation of RNA by urea. After lysing the cells, the total RNA of the cells was fractionated by increasing the GO-modified magnetic bead concentration to 10 mg / mL (Fig. 9B). When the GO-modified magnetic beads were used at a concentration of 10 mg / ml, the amount of RNA extracted was about 1.7 times larger than that of the control (TRIZOL). In addition, the amount of 5S ribosomal RNA was higher than that of 18S and 28S ribosomal RNA in the total RNA extracted with the GO-modified magnetic beads, whereby the GO-modified magnetic beads used guanidinium thiocyanate It was confirmed that it is suitable for extracting small RNA molecules compared to a conventional RNA extraction method. Thus, the above method using GO-modified magnetic beads could be utilized for extraction of small molecule RNA such as miRNA present in a cell. Since RNA isolation using GO-modified magnetic beads as a mobile solid phase particle candidate has been successfully performed, the method will be utilized as a simple and convenient method for purifying single-stranded DNA or RNA in the biotechnology field.

4. Conclusion

A GO-modified magnetic bead was developed to separate RNA from lysosomal cells. First, magnetic beads were synthesized by seed emulsion polymerization and subsequent acetic acid / H ₂ SO ₄ sulfonation. The prepared sulfonated macroporous beads were amidated in the presence of Fe ^{2 +} / Fe ^{3 +} under basic conditions, and after silica coating, aminated with APTS by sol-gel process. The GO was then activated with EDC / NHS and conjugated with aminated and silica coated magnetic beads. SEM and TEM photographs, EDX, ATR-FTIR, and hysteresis curve techniques confirmed the presence of GO on the surface of silica-coated magnetic beads. The prepared material can be utilized to isolate RNA from the lysed cells. The GO-modified magnetic beads can be used as mobile solid-phase particle candidates used for direct isolation of RNA from biomaterials. The amount of RNA extracted with the GO-modified magnetic beads (10 mg / ml) was 1.7 times higher than that of the control (TRIZOL). Thus, GO-modified magnetic beads will be used as a simple and convenient way to purify single-stranded DNA or RNA in the biotechnology field.

Claims (12)

(a) sulfonating the porous polystyrene-divinylbenzene beads;
(b) allowing the sulfonated beads obtained in step (a) to adsorb one or more selected from the group consisting of ferrous salts, ferric salts, and mixtures thereof;
(b-1) aminating and silica coating the magnetized beads obtained in step (b); And
(c) reacting 1 part by weight of the aminated and silica-coated magnetic beads obtained in the step (b-1) at a ratio of 3 parts by weight or more of the graphene oxide
A magnetic bead having a silica coating layer modified with graphene oxide. The magnetic bead according to claim 1, wherein the bead has a diameter of 0.001 - 10,000 μm. The magnetic bead according to claim 1, wherein the bead has a molecular weight of from 10 ³ to 10 ¹² . The magnetic bead according to claim 1, wherein the magnetic beads have a magnetization value of 0.1 to 1,000 emu / g. delete The magnetic bead according to claim 1, wherein the silica coating layer has a thickness of 10 ^-4 - 10 ⁴ μm. The magnetic bead according to claim 1, wherein the graphene oxide has a thickness of 10 ^-4 - 10 ⁴ μm. delete delete (I) mixing and incubating a sample containing a nucleic acid and the magnetic beads of any one of claims 1 to 4 and 6 to 7;
(Ii) separating the nucleic acid-bead complex from the mixture of step (i) into a magnet; And
(Iii) adding urea to the nucleic acid-bead complex of step (ii) to dissociate the nucleic acid and the bead
&Lt; / RTI &gt; 11. A separation method according to claim 10, characterized in that the magnetic beads of step (i) are mixed at a concentration of 10 ^-3 to 10 ³ mg / ml. [Claim 11] The method according to claim 10, wherein the single stranded nucleic acid is at least one selected from the group consisting of 5S ribosomal RNA, 18S ribosomal RNA, and 28S ribosomal RNA.

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