KR20210152114A - Method of preparing silica-coated iron oxide nanobeads and its use for separating nucleic acids from sample under magnetic field - Google Patents

Abstract

The present invention relates to Fe_3O_4@SiO_2 core-shell nanobeads having a silica shell thickness of about 10 nm and a diameter of about 50-60 nm, and a method for preparing the same. The nanobeads prepared by the method according to the present invention provide high saturation magnetization, and the silica shell exhibits high binding force to nucleic acids. In addition, as a result of in vitro experiments, the nanobeads has biocompatibility without cytotoxicity, and thus can be effectively used for separation of nucleic acids from various samples.

Description

Method of preparing silica-coated iron oxide nanobeads and its use for separating nucleic acids from sample under magnetic field

The present application relates to a technology for isolating or purifying a nucleic acid from a sample.

Superparamagnetic iron oxide nanobeads are considered promising nanomaterials in biomedical science because they can be applied to various biomedical fields, such as magnetic resonance imaging, hyperthermia for cancer treatment, and magnetic separation of biological molecules. The high saturation magnetization of iron oxide nanobeads is essential for these applications.

Isolation of DNA and RNA from biological samples is the most important and essential first step in experiments in the field of molecular biology. In particular, in the field of clinical medicine, isolated nucleic acids are used in various fields, such as diagnosing whether a patient is infected, diagnosing genetic diseases, and identifying hereditary characteristics. Various methods for separating nucleic acids, such as centrifugation, ethanol precipitation, and chromatography, have been developed in the past, but each method has advantages and disadvantages. The separation method using magnetism is a quick and convenient method compared to the conventional method, but there is a problem in that the saturation magnetization of the iron oxide nanobeads is not high.

Republic of Korea registered patent 1646610 (published on August 9, 2016)

An object of the present application is to provide a magnetic nanobead having a high saturation magnetization and exhibiting a high binding force to a nucleic acid.

In one aspect, the present application provides a method for preparing silica-coated magnetic nanobeads having a particle diameter of 50 to 60 nm and a silica shell thickness of 10 nm, Fe ₃ O _{4 core, comprising the following steps.}

In one embodiment, the method according to the present invention comprises the steps of (a) reacting 0.025 _{mM FeSO 4} ·7H ₂ O in water with 500 mM NaOH aqueous solution at room temperature with stirring; (b) then allowing the solution to stand at 4° C. to produce a precipitate of _{Fe 3} O _{4 nanobeads;} (c) after generating the precipitate, removing the supernatant from the solution, washing the precipitate with water, and suspending in water to prepare a _{Fe 3} O _{4 nanobead suspension;} (d) _{adding the Fe 3} O ₄ nanobead suspension to the SiO ₂ nanobead solution in a volume ratio of 1:3, wherein the Fe ₃ O ₄ nanobeads to the SiO ₂ nanobeads are included in a weight ratio of 1:3, ultrasonicating the solution at room temperature; (e) allowing the sonicated solution to stand at 4° C. to produce a precipitate of _{silica-coated Fe 3} O _{4 nanobeads;} and (f) after the precipitate of step (e) is generated, removing the supernatant, and washing the precipitate with ethanol and water, to obtain _{silica-coated Fe 3} O _{4 nanobeads.}

_{In one embodiment, the SiO 2} nanobead solution used in the method according to the present application is prepared by mixing ethanol, tertiary distilled water, 18M NH ₄ OH and TEOS tetraethoxysilane (TEOS) in a volume ratio of 5: 1: 1.7: 0.6. .

In another aspect, there is provided a silica-coated magnetic nanobead having _{a Fe 3} O ₄ core prepared by the method according to the present disclosure.

In another aspect, the present application provides a method for isolating a nucleic acid, characterized in that the magnetic nanobead prepared according to the method of the present application is used as a nucleic acid-binding carrier.

Nucleic acids separable using the method or nanobeads according to the present disclosure include DNA, or RNA.

In another aspect, the present application provides a composition or kit for nucleic acid isolation comprising a silica-coated magnetic nanobead having _{a Fe 3} O ₄ core prepared by the method of the present application.

_{Fe 3} O ₄ @SiO ₂ core-shell nanobeads prepared according to the method of the present application are magnetic particles having a silica shell thickness of ~10 nm and a diameter of 50-60 nm. The nanobeads prepared by the method according to the present application have a large iron oxide core to provide high saturation magnetization (= 33.80 emu/g), and the silica shell exhibits high binding force to nucleic acids. In addition, as a result of in vitro experiments, it has biocompatibility without cytotoxicity, and thus can be effectively used for separation of nucleic acids from various samples.

1 schematically shows the synthesis of Fe ₃ O ₄ @SiO ₂ core-shell nanobeads.
Figure 2 is Fe ₃ O ₄ @SiO ₂ core-shell nanobeads a. HRTEM images; b. HAADF-STEM image, c. Fe; d. O; e. Si element and f. It is a mapping image of all elements (Merge).
Figure 3 is a bare Fe ₃ O ₄ nano-beads (see below), and Fe ₃ O ₄ @SiO ₂ shows a core-shell X-Ray diffraction pattern of the Fe ₃ O ₄ nano-beads bare nano-beads (above). The assigned values are: (hkl) Miller indices: 2θ(hkl) = 18.31°(111), 30.13°(220), 35.37°(311), 37.02°(222), 43.14°(400), 53.39° (422), 56.85°(511), 62.57°(440), 70.82°(620), 73.88°(533), 74.96°(622), 78.93°(444), 86.78°(642), 89.70°(731) ), 94.48° (800).
4 shows FT-IR absorption spectra _{of bare Fe 3} O ₄ nanobeads (top) and _{bare Fe 3} O _{4 nanobeads of Fe 3} O ₄ @SiO ₂ core-shell nanobeads (bottom). The vertical dotted lines indicate absorption peaks due to Fe-O and Si-O-Si stretching vibrations, respectively.
5 shows the MH curves of bare Fe ₃ O ₄ nanobeads and Fe ₃ O ₄ @SiO ₂ core-shell nanobeads.
6 shows the in vitro cell viability of DU145 and NCTC1469 cells treated with _{Fe 3} O ₄ @SiO _{2 core-shell nanobeads.}
7 is a result of performing a LAMP reaction, a type of amplification reaction, using a nucleic acid isolated using _{Fe 3} O ₄ @SiO ₂ core-shell nanobeads prepared according to the present application: 1: Sample No. 3 without LAMP and 2-7 LAMP reaction progress [2: Sample No. One; 3: Sample No. 2; 4: Sample No. 3; 5: Sample No. 4; 6: Sample No. 5; 7: Control (containing only LAMP reaction solution without nucleic acid)].
Figure 8 shows the fluorescence curve in real-time amplification according to the cycle (1 cycle = 30 s): a. Nucleic acid isolated from A549 non-small lung cancer cell sample, b. nucleic acid isolated from a dengue virus sample, and c. Control (containing only LAMP reaction solution without nucleic acid). RFU = relative fluorescence unit.

The present application is based on the development of a method for manufacturing silica-coated magnetic nanobeads having _{a Fe 3} O ₄ core that provides high saturation magnetization and a silica shell exhibits high binding force to nucleic acids.

Accordingly, in one embodiment, the present application provides a silica-coated magnetic nanobead having a _{Fe 3} O ₄ core having a particle diameter of 50 to 60 nm, a silica shell thickness of 10 nm, and a saturation magnetization of 33.80 emu/g.

In another aspect, the present application provides a method for manufacturing silica-coated magnetic nanobeads having _{an Fe 3} O ₄ core according to the present application, comprising the following steps.

In one embodiment, the method comprises the steps of (a) reacting 0.025 _{mM FeSO 4} ·7H ₂ O in water and 500 mM NaOH aqueous solution at room temperature with stirring; (b) then allowing the solution to stand at 4° C. to produce a precipitate of _{Fe 3} O _{4 nanobeads;} (c) after generating the precipitate, removing the supernatant from the solution, washing the precipitate with water, and suspending in water to prepare a _{Fe 3} O _{4 nanobead suspension;} (d) _{adding the Fe 3} O ₄ nanobead suspension to the SiO ₂ nanobead solution in a volume ratio of 1:3, wherein the Fe ₃ O ₄ nanobeads to the SiO ₂ nanobeads are included in a weight ratio of 1:3, ultrasonicating the solution at room temperature; (e) allowing the sonicated solution to stand at 4° C. to produce a precipitate of _{silica-coated Fe 3} O _{4 nanobeads;} and (f) after the precipitate of step (e) is generated, removing the supernatant, and washing the precipitate with ethanol and water, to obtain _{silica-coated Fe 3} O _{4 nanobeads.}

_{The SiO 2} nanobead solution used for silica coating in the method according to the present application can be prepared by the Stober method as described in the examples herein. In one embodiment, ethanol, tertiary distilled water, 18M NH ₄ OH and TEOS (tetraethoxysilane) are mixed in a volume ratio of 5: 1: 1.7: 0.6.

_{The silica-coated magnetic nanobeads having a Fe 3} O ₄ core prepared according to the present method have a particle diameter of about 50 to 60 nm, a silica shell thickness of about 10 nm, and exhibit a high saturation magnetization of 33.80 emu/g. In addition, the coercivities and remanences are almost zero and have superparamagnetic properties (see FIGS. 2 to 5 and the like herein).

_{The silica-coated magnetic nanobeads having a Fe 3} O ₄ core according to the present application can be used to purely separate nucleic acids from samples containing nucleic acids such as cells and tissues, and it is particularly important to have biocompatibility characteristics. As shown in FIG. 6 , the magnetic nanoparticles prepared according to the method of the present application do not exhibit cytotoxicity, and thus have biocompatibility.

In another aspect, the present application relates to a method for isolating and purifying a nucleic acid from a biological sample or an in vitro reaction containing nucleic acids such as cells and tissues using silica-coated magnetic nanobeads having _{an Fe 3} O _{4 core according to the present disclosure.}

The nucleic acid separation method using silica-coated magnetic particles was conducted using a chaotropic reagent (R. Boom et al., J. Clin. Microbiol., Vol 28(3), p495-503 (1990)). binds to the nucleic acid, and is performed by separating the silica-coated magnetic particles from the rest of the solution using an external magnetic force.

_{A nucleic acid separation method using a silica-coated magnetic nanobead having a Fe 3} O ₄ core according to the present application is a sample requiring separation and purification of nucleic acids (eg, a biological sample such as prokaryotic or eukaryotic cells or tissues, or enzymes for a specific reaction) In the step of providing a treated in vitro reactant, etc.), for example, the sample includes a lysate containing a nucleic acid by disrupting cells, or an in vitro amplification reaction product or a synthesized nucleic acid product requiring purification. mixing the sample and the magnetic nanobeads according to the present application in an appropriate binding buffer, whereby the nucleic acids in the sample are bound to the magnetic nanobeads; Then, in the step of providing an external magnetic force to the mixture, the magnetic nanobeads to which nucleic acids are bound by providing the magnetic force are collected by moving toward the magnetic force of the container containing the mixture; then removing the supernatant except for the magnetic nanobeads of the mixture; after removing the external magnetic force, washing using a washing solution such as distilled water for the magnetic nanobeads; and eluting the nucleic acid bound to the washed magnetic nanobeads.

The binding buffer used in the nucleic acid separation and purification method using the silica-coated magnetic nanobeads having _{an Fe 3} O ₄ core according to the present application is a chaotropic reagent, for example, guanidine salt, urea, chloride, iodide, perchloric acid. salt, (iso) thiocyanate, etc. are included, and specific compounds include sodium perchlorate, guanidine hydrochloride, guanidine isothiocyanate, and potassium iodide. , potassium thiocyanate, sodium chloride, sodium isothiocyanate, magnesium chloride, sodium iodide, and the like. Chaotropic reagents are used in concentrations of 1 to 8 M (mol/L).

As the elution buffer used in the nucleic acid separation and purification method using the silica-coated magnetic nanobeads having _{an Fe 3} O ₄ core according to the present application, for example, a Tris-(hydroxymethyl)amino methane buffer may be used.

_{The nucleic acid separation and purification method using a silica-coated magnetic nanobead having a Fe 3} O ₄ core according to the present disclosure can separate nucleic acids from a sample and at the same time be purified from substances such as other intracellular proteins.

In the method according to the present application, the nucleic acid includes DNA and RNA of a bio-derived or in vitro reaction product, the former being plasmid DNA, genomic DNA, cDNA, polymerase chain reaction (PCR) reaction product, oligonucleotide or DNA primer (primers), the latter including mRNA, siRNA, and miRNA.

The present application also relates to a composition or kit for isolation and purification of nucleic acids comprising silica-coated magnetic nanobeads having _{an Fe 3} O ₄ core according to the present application.

The composition or kit according to the present disclosure may include a chaotropic reagent for binding the nucleic acid to the magnetic silica nanoparticles, an elution reagent, and/or a reagent for washing and/or instructions for use.

_{Using the silica-coated magnetic nanobeads having a Fe 3} O ₄ core according to the present application, the isolated nucleic acid can be used in various reactions using the nucleic acid.

In one embodiment according to the present application, _{using the silica-coated magnetic nanobeads having an Fe 3} O ₄ core according to the present application, it was possible to isolate DNA from cells with high purity.

Subsequently, as a result of using the isolated DNA in the LAMP reaction, which is a type of nucleic acid amplification reaction, the template was successfully amplified, indicating excellent separation and purification of nucleic acids by the magnetic nanobead according to the present application and a method using the same (Fig. 7 to 8).

Hereinafter, examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the present invention is not limited to the following examples.

Example

Experimental materials and methods

1. FeSO ₄ 7H2O (≥ 99%), NaOH (> 99.9%), ethanol (≥ 99.5%), NH ₄ OH (≥ 99.99%), and tetraethyl orthosilicate (TEOS) (98 %) were purchased from Sigma-Aldrich and used as purchased. Ethanol (99% Duksan, South Korea) was used for initial washing of the nanobeads. Tertiary distilled water was used for the final wash and was used to prepare aqueous nanobead suspension samples.

2. Methods used to characterize the nanobeads synthesized herein

HRTEM images and element mapping images (Titan G2 ChemiSTEM CS Probe, FEI) were measured to determine the diameter, shape, and core-shell structure of _{Fe 3} O ₄ @SiO _{2 nanobead particles.} To determine the crystal structure of the powder sample, an XRD pattern (X'PERT PRO MRD, Philips) with unfiltered CuKα radiation (λ = 0.154184 Å) was recorded. An FT-IR absorbance spectrum (Galaxy 7020A, Mattson Instruments, Inc) was analyzed to investigate the silica coating on the _{Fe 3} O _{4 @ surface.} The curve of magnetization (M, Magnetization) versus applied field (H, applied field) (ie, MH curve) was measured using a VSM (7407-S, Lake Shore) at 300 K, and the magnetic characteristics of the nanobeads according to the present application were measured. used to clarify. In order to measure the iron (Fe) concentration in the solution sample, an Inductively Coupled Plasma Atomic Emission Spectrometer (ICPAES) (Optima 7300DV & Avio500, Perkin Elmer) was used. The biocompatibility of the nanobeads according to the present application was measured using a Luminescent Cell Viability Assay (CellTiter-Glo, Promega) for the cell viability of human prostate cancer cells (DU145) and normal mouse hepatocytes (NCTC1469).

3. Samples: Cancer Cells, Viruses and Bacteria

Table 4 shows five types of samples for isolating nucleic acids. Five types of samples were as follows: A549 non-small lung cancer cells (Sample No. 1), bacillus cereus (ATCC® 14579TM) (Sample No. 2) and campylobacter jejuni (Sample No. 1). 3), and dengue (Sample No. 4) and influenza A (Sample No. 5) viruses. A specific amount of each sample was mixed with 200 μl of human blood or 90 μl of phosphate buffered saline or 90 μl of serum in a 2.0 mL microtube (see Table 4). To disrupt cells, viruses and bacteria, 200 μl of disruption buffer (10 mM Tris-HCl + 1 mM ethylenediaminetetraacetic acid (EDTA) + 0.5% sodium dodecyl sulfate) was added to each sample. The sample was vortexed for 10 seconds, incubated at 60° C. for 5 minutes, and 200 μl of isopropyl alcohol was added.

4. Magnetic Separation of Nucleic Acids from Samples

A 2mL micro-tube containing the sample was placed in an 8-neck microtube rack, 20 μl of a magnetic nanobead solution was added to the sample, and vortexed for 10 seconds. A magnet was then attached to the back of the rack for 1 minute. The solution in the tube was decanted and the magnet removed from the rack. 800ul of Wash Buffer-1 (MagListo-2, Bioneer, USA) was added to the sample and vortexed for 10 seconds. The magnet was attached to the back side of the rack again for 1 minute, the solution in the tube was poured, and the magnet was detached. The washing process using this buffer-1 was repeated 3 times. The same washing procedure was repeated three times with buffer-2 (MagListo-2, Bioneer, USA). At this stage the nucleic acids in the sample were considered purified. Then, in order to separate the nucleic acids attached to the surface of the nanobeads, 80 μl of an elution buffer (MagListo-2, Bioneer, USA) was added to the sample and vortexed for 10 seconds. A magnet was attached to the back of the rack, and the solution sample containing the nucleic acid was transferred to a new micro-tube.

5. Analysis of Nucleic Acids Isolated from Samples

Confirmation of whether the nucleic acid was successfully isolated from the sample was performed with the following four types of experiments.

First, the concentration of the isolated nucleic acid was determined by measuring the absorbance (=A260) at 260 nm using a UV-visible absorption spectrometer (QIAxpert, Qiagen, Germany). The purity of the isolated nucleic acid was determined by measuring the A260/A230 and A260/A280 ratio, where A230 is the absorbance at 230 nm of organic compounds such as isopropyl alcohol and EDTA, and A280 is the absorbance at 280 nm of the protein. The absorbance of the isolated nucleic acid is considered high when the values of A260/A230 and A260/A280 are between 1.9 and 2.2.

Next, the isolated nucleic acid was confirmed by performing electrophoresis on an agarose gel.

ki, T. Henkel, W. Fritzsche, Appl. Microbiol. Biotechnol. 104, 405-415 (2020)). In addition, it was confirmed by the LAMP reaction of the isolated nucleic acid. Specifically, the LAMP reaction of the isolated nucleic acid (T. Notomi, H. Okayama, H. Masubuchi, T. Yonekawa, K. Watanabe, N. Amino, T. Hase, Nucleic Acids Res. 28, e63 (2000)) was isolated This was performed by adding 2 μL of the nucleic acid sample to 23 μL of LAMP solution containing 2X buffer, Bst DNA or RNA polymerase and primers. When the nucleic acid concentration exceeds 10 pg/μL due to amplification during LAMP, the solution color changes from purple to blue (23. C. Reuter, N. Slesiona, S. Hentschel, O. Aehlig, A. Breitenstein, A. Cs

ki, T. Henkel, W. Fritzsche, Appl. Microbiol. Biotechnol. 104, 405-415 (2020)).

Finally, nucleic acid amplification during the LAMP reaction was confirmed by recording real-time fluorescence curve versus cycle (1 cycle = 30 sec) using real-time nucleic acid amplification equipment (TP650, Takara, Japan).

The experimental method and materials of FIG. 7 are as follows.

Nucleic acid extracted from Bacillus cereus (ATCC® 14579™) was used as the template, the target gene is Bacillus cereus EntD, and primers and reaction conditions for amplification are as follows. FIP and BIP primers were used at 1.6 uM, respectively, and F3 and B3 at 0.2 uM, respectively. The reaction was carried out in a total volume of 25ul (nucleic acid 2μl, 2X Reaction buffer 12.5μl, Bst Polymerase 1μl, primer 2μl, distilled water 7.5ul). After 30 minutes at 63° C., it was performed at 80° C. for 3 minutes.

[Table 1]

The experimental method and materials of FIG. 8 are as follows.

Nucleic acids extracted from A549 (ATCC® CCL-185) and dengue virus (NCCP 43248) were used as the template, and the target gene is wdr1 gene (RNA target) for A549 cells, and polyprotein gene (RNA target) for dengue virus. Primers and reaction conditions are shown in the following table. FIP and BIP primers were used at 1.6 uM, respectively, and F3 and B3 at 0.2 uM, respectively. The reaction was performed in a total volume of 25ul (nucleic acid 2μl, 2X Reaction buffer 12.5μl, Bst Polymerase 1μl, primer 2μl, distilled water 7.5ul). After 30 minutes at 58°C, it was performed at 80°C for 3 minutes.

[Table 2]

[Table 3]

Example 1. Fe ₃ O ₄ @SiO ₂ Core-shell nanobeads (core = Fe ₃ O ₄ and shell=SiO ₂ ) synthesis

A single Fe ²⁺ precursor was used _{to prepare Fe 3} O ₄ nanobeads (see FIG. 1 ). The reaction equation is:

3Fe ²⁺ (aq) + ^{8OH -} (aq) → 3Fe(OH) ₂ (s) + 2OH- (aq) → Fe ₃ O ₄ (s) + 4H ₂ O(ℓ)

0.5 mmol of FeSO ₄ ·7H ₂ O was added to 20 mL of tertiary distilled water in a 50 mL beaker at room temperature under atmospheric conditions with magnetic stirring. Then, 10 mmol of NaOH dissolved in 20 mL of tertiary distilled water was _{slowly added to a FeSO 4} ·7H ₂ O solution, and the pH of the solution was >8. The reaction mixture was magnetically stirred for 30 minutes, and transferred to a 500 mL beaker containing 400 mL of tertiary distilled water. The product solution was stored in a refrigerator (˜4° C.) until _{Fe 3} O _{4 nanobeads were precipitated on the bottom.} The top clear solution was then decanted containing Na+, OH-, and unreacted precursor. The precipitate was washed three times with tertiary distilled water.

ber 방법으로 제조하였다(W. St

ber, A. Fink,E. Bohn, J. Colloid Interface Sci. 26,62-69(1968)). TEOS(Tetraethyl orthosilicate)는 물의 추가시 SiO₂로 변환된다. 이를 위해, 50mL의 에탄올, 10mL의 3차 증류수, 1.7mL의 18M NH₄OH, 및 0.6mL의 TEOS를 500mL 비이커에서 혼합하였다. 상기 혼합 용액을 대기 조건하의 실온에서 1시간 동한 자기 교반하였다. 이어 상기 20ml의 Fe₃O₄ 나노비드 용액을 상기 60ml의 SiO₂ 나노비드 용액(pH=8~9)에 서서히 추가하였다. 상기 혼합물을 대기 조건하에서 5시간 동안 계속 초음파처리하였다. NH4⁺, OH^-, 및 자유 SiO₂ 나노비드를 상기 프러덕트 용액으로부터 제거하기 위해, 상기 프러덕트 용액을 400mL의 에탄올로 희석한 후, Fe₃O₄ 나노비드가 비이커 바닥에 침전될때까지 냉장고(~4℃)에 보관하였다. 상층액을 제거하였다. 에탄올을 이용한 세척은 3회 반복되었다. 나노비드에서 에탄올을 제거하기 위해, 3차 증류수를 이용한 동일한 세척과정을 3회 반복하였다. 수득한 나노비드를 두 부분으로 나누었다: 하나는 공기중에서 건조하여 분말을 수득하였으며, 이는 특징 규명 실험을 위한 실험에 사용되었으며, 다른 하나는 3차 증류수로 희석하여 나노비드 용액 시료(> 15 mM Fe)를 제조하였다. Following In order to coat _{Fe 3} O ₄ nanobeads with silica (ie SiO _{2 ), SiO 2} nanobeads are coated with St

ber method (W. St

ber, A. Fink, E. Bohn, J. Colloid Interface Sci. 26,62-69 (1968)). Tetraethyl orthosilicate (TEOS) is converted to _{SiO 2 upon addition of water.} For this, 50 mL of ethanol, 10 mL of tertiary distilled water, 1.7 mL of 18M NH ₄ OH, and 0.6 mL of TEOS were mixed in a 500 mL beaker. The mixed solution was magnetically stirred for 1 hour at room temperature under atmospheric conditions. Then, the 20ml Fe ₃ O ₄ nanobead solution 60ml of SiO ₂ nanobead solution (pH=8-9) was added slowly. The mixture was sonicated continuously for 5 hours under atmospheric conditions. NH4 ^+, OH ^- refrigerator until, and the free SiO ₂ nano-beads in order to remove from the peureo duct solution, after diluting the peureo duct solution in a 400mL ethanol, Fe ₃ O ₄ nano-beads to settle to the bottom beaker ( ~4°C). The supernatant was removed. Washing with ethanol was repeated 3 times. In order to remove the ethanol from the nanobeads, the same washing process using tertiary distilled water was repeated three times. The obtained nanobeads were divided into two parts: one was dried in air to obtain a powder, which was used for the experiment for characterization experiments, and the other was diluted with tertiary distilled water to obtain a nanobead solution sample (> 15 mM Fe ) was prepared.

[Table 4] Samples, characterizations, and UV-absorbance results

Example 2. Characterization of the nanobeads synthesized herein

2-1. HRTEM analysis results

As shown in Fig. 2a, Fe ₃ O ₄ @SiO ₂ nanobeads had a nearly spherical shape with a particle diameter of 50 to 60 nm, and a core-shell having _{a silica shell and a Fe 3} O _{4 core with contrast.} structure was shown. The shell thickness was found to be approximately 10 nm. In the method according to the present application, a single precursor Fe ²⁺ was used for synthesis to prepare large diameter particles of _{Fe 3} O _{4 nanobeads.} High-angle annular dark field-scanning transmission electron microscopy (HAADF-STEM) images are shown in FIG. 2B . Elemental (Fe, O, and Si) mappings on HAADF-STEM images are described in Figures 2c-2f. As a result of element mapping, Fe was distributed in the core (FIG. 2c), O was distributed in both the core and the shell (FIG. 2D), and Si was mainly distributed in the shell (FIG. 2E). The merged image clearly shows the core-shell structure (Fig. 2f).

2-2. XRD analysis result

3 shows XRD patterns of bare Fe ₃ O ₄ nanobeads (bottom) and Fe ₃ O ₄ @SiO ₂ core-shell nanobeads (top). All peaks appearing in the XRD pattern of bare Fe ₃ O ₄ nanobeads can be assigned to (hkl) Miller index corresponding to _{cubic Fe 3} O _{4 .} Fe ₃ O ₄ @SiO ₂ core-shell nanobeads exhibited reduced peak intensity due to the _{amorphous SiO 2 coating.} The estimated cell constant of Fe ₃ O ₄ nanobeads is 8.37 ± 0.05 Å, which is consistent with the reported values (T. Tegafaw, W. Xu, SH Lee, KS Chae, Y. Chang, GH Lee, Int. J. Mod. Phys. B 31, 1750014 (2017)).

2-3. FT-IR analysis

, N. Filipovi

-Vincekovi

, L. Sekovani

, Braz. J. Chem. Eng. 28, 89-94 (2011)). 이러한 결과는 Fe₃O₄ 나노 비드의 표면이 SiO₂로 성공적으로 코팅되었음을 나타내는 것이다. Fe ₃ O ₄ to investigate the SiO ₂ coating of the nano-beads bear a surface Fe ₃ O ₄ nano-beads (top) and Fe ₃ O ₄ @SiO ₂ core-examined the FT-IR absorption spectrum of the shell (bottom) (FIG. 4). ^{The characteristic absorption peak at 550 cm −1} in the FT-IR absorption spectrum of bare Fe ₃ O ₄ and Fe ₃ O ₄ @SiO ₂ core-shell nanobeads was due to the Fe-O stretching vibration (T. Tegafaw, W. (. Xu, SH Lee, KS Chae, Y. Chang, GH Lee, Int. J. Mod. Phys. B 31, 1750014 (2017)). ^{The peak at 1090 cm -1} in the FT-IR absorption spectrum of Fe ₃ O ₄ @SiO ₂ core-shell nanobeads was due to Si-O-Si stretch vibration (S. Musi).

, N. Filipovi

-Vincekovi

, L. Sekovani

, Braz. J. Chem. Eng. 28, 89-94 (2011)). These results indicate that the surface _{of the Fe 3} O ₄ nanobeads was successfully coated _{with SiO 2 .}

2-4. VSM analysis results

The magnetic properties of bare Fe ₃ O ₄ and Fe ₃ O ₄ @SiO ₂ core-shell nanobeads were determined by recording MH curves at 300 K. As shown in FIG. 5 , the coercivities and remanences are almost zero, indicating that the nanobeads synthesized herein are mainly superparamagnetic. The saturation magnetization (Ms) of bare Fe ₃ O ₄ nanobeads was 78.5 emu/g, which was higher than 33.80 emu/g of _{Fe 3} O ₄ @SiO ₂ nanobeads due to the _{non-magnetic SiO 2 coating.} The number is high enough to be powerful for magnetic separation.

2-5. In vitro cytotoxicity

Fe ₃ O ₄ @SiO ₂ core shell nanobeads showed very low cytotoxicity of up to 200 μM Fe in DU145 and NCTC1469 cells ( FIG. 6 ). This indicates the excellent biocompatibility of the nanobeads prepared according to the present method.

2-6. Analysis result of isolated nucleic acid

Successful nucleic acid isolation using Fe ₃ O ₄ @ SiO ₂ core-shell nanobeads was confirmed through various experiments (Table 4) as described below.

The solution color changed from purple to blue during LAMP in all samples tested ( FIG. 7 ). This indicates that the nucleic acid was successfully amplified in all samples. Therefore, further experiments were then performed only on the selected samples.

Concentrations of isolated nucleic acids were determined for Sample Nos. 1, 2 and 4 from UV-absorbance measurements of A260. Concentrations were known for all three samples (Table 4). The A260/A230 ratio was high for all three samples, indicating little contamination by organic molecules in the isolated nucleic acids. However, the A260 / A280 ratio was high only for Sample No. 1, which is to a degree that the isolated nucleic acid was somewhat contaminated with protein for Sample Nos. 2 and 4, but there was no problem in detecting the nucleic acid.

Nucleic acid segregation was confirmed by recording real-time fluorescence curves for sample numbers 1 and 4 during LAMP. A fluorescence signal was observed in both samples (FIG. 8), and no fluorescence was observed in the control solution containing no nucleic acid.

All the above results indicate successful nucleic acid separation by _{Fe 3} O ₄ @ SiO _{2 core-shell nanobeads from all samples.}

Although the exemplary embodiments of the present application have been described in detail above, the scope of the present application is not limited thereto, and various modifications and improvements by those skilled in the art using the basic concept of the present application as defined in the following claims are also included in the scope of the present application. will belong to

All technical terms used in the present invention, unless otherwise defined, have the meaning as commonly understood by one of ordinary skill in the art of the present invention. The contents of all publications herein incorporated by reference are incorporated herein by reference.

Claims (7)

A method for producing silica-coated magnetic nanobeads having a particle diameter of 50 to 60 nm and a silica shell thickness of 10 nm, _{having a Fe 3} O _{4 core, comprising the steps of:}
(a) reacting 0.025 _{mM FeSO 4} ·7H ₂ O in water and 500 mM NaOH aqueous solution at room temperature with stirring;
(b) then allowing the solution to stand at 4° C. to produce a precipitate of _{Fe 3} O _{4 nanobeads;}
(c) after generating the precipitate, removing the supernatant from the solution, washing the precipitate with water, and suspending in water to prepare a _{Fe 3} O _{4 nanobead suspension;}
(d) _{adding the Fe 3} O ₄ nanobead suspension to the SiO ₂ nanobead solution in a volume ratio of 1:3, wherein the Fe ₃ O ₄ nanobeads to the SiO ₂ nanobeads are included in a weight ratio of 1:3, ultrasonicating the solution at room temperature;
(e) allowing the sonicated solution to stand at 4° C. to produce a precipitate of _{silica-coated Fe 3} O _{4 nanobeads;} and
(f) After the precipitate of step (e) is generated, removing the supernatant, and washing the precipitate with ethanol and water, to obtain _{silica-coated Fe 3} O _{4 nanobeads.}
The method of claim 1,
The SiO ₂ nanobead solution is prepared by mixing ethanol, tertiary distilled water, 18M NH ₄ OH and TEOS TEOS (tetraethoxysilane) in a volume ratio of 5: 1: 1.7: 0.6, Fe ₃ O ₄ Silica coating having a core A method of manufacturing a magnetic nanobead.
A silica-coated magnetic nanobead having _{a Fe 3} O ₄ core prepared by the method according to claim 1 or 2 .
A method for isolating a nucleic acid, characterized in that the magnetic nanobead according to claim 3 is used as a nucleic acid-binding carrier.
5. The method of claim 4
The nucleic acid is DNA or RNA, a nucleic acid isolation method.
A composition for separating nucleic acids comprising silica-coated magnetic nanobeads having _{a Fe 3} O ₄ core prepared by the method according to claim 1 or 2.
A kit for separating nucleic acids comprising silica-coated magnetic nanobeads having _{a Fe 3} O ₄ core prepared by the method according to claim 1 or 2.

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