KR20140055563A - Magnetic core/shell nanoparticle comprising immobilized enzyme or biomaterial and preparation method thereof - Google Patents

Abstract

The present invention provides a magnetic core/shell nanoparticle comprising: a) a core part that is formed as magnetic material and boron are bonded; b) a silica shell part that wraps the core; c) an outermost shell part that warps the shell and includes silica; d) an outermost shell surface part that is obtained by improving the surface of the outermost shell through one or more substituents selected from the group consisting of -NH_2, -COOH, -NHS, and -biotin; and e) an enzyme or a biomaterial that is fixed to the shell surface part. The core/shell nanoparticle including the immobilized enzyme or biomaterial according to the present invention has the surface that is improved by means of amine groups and the like. The immobilization is optimized by means of a covalent bond of the enzyme or biomaterial and a carboxyl group. Even though the nanoparticle is used several times, the activity of the enzyme or biomaterial can be maintained at a high level.

Description

Magnetic Core / Shell Nanoparticles Containing Immobilized Enzymes or Biomaterials and Methods of Producing the Magnetic Core / Shell Nanoparticles

The present invention relates to a magnetic core / shell nanoparticle containing an immobilized enzyme or a biomaterial and a method for preparing the same, and more particularly, to a magnetic core / shell nanoparticle comprising a magnetic core / shell nanoparticle modified by effectively immobilizing an enzyme or a biomaterial Core / cell nanoparticles and a method of manufacturing the same.

Studies on the conjugation of enzymes for industrial use have been under way for the development of immobilized enzymes combined with various carriers such as continuous processing of enzymes, efficient separation, and cost reduction by reuse. These enzymes have been used to immobilize enzymes on various surfaces of particles such as magnetic, gold, silver, and silica, and have great potential for applications such as biosensors and biofuel cells.

Magnetic particles (especially iron oxide) are widely used in biomedical applications such as biochemical product dispersion, magnetic resonance imaging (MRI), target drug delivery, biosensors, etc., and are chemically stable, well dispersed in a liquid environment, It has been used in many applications. However, when magnetic particles are exposed directly to the biological environment, aggregation occurs due to anisotropic dipole attraction and biodegradation.

Cobalt particles have 3 to 4 times the saturation magnetization of iron oxide, and when coated with a surfactant or polymer on the wall surface, they become non-magnetic cobalt oxide. However, cobalt particles are not well dispersed due to ferromagnetic cohesion and oxidation occurs unstably in the air phase.

Korean Patent Laid-Open Publication No. 2012-0108492 and Korean Laid-Open Patent Application No. 2012-0101961 disclose techniques for nanoparticles, but no technology for preventing aggregation phenomenon and oxidation process is described at all.

To solve these problems, the present inventors have synthesized magnetic particles by coating silica on surfaces of cobalt-boron bonded particles to prevent aggregation and oxidation. The enzyme and the biomaterial were immobilized by modifying the surface of the particle. For this immobilized enzyme, optimal conditions and reuse were studied.

Korean Patent Publication No. 2012-0108492 Korean Patent No. 2012-0101961

It is an object of the present invention to provide a magnetic core / shell nanoparticle immobilized with an enzyme or a biomaterial so as to be reusable.

Another object of the present invention is to provide a method for producing the nanoparticles.

The present invention provides a magnetic recording medium comprising: a) a core portion formed by coupling a magnetic material with boron; b) a silica shell portion surrounding the core; c) an outermost shell portion surrounding the shell portion and comprising silica; d) an outermost shell surface portion modified by a substituent selected from the group consisting of -NH ₂ , -COOH, -NHS and biotin on the surface of said outermost shell; And e) one or more enzymes or biomaterials selected from the group consisting of glucosoxidase, lipase, laccase, catalase, microorganism, antigen and antibody, immobilized on the surface of the shell, The present invention also provides a magnetic core / shell nanoparticle.

According to an embodiment of the present invention, the magnetic material may be cobalt.

According to another example of the present invention, the particle size may be 10 nm to 1000 nm.

According to another embodiment of the present invention, the substituent may be -NH ₂ .

The present invention also relates to a method for producing a magnetic material comprising the steps of: a) reacting a magnetic material with a boron hydride to form a magnetic material-boron bonded core portion; b) coating the core with silica to form a shell portion surrounding the core; c) adding silica to the particles formed from the core and the shell to form an outermost shell portion; d) modifying the surface of said outermost shell with a substituent selected from the group consisting of -NH ₂ , -COOH, -NHS and biotin; And e) contacting the surface of the shell with at least one enzyme selected from the group consisting of glucosoxidase, lipase, laccase, catalase, microorganism, And immobilizing the magnetic core / shell nanoparticles. The present invention also provides a method for producing magnetic core / shell nanoparticles.

According to an embodiment of the present invention, the immobilization buffer in step e) may be sodium phosphate buffer.

According to another embodiment of the present invention, the sodium phosphate buffer may be 0.025 to 0.2M.

According to another embodiment of the present invention, the step e) may be carried out at a pH of 5 to 8 and at a temperature of 5 to 45 ° C.

The core / shell nanoparticles immobilized with the enzyme or the biomaterial according to the present invention can be modified by modifying the surface of the particles with an amine group or the like to optimize the immobilization of the enzyme or the biomaterial by covalent bonding with the carboxyl group, The activity of the enzyme or the biomaterial can be maintained at a high level even when it is reused.

FIG. 1 shows a process of manufacturing magnetic core / shell nanoparticles according to an embodiment of the present invention.
FIG. 2 is a photograph of a magnetic core / shell nanoparticle according to an embodiment of the present invention taken with a TEM (transmission electron microscope).
FIG. 3 shows measurement of a pattern (Co2P binding energy) of magnetic core / shell nanoparticles according to an embodiment of the present invention using an X-ray photoelectron spectroscope (XPS).
FIG. 4 is a graph showing the measurement of a pattern (binding energy of B1S) of magnetic core / shell nanoparticles according to an embodiment of the present invention using X-ray photoelectron spectroscopy (XPS).
FIG. 5 is a graph showing a pattern (bond energy of Si2p) of magnetic core / shell nanoparticles according to an embodiment of the present invention measured using an X-ray photoelectron spectroscope (XPS).
FIG. 6 is a graph showing a pattern (binding energy of N1s) of magnetic core / shell nanoparticles according to an embodiment of the present invention measured using an X-ray photoelectron spectroscope (XPS).
Fig. 7 (a) shows a state in which magnetic particles are dispersed, and Fig. 7 (b) shows a state in which magnetic particles are gathered due to magnetism.
FIG. 8 is a field-dependent magnetization curve measured at 300 K with a vibrating sample magnetometer (VSM) for magnetic core / shell nanoparticles according to an embodiment of the present invention.
FIG. 9 shows enzyme activity of magnetic core / shell nanoparticles according to an embodiment of the present invention, according to pH.
FIG. 10 shows the enzyme activity of the magnetic core / shell nanoparticles according to an embodiment of the present invention, according to the temperature.
11 is a graph showing the enzyme activity of the magnetic core / shell nanoparticles according to an embodiment of the present invention, according to the concentration of phos- phate buffer.
FIG. 12 shows the enzyme activity of the immobilized enzyme when the magnetic core / shell nanoparticle according to an embodiment of the present invention is repeatedly used up to seven times (Nmuber of cycle). (non-blocking, before substrate treatment, Blocking with 0.1 M D-glucose, after substrate treatment)

Hereinafter, the present invention will be described in detail.

The present invention relates to a magnetic nanoparticle in which an enzyme or a biomaterial such as glucose oxidase (GOD) is immobilized on a core / shell particle composed of a cobalt core containing a magnetic substance and a silica shell, And provides a manufacturing method thereof.

More specifically, the present invention provides a magnetic recording medium comprising: a) a core portion formed by coupling a magnetic material and boron; b) a silica shell portion surrounding the core; c) an outermost shell portion surrounding the shell portion and comprising silica; d) an outermost shell surface portion modified by a substituent selected from the group consisting of -NH ₂ , -COOH, -NHS and biotin on the surface of said outermost shell; And e) a core / shell nanoparticle immobilized on the surface of the shell, wherein the core / shell nanoparticle comprises an enzyme or a biomaterial.

Examples of the magnetic material include Co, Mn, Fe, Ni, Gd and Mo, and cobalt (Co) is preferably used.

Further, the particle size is preferably 10 nm to 1000 nm, more preferably 100 nm to 200 nm.

Substituents for modifying the surface of the outermost shell include -NH ₂ , -COOH, -NHS, and biotin. Preferably, the outermost shell is modified with an amine. That is, for example, when the outermost surface is modified with -NH ₂ , -COOH, -NHS, -Biotin or the like, the enzyme or the biomaterial can be stably immobilized.

The enzyme or the biomaterial is not particularly limited as long as it is recognized by the person skilled in the art as an enzyme or a biomaterial. Preferably, an enzyme selected from glucose oxidase, lipase, laccase, or catalase, or a biomaterial selected from a microorganism, an antigen, or an antibody may be used. The microorganisms, antigens and antibodies are generally recognized as microorganisms, antigens or antibodies. Any microorganism, antigen, or antibody having a structure composed of proteins may be used in the present invention, and it is preferable that the microorganism, antigen and antibody have a substituent such as an amine group, a carboxyl group or an aldehyde.

Most preferably, glucoose oxidase can be used.

On the other hand, the present invention provides a method for producing a magnetic material, comprising the steps of: a) reacting a magnetic material with a boron hydride to form a magnetic material-boron bonded core; b) coating the core with silica to form a shell portion surrounding the core; c) adding silica to the particles formed from the core and the shell to form an outermost shell portion; d) modifying the surface of said outermost shell with a substituent selected from the group consisting of -NH ₂ , -COOH, -NHS and biotin; And e) immobilizing an enzyme or a biomaterial on the surface of the shell using an immobilizing buffer. The present invention also provides a method for producing magnetic core / shell nanoparticles.

The reason for forming the core part by bonding the magnetic material with boron in the step a) is that when boron is used in combination with the magnetic material than when it is used alone, there is an effect such as bonding between magnetic molecules, formation of particles, and the like.

Examples of the magnetic material include Co, Mn, Fe, Ni, Gd and Mo, and cobalt (Co) is preferably used.

The substituent for modifying the surface of the outermost shell in the step d) is -NH ₂ , -COOH, -NHS, and biotin. The shell may be modified with an amine. That is, for example, when the outermost surface is modified with -NH ₂ , -COOH, -NHS, -Biotin or the like, the enzyme or the biomaterial can be stably immobilized.

The enzyme or the biomaterial of step e) is not particularly limited as long as it is recognized by the person skilled in the art as an enzyme or a biomaterial. Preferably, an enzyme selected from glucose oxidase, lipase, laccase, or catalase, or a biomaterial selected from a microorganism, an antigen, or an antibody may be used. The microorganisms, antigens and antibodies are generally recognized as microorganisms, antigens or antibodies. Any microorganism, antigen, or antibody having a structure composed of proteins may be used in the present invention, and it is preferable that the microorganism, antigen and antibody have a substituent such as an amine group, a carboxyl group or an aldehyde.

Most preferably, glucoose oxidase can be used.

In addition, the immobilization buffer may be sodium phosphate buffer, but is not limited thereto.

The sodium phosphate buffer is preferably used at a concentration of 0.025 to 0.2 M. When sodium phosphate buffer is used within the above range, the activity of the enzyme or the biomaterial immobilized on the magnetic core / shell nanoparticles is maximized great.

In addition, the step e) is preferably performed at a pH of 5 to 8 and at a temperature of 5 to 45 ° C. When the reaction is performed within the above range, the enzyme or the biomaterial immobilized on the magnetic core / shell nanoparticle Is the most active.

The magnetic core / shell nanoparticle of the present invention is composed of a combination of a cobalt ion and a boron which are magnetic substances, a weakly surfactant citric acid, and a chemical reaction with a boron chemical hydride, To maintain the ferromagnetic properties and to be well dispersed in water-soluble and organic solvents.

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

[Example]

Example 1

<Formation of Cobalt-Boron Particles and Formation of Magnetic Core / Shell Nanoparticles According to Coating of Silica Molecules (Mass Production)>

As shown in Fig. 1-A, 1000 mL of a mixed solution of 0.2 M NaBH ₄ and 0.4 mM citric acid and 2 M of CoCl ₂ 6H ₂ O_2 mL were mixed at room temperature and mixed at 65 ° C for 8 minutes on a nitrogen gas Reaction is given. The formation of cobalt-boron particles grows into a double layer by the repulsion between ions of cobalt particles, and -B (OH) ₄ is bonded by adsorption. Also, the size of the cobalt-boron (Co-B) particles is related to the amount of citrate.

As shown in FIG. 1-B, the cobalt-boron particles grown as cores are coated on their surfaces by the stove method. 2000 ml of ethanol, 1 ml of TEOS (tetraethyl orthosilicate) and 1 ml of ammonia (28%) are added to the mixed solution of cobalt-boron particles and reacted for 2 hours, then washed and then dried in an oven. Citric acid acts as a weak surfactant to form a single membrane, and the silica molecules are hydrolyzed and condensed in the membrane to form a silica layer in the form of a shell. As the concentration of citrate increases, the thickness of the shell decreases . Therefore, the shell was formed to have a thin thickness in order to maintain magnetic properties.

Example 2

<The surface of the particle Reformed  Formation of magnetic core / shell nanoparticles &gt;

1-C, the particles obtained in Example 1 were mixed with 100 ml of acetone and then dispersed. 18 ml of 3-APTS (3-aminopropyltriethoxysilane) C for 2 hours. As the silane groups of 3-APTS are bonded by hydrolysis, the surface of the particles is easily reformed into amine groups.

Example 3

<Surface Reformed  On the particle surface Glucose Oxides  Formation of immobilized, magnetic core / shell nanoparticles &gt;

As shown in FIG. 1-D, 0.2 g of the surface-modified particles and 0.025 g of sodium phosphate buffer (0.025, 0.05, 0.1 , 0.2 M, pH 5.0, 6.0, 7.0, 8.0). Then, 1 mg / 5 ml of glucose oxidase (GOD) and 20 mM EDC were added to the buffer mixed with the particles, and the reaction was carried out at 5, 15, 25, 35 and 45 ° C for 15 hours The covalent bond between the amine group of the particle and the enzyme or biomaterial is immobilized.

As a result, it was possible to mass-produce particles with a core of cobalt-boron particles and a shell of silica. After modifying the enzyme or a variety of biomaterials to immobilize the thus synthesized particle surface, the magnetic particles were treated with an enzyme or a biomaterial Immobilized. The particles immobilized with the enzyme or the biomaterial of the present invention can be utilized in various applications.

[ Test Example  One] TEM  Image measurement

The particles obtained in Example 1 were photographed by TEM (transmission electron microscope) and shown in Fig.

As a result, it can be seen that the particles of Example 1 are spherical bodies having a core / shell structure, and the surfaces are discontinuously distributed in the shell of the particles.

The TEM results of FIG. 2 show that a silica layer having a thickness of about 5 nm is formed when silica is coated on the center of the cobalt as shown in FIG. 1-B, and the size of the entire magnetic particles is about 100 nm Respectively.

[ Test Example  2] XPS  Pattern measurement

The magnetic micron particles of Example 1 were measured for patterns using an X-ray photoelectron spectroscope (XPS) and shown in FIG. 3 to FIG.

As a result, it was confirmed that all the cobalt particles have binding energy of Co2P (780.9, 796.6 eV) and B1S (191.7, 198.0 eV) in the XPS range. In addition, it was confirmed that Si2p (102.0 eV) for Co-B particle-coated silica and N1s (399.7 eV) after surface modification with amine have binding energy.

It can be seen that Co-B / SiO2 / NH2 particle formation and surface modification with magnetism exist.

[ Test Example  3] Magnetization ( 자화화 ) Measure

The field-dependent magnetization curves of the particles obtained in Examples 1 and 2 measured at 300 K with a VSM (vibrating sample magnetometer) are shown in FIG.

The magnetic hysteresis curve of 300K shows little coercivity and does not hunt in a strong magnetic field, indicating that it has magnetic properties.

It was confirmed that the saturation moment per unit mass_Ms and the coercivity were respectively 15.02 emu / g and 52.38 Oe on 300K.

[Test Example 4] The concentration of the immobilization buffer, pH  And immobilization reaction temperature Glucose Oxides  Enzyme activity measurement

The enzyme activity according to the concentration, pH, and immobilization reaction temperature of the immobilization buffer of Example 3 is shown in Figs. GOD assay kit (K-GOLX 01/05, Megazyme Ltd., Ireland) was used to measure the activity of glucose oxidase (GOD).

When the concentration and pH of the immobilized buffer, sodium phosphate buffer, were increased from 0.025 to 0.2M and the pH was increased from 5.0 to 8.0, the highest activity of the enzyme was found to be high when the concentration was 0.1 M and pH 7.0 Respectively. When the immobilization temperature of magnetic particles and enzyme was increased from 5 to 45 ° C, the highest activity of enzyme was observed at 5 ° C.

[Test Example 5] Reuse of immobilized enzyme

In order to measure the activity of the optimized immobilized enzyme of Example 3, when the enzyme was immobilized without reacting with the substrate (non-blocking) and when immobilized after reaction with the substrate (glucose) Respectively. Specifically, the activity of the optimized immobilized enzyme of Example 3 when repeatedly used up to 7 times before the non-blocking (before the substrate treatment) and after (Blockig with 0.1 M D-glucose; after the substrate treatment) activity was measured in the same manner as in Test Example 4 and shown in FIG.

As shown in FIG. 12, when the enzyme activity was measured seven times, it was confirmed that the activity was maintained up to 50%. In addition, there was no significant difference in the activity of the enzyme from 1 to 3 before and after the pretreatment of the substrate, but from 4th time after the non-blocking treatment (Blockig with 0.1 M D -glucose; after substrate treatment).

Claims (8)

a) a core portion formed by coupling a magnetic material and boron;
b) a silica shell portion surrounding the core;
c) an outermost shell portion surrounding the shell portion and comprising silica;
d) an outermost shell surface portion modified by a substituent selected from the group consisting of -NH ₂ , -COOH, -NHS and biotin on the surface of said outermost shell; And
e) one or more enzymes or biomaterial immobilized on the surface of the shell, selected from the group consisting of glucosoxidase, lipase, laccase, catalase, microorganism, antigen and antibody Magnetic core / shell nanoparticles. The method according to claim 1,
Wherein the magnetic material is cobalt. The method according to claim 1,
Wherein the particle size is from 10 nm to 1000 nm. The method according to claim 1,
Wherein the substituent is -NH ₂ . a) reacting a magnetic material with a boron hydride to form a magnetic material-boron bonded core portion;
b) coating the core with silica to form a shell portion surrounding the core;
c) adding silica to the particles formed from the core and the shell to form an outermost shell portion;
d) modifying the surface of said outermost shell with at least one substituent selected from the group consisting of -NH ₂ , -COOH, -NHS and biotin; And
e) immobilizing an enzyme or a biomaterial selected from the group consisting of glucosoxidase, lipase, laccase, catalase, microorganism, antigen and antibody on the surface of the shell using an immobilization buffer; Wherein the magnetic core / shell nanoparticles have an average particle size of less than 100 nm. The method of claim 5,
wherein the immobilization buffer of step (e) is sodium phosphate buffer. The method of claim 6,
Wherein the sodium phosphate buffer is 0.025 to 0.2M. The method of claim 5,
Wherein the step e) is carried out at a pH of 5 to 8 and at a temperature of 5 to 45 ° C.

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