KR20120119931A - Method for manufacturing the functional silica magnetic nano-paticle immobilized the cystene protein G and the functional silica magnetic nano-paticle is manufactured thereby - Google Patents

Abstract

PURPOSE: A method for preparing cysteine protein G-fixed magnetic silica nanoparticles is provided to improve sensitivity and to simplify the process by one-step. CONSTITUTION: A cysteine protein G-fixed magnetic silica nanoparticle is prepared by substituting chlorosilane onto the surface of silica-coated magnetic silica nanoparticles and conjugating cysteine protein G to a chloro group of the nanoparticles. The nanomagentic material is magnetite(Fe_3O_4), hematite(alpha-Fe_2O_3), maghemite(gamma-Fe_2O_3), nickel, cobalt, or compounds thereof. The cysteine protein G is a cysteine functional group-tagged protein G multimer.

Description

Method for manufacturing the functional silica magnetic nano-paticle immobilized the cystene protein G and the functional silica magnetic nano-paticle is manufactured thereby}

The present invention relates to a silica magnetic nanoparticle having protein G immobilized thereon and a method for producing the same, and more particularly, by introducing a chloro silane to the surface of the _{silica-coated magnetic nanoparticle (Fe 3} O ₄ @SiO ₂₎ It relates to a method of immobilizing cysteine-based protein G on the surface of the prepared functional silica magnetic nanoparticles (Cl-Fe ₃ O ₄ @SiO _{2 ).}

In applications such as immune sensors and diagnostic kits using antigen-antibody reactions, antibodies are mostly used in the form of immobilized on the surface of various inorganic solid materials. The technology to immobilize the antibody on various solid substrates while controlling the shape and orientation of the monolayer film so that the active site of the antibody can be easily exposed to the surface without impairing the inherent binding characteristics and activity of the antibody is directly related to the detection sensitivity of the chip or sensor. It is very important because it is connected to. In particular, since the binding site to the antigen in the antibody exists in the Fab region, immobilizing the antibody to expose the Fab region of the antibody to the outside is very important to increase the performance and sensitivity of the antibody. In spite of the immobilization of, the function is lost, so there is no effect on the sensor and detection regardless of the amount of immobilization. In the end, when the antibody is immobilized, not only the amount of immobilization is important, but the immobilization in which orientation is secured becomes an important factor.

Another factor to consider is that in order to measure the antigen-antibody reaction with high sensitivity from the monomolecular membrane of the antibody, the antigen-antibody binding reaction must be made at a high level under conditions that minimize steric hindrance. Steric restriction is mainly caused by the density of antibody molecules immobilized on a solid surface, and when the density is high, the antigen binding efficiency is severely degraded. Therefore, a technology for controlling the density of antibody molecules on a solid surface or a new monomolecular membrane manufacturing technology that can minimize the effect of the immobilization density of antibody molecules on the antigen-antibody binding reaction must be developed. Recently, a lot of research has been conducted to develop such a technology, but the results are still incomplete and are only at a rudimentary level.

Looking at the existing studies, it is moving from immobilization of antibodies using simple adsorption to a method of controlling orientation, and the basis of which is to immobilize using an aldehyde or carboxyl group on a solid surface using amino groups that are abundant in the Fc region of the antibody. That's the way. In this method, the amount of immobilization of the antibody increases, but the steric limitation specified above appears, and there is a disadvantage in that the efficiency of orientation is not greater than that of using a specific tag or ligand in terms of orientation. Another commonly used method is streptavidin-biotin binding. Streptavidin has a conformational structure capable of binding to four low-molecular vitamins, biotin, and the amine group of biotin is the fixed region of the antibody ( Since the carbohydrate portion located at Fc) can be oxidized and attached, streptavidin immobilized on the surface of the solid material and biotin attached to the antibody are combined to perform orientation immobilization. However, streptavidin and biotin are relatively expensive samples, so they act as a disadvantage for mass production and commercialization.

On the other hand, a method of securing the orientation of the antibody by using protein A and protein G that specifically binds to the Fc region of the antibody has also been introduced. Antibody immobilization technology using microbial-derived antibody-binding proteins (protein A, protein G, protein A/G, or protein L) that specifically binds to the antibody strongly binds to a specific site of the antibody that does not participate in the antigen-antibody reaction. The antibody is immobilized on a solid substrate to facilitate antigen access (Danczyk R et al., Biotechnol Bioeng 84:215-223, 2003). Since chemical modification is not required for the binding of the proteins to the antibody, there is an advantage of not impairing the intrinsic function of the antibody. However, in the process of immobilizing the antibody-binding protein on a solid substrate, it is difficult to control the orientation of the protein, which has been a problem in optimizing the immobilization efficiency of the antibody.

The recently published method is to fix a recombinant fusion protein expressed by a microorganism transformed with a recombinant vector containing a gene encoding a gold-binding protein and a gene encoding protein A and G (protein A, G) on a solid phase. A method of securing the orientation of the antibody was introduced (Korean Patent Registration No. 10-0965480).

Sulfo succinimidyl 4-(N-maleimi-domethyl) cyclohexane-1-carboxylate (sulfo-SMCC), a cross-linking reagent, was used as a method for immobilizing the existing protein G on the surface of magnetic nanoparticles, but this reaction is complicated. There is a problem in that expensive reagents are used, which increases the production cost.

The problem to be solved by the present invention is to improve the sensitivity for high-sensitivity detection in immunodetection, and to improve sensitivity through securing the orientation of the antibody as well as overcome steric limitations through structural arrangement. It is to provide a one-step, low-cost manufacturing method of magnetic silica nanoparticles in which cysteine-based protein G, which is effectively bound, is immobilized with high efficiency.

To solve the above problems, the functional silica magnetic nanoparticles immobilized with cysteine protein G of the present invention use a nanomagnetic oxide as a core, and the surface of the nanomagnetic oxide is coated with silica, and then again, silica is used as a chlorosilane. It is characterized in that the surface treatment is performed by binding cysteine-based protein G to the chloro group of the silica-coated magnetic nanoparticles substituted with the chloro-based silane and immobilized thereon.

Here, the nanomagnetic material is from magnetite (Fe ₃ O ₄ ), hematite (α-Fe ₂ O ₃ ), maghemite (γ-Fe ₂ O ₃ ), nickel, cobalt, and compounds thereof. It characterized in that it is one selected from the group consisting of.

Here, the magnetic nanoparticles are characterized in that they have a size of 30 ~ 200nm.

Here, the protein G multimer is characterized in that it is a tri- protein G multimer in which a cysteine functional group is tagged at the terminal.

Here, the chloro-based silane is ((chloromethyl)phenylethyl -trimethoxysilane),
chloromethylphenethyltris(trimethylsiloxy)silane, (p-Chloromethyl)phenyltrimethoxysilane,
chloromethyltriethoxysilane, chloromethyltrimethoxysilane, chloromethyltrimethylsilane,
chloromethyltris(trimethylsiloxy)silane, chlorophenyltriethoxysilane,
p-Chlorophenyltriethoxysilane, p-Chlorophenyltrimethylsilane, 3-Chloropropyltriethoxysilane,
3-Chloropropyltrimethoxysilane, 3-Chloropropyltrimethylsilane, 3-Chloropropyltris
(trimethylsiloxy) -silane, chlorosilane, 2-(4-Chlorosulfonylphenyl)ethyltrimethoxy -silane,
(dichloromethyl)trimethylsilane, triethylchlorosilane, trimethylchlorosilane,
2-(Trimethylsilyl)ethoxymethyl chloride, tris -(trimethylsiloxy)chlorosilane,
It is characterized in that it is selected from the group consisting of vinyldiphenylchlorosilane and trivinyl-chlorosilane.

In addition, in the present invention, magnetic silica nanoparticles coated with silica on the nanomagnetic oxide as a core are prepared, chloro-based silane is substituted on the surface of the silica-coated nanoparticles, and silica-coated magnetic nanoparticles in which the chloro group is substituted. It provides a method for producing functional silica magnetic nanoparticles to which cysteine protein G is immobilized, comprising a process of immobilizing cysteine protein G by binding to a chloro group of the particle.

The chloro-based silane is
((Chloromethyl)phenylethyltrimethoxy silane); ((chloromethyl)phenylethyl -trimethoxysilane);
chloromethylphenethyltris(trimethylsiloxy)silane; (p-Chloromethyl)phenyltrimethoxysilane;
chloromethyltriethoxysilane; chloromethyltrimethoxysilane; chloromethyltrimethylsilane;
chloromethyl tris(trimethylsiloxy)silane; chlorophenyltriethoxysilane;
p-Chlorophenyltriethoxysilane; p-Chlorophenyltrimethylsilane; 3-Chloropropyltriethoxysilane;
3-Chloropropyltrimethoxysilane; 3-Chloropropyltrimethylsilane; 3-Chloropropyltris
(trimethylsiloxy)-silane; chlorosilane; 2-(4-Chlorosulfonylphenyl)ethyltrimethoxy-silane;
(dichloromethyl)trimethylsilane; triethylchlorosilane; trimethylchlorosilane;
2-(Trimethylsilyl)ethoxymethyl chloride; tris-(trimethylsiloxy)chlorosilane;
vinyldiphenylchlorosilane; selected from the group consisting of trivinyl-chlorosilane
It is characterized.

Here, the nanomagnetic material is one selected from the group consisting of magnetite (Fe3O4), hematite (α-Fe2O3), maghemite (γ-Fe2O3), nickel, cobalt, and compounds thereof. do.

The protein G multimer is characterized in that it is a tri-protein G multimer in which a cysteine functional group is tagged at the terminal.

The cysteine protein G immobilized on the chloro-silane-treated magnetic nanoparticles provided in the present invention can improve the sensitivity, which is the most important in immunodetection, as a method capable of overcoming steric limitations while securing the orientation of the antibody. In other words, in the case of conventional protein G, even if many proteins G are bound to the surface, most of the protein G cannot be used for antibody immobilization due to steric limitations, but due to steric restriction, the sensitivity is lowered, but chlorosilane-treated magnetic nanoparticles The use of the cysteine-based protein G immobilized on has an effect that can significantly increase the sensitivity by immobilizing antibodies through a three-dimensional array rather than a two-dimensional array, while overcoming steric limitations.

In addition, since the manufacturing process is simple as one-step and does not use cross-linking reagents, the manufacturing cost can be lowered.

1 is a diagram illustrating a process of manufacturing silica-coated magnetic nanoparticles expressing a chloro-based silane according to an embodiment of the present invention.
2 is a diagram illustrating a process of immobilizing cysteine protein G on silica-coated magnetic nanoparticles expressing chloro-based silane according to an embodiment of the present invention.
Figure 3 shows the FT-IR spectra before and after the chlorosilane is substituted on the surface of the silica-coated magnetic nanoparticles according to the present invention.
Figure 4 shows the results of the binding efficiency according to each concentration of the cysteine-based protein G according to the present invention.
Figure 5 shows the results of SDS-polyacrylic aminogel electrophoresis analysis on the binding of cysteine-based protein G according to the present invention.

Hereinafter, the present invention will be described in more detail.

1 is a diagram illustrating a manufacturing process of silica-coated magnetic nanoparticles expressing a chloro-based silane according to an embodiment of the present invention. FIG. 2 is a chloro-based silane expression according to an embodiment of the present invention. It shows a series of processes of immobilizing cysteine-based protein G on the silica-coated magnetic nanoparticles, and FIG. 3 shows FT-IR spectra before and after substitution treatment with chloro-based silane on the surface of the silica-coated magnetic particles according to the present invention. 4 shows the results of binding efficiency according to each concentration of cysteine-based protein G according to the present invention, and FIG. 5 is an SDS-polyacrylic aminogel electrophoresis for binding of cysteine-based protein G according to the present invention. It shows the results of the electrophoretic analysis.

The present invention provides a multimer composed of protein G having affinity to the Fc region of an antibody, and the cysteine protein G is produced by cloning a protein G coding gene into an appropriate expression vector through gene recombination technology. For the protein G triplet, an expression vector in which a gene encoding a protein Gdp was repeatedly ligated three times was prepared, and an appropriate microorganism was transformed using the expression vector, and the transformed microorganism was cultivated to produce a protein G triplet. After that, the outer cell wall is destroyed and the synthesized protein G triplet is separated through a column. At this time, it is expressed so that a cysteine residue is attached to the bottom of the protein G.

The silica-coated magnetic nanoparticles used in the present invention are prepared by coating silica on the surface of the magnetic nanoparticles as shown in FIG. 1 of the accompanying drawings.

After dispersing the silica-coated magnetic nanoparticles substituted with chlorosilane, they are mixed with cysteine-based protein G to react, and then separated and washed through a permanent magnet. Through this, silica-coated magnetic nanoparticles to which cysteine-based protein G is immobilized are produced.

In the present invention, a method of using a magnetic iron oxide as a core, coating a magnetic iron oxide with silica, deriving and replacing a chlorosilane on the surface, and immobilizing cysteine protein G on the substituted chlorosilane. to provide.

The nanomagnetic material may be any material as long as it reacts to magnetism, but preferably magnetite (Fe ₃ O ₄ , hematite; α-Fe ₂ O ₃ ), maghemite (γ-Fe) It is preferable to use one selected from the group consisting of ₂ O _{3 ), nickel, cobalt, and compounds thereof.}

It is preferable to use the magnetic nanoparticles having a size of 30 ~ 200nm.

The cysteine-based protein G is characterized in that a cysteine group is bonded to the terminal thereof and is a tri-protein G multimer.

According to the present invention, the silica-coated magnetic nanoparticles to which the disclosed cysteine-based protein G is immobilized as illustrated in FIG. 2 is coated with silica on the surface of the magnetic nanoparticles as illustrated in FIG. 1. Cysteine protein G is fixed to magnetic silica nanoparticles by a process consisting of substituting chlorosilane on the surface of nanoparticles and immobilizing the cysteine protein G on silica-coated magnetic nanoparticles with chlorosilane attached to the surface Are combined.

Hereinafter, the present invention will be described in more detail through preferred embodiments. However, the present invention is not limited to the following experimental examples, and it is obvious that it can be modified in various forms according to the enemy selected configurations as described above.

Example 1: Preparation of chlorosilane-substituted silica-coated magnetic nanoparticles (Cl-Fe ₃ O ₄ @SiO ₂ )

Example 1-1: Coating of silica layer

To prepare silica-coated magnetic nanoparticles using magnetite obtained by precipitation, a solution of 0.22 g of Igepal CO-520, a surfactant, dissolved in 4.5 mL of cyclohexane, was prepared, and magnetite and olieic acid were added to the cyclohexane solution. After dispersing by sonicating for about a period of time, it was put in a cyclohexane solution containing Igepal and stirred. At this time, ammonium hydroxide and TEOS (tetraethyl orthosilicate) are added and stirred for an additional 20 hours, followed by washing with methanol to finally obtain silica-coated magnetic nanoparticles.

Example 1-2: Synthesis of functional groups on silica-coated magnetic nanoparticles (Fe ₃ O ₄ @SiO ₂₎

Silica-coated magnetic nanoparticles are dispersed in Toluene, ultrasonicated for about 1 hour, dispersed, and stirred at room temperature for about 2 hours. After stirring, ((Chloromethyl)phenylethyl-trimethoxysilane) was added and refluxed at 100℃ for 8 hours in a nitrogen atmosphere. After washing with Toluene, silica-coated magnetic nanoparticles (hereinafter, _{abbreviated as "Cl-Fe 3} O ₄ @SiO ₂ ") with a chlorine group substituted are finally obtained. Characteristics of silica-coated magnetic nanoparticles substituted with chlorine groups FT-IR (Fig. 3) was analyzed to confirm.

Example 2: Synthesis and purification of cysteine-tagged multimer protein G in _{Cl-Fe 3} O ₄ @SiO _{2 (see FIG. 2)}

0.1% Tween 20 was added to 5 mg of Cl-Fe ₃ O ₄ @SiO _{2 so} that the concentration of cysteine protein G received from Junggyeom Co., Ltd. was 100, 125, 150, 175, 200, 300㎍/㎖, respectively. Resuspended in 10 mM PBS buffer at the concentration. Each sample is stirred and incubated by rotary shaking for 1 hour. After the resuspension was performed, magnetically for 10 minutes, Cl-Fe ₃ O ₄ @SiO ₂ particles to which the cysteine protein G was immobilized were purified using a magnet. After purification, a 1.5 ml 10 mM PBS buffer (pH Wash 3 times with 7.4).

In the above process, cysteine-based protein G is immobilized on the magnetic nanoparticles, as shown in FIG. 2 of the accompanying drawings. The BCA test was performed to confirm the efficiency of each concentration of cysteine-based protein G _{immobilized on Cl-Fe 3} O ₄ @SiO _{2 particles.} 4 shows the results of binding efficiency according to each concentration of cysteine-based protein G. FIG. 5 shows that cysteine protein G is immobilized on _{Cl-Fe 3} O ₄ @SiO ₂ particles when the concentration of cysteine protein G is 100, 300 μg/ml in 5 mg of Cl-Fe ₃ O ₄ @SiO _{2 particles.} This is the result of SDS-PAGE analysis of the supernatant that was washed out without being washed.

_{As a result of the above, in the present invention, functional magnetic nanoparticles Cl-Fe 3} O ₄ @SiO ₂ for immobilizing cysteine-based protein G were prepared more economically and simpler than the conventional method for making functional particles for immobilizing protein G, It was confirmed that the immobilization of cysteine protein G of the prepared Cl-Fe ₃ O ₄ @SiO _{2 particles was excellent. When 5 mg of Cl-Fe 3} O ₄ @SiO ₂ particles were used, the concentration of cysteine protein G was 100 μg. When it was /ml, it was confirmed that it became saturated.

Claims (9)

As a core, a chloro-based silane is substituted on the surface of the magnetic silica nanoparticles coated with silica on the nanomagnetic oxide, and the cysteine-based protein G is bonded to the chloro group of the silica-coated magnetic nanoparticles substituted with the chloro-based silane. Functional silica magnetic nanoparticles immobilized with cysteine protein G, characterized in that there is.
The method of claim 1,
The nanomagnetic material is one selected from the group consisting of magnetite (Fe3O4), hematite (α-Fe2O3), maghemite (γ-Fe2O3), nickel, cobalt, and their compounds. Silica magnetic nanoparticles.
The method of claim 1,
The cysteine-based protein G is a functional silica magnetic nanoparticle immobilized with cysteine-based protein G, characterized in that it is a tri-protein G multimer in which a cysteine functional group is tagged at the terminal.
The method of claim 1,
The magnetic nanoparticles are functional silica magnetic nanoparticles immobilized with cysteine-based protein G, characterized in that they have a size of 30 to 200 nm.
The method of claim 1,
The chloro-based silane is ((Chloromethyl)phenylethyl-trimethoxy silane);
((chloromethyl)phenylethyl-trimethoxysilane); chloromethylphenethyltris(trimethylsiloxy)silane;
(p-Chloromethyl)phenyltrimethoxysilane; chloromethyltriethoxysilane; chloromethyltrimethoxysilane;
chloromethyltrimethylsilane; chloromethyltris(trimethylsiloxy)silane; chlorophenyltriethoxysilane;
p-Chlorophenyltriethoxysilane; p-Chlorophenyltrimethylsilane; 3-Chloropropyltriethoxysilane;
3-Chloropropyltrimethoxysilane; 3-Chloropropyltrimethylsilane; 3-Chloropropyltris(trimethylsiloxy)-
silane; chlorosilane; 2-(4-Chlorosulfonylphenyl)ethyltrimethoxy-silane;
(dichloromethyl)trimethylsilane; triethylchlorosilane; trimethylchlorosilane;
2-(Trimethylsilyl)ethoxymethyl chloride; tris-(trimethylsiloxy)chlorosilane;
vinyldiphenylchlorosilane; trivinyl-chlorosilane, characterized in that selected from the group consisting of
Functional silica magnetic nanoparticles immobilized with cysteine protein G.
Prepare magnetic silica nanoparticles coated with silica on the nanomagnetic oxide as a core, and substitute chloro-based silane on the surface of the silica-coated nanoparticles,
A method for producing functional silica magnetic nanoparticles immobilized with cysteine-based protein G, comprising the step of immobilizing cysteine-based protein G by binding to a chloro group of the silica-coated magnetic nanoparticles substituted with the chloro group.
The method of claim 6,
The chloro-based silane is ((Chloromethyl)phenylethyl-trimethoxy silane); ((chloromethyl)phenylethyl-trimethoxysilane); chloromethylphenethyltris(trimethylsiloxy)silane;
(p-Chloromethyl)phenyltrimethoxysilane; chloromethyltriethoxysilane;
chloromethyltrimethoxysilane; chloromethyltrimethylsilane; chloromethyltris(trimethylsiloxy)
silane; chlorophenyltriethoxysilane; p-Chlorophenyltriethoxysilane;
p-Chlorophenyltrimethylsilane; 3-Chloropropyltriethoxysilane; 3-Chloropropyltrimethoxysilane;
3-Chloropropyltrimethylsilane; 3-Chloropropyltris(trimethylsiloxy)-silane; chlorosilane;
2-(4-Chlorosulfonylphenyl)ethyltrimethoxy-silane; (dichloromethyl)trimethylsilane;
triethylchlorosilane; trimethylchlorosilane; 2-(Trimethylsilyl)ethoxymethyl chloride;
tris-(trimethylsiloxy)chlorosilane; vinyldiphenylchlorosilane; with trivinyl-chlorosilane
Cysteine-based protein G-immobilized functional silica, characterized in that selected from the group consisting of
Method for producing magnetic nanoparticles.
The method of claim 6,
The nanomagnetic material is cysteine, characterized in that it is one selected from the group consisting of magnetite (Fe3O4), hematite (α-Fe2O3), maghemite (γ-Fe2O3), nickel, cobalt, and compounds thereof. Method for producing functional silica magnetic nanoparticles with immobilized protein G.
The method of claim 6,
The protein G multimer is a method for producing functional silica magnetic nanoparticles immobilized with cysteine protein G, characterized in that the protein G multimer is a tri- protein G multimer in which a cysteine functional group is tagged at the terminal.

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