KR20190095980A - Janus particle and method of the same - Google Patents

Abstract

The present invention relates to Janus particles that can be used for DNA screening and a manufacturing method thereof. Janus particles according to an embodiment of the present invention include body particles; a self-assembled layer formed on the surface of the body particles; an inorganic metal layer formed on one surface of the self-assembled layer; and a target material linker portion coupled to the other surface other than the one surface.

Description

Janus particle and method of manufacturing same

The present invention relates to a Janus particle and a method for preparing the same, and a Janus particle and a method for producing the same, which can be used for DNA screening through a Janus particle capable of immobilizing DNA and protein.

Nano Technology (NT) is a technology that controls and controls materials at the atomic or molecular level, and is suitable for the creation of new materials or new devices. Therefore, their applications include electronics, materials, communications, machinery, medicine, agriculture, energy. And environmental fields.

Such nanotechnology is currently developing variously and is classified into three fields. First, it relates to a technology for synthesizing new materials and materials of extremely small sizes with nanomaterials. Secondly, it is a nano device and relates to a technology for manufacturing a device having a certain function by combining or arranging nano-sized materials. Third, it relates to nano-bio technology that applies nanotechnology to the biotechnology field.

In nano-bio technology, nanoparticles have various characteristics such as detection, quantification and separation of biomolecules related to various diseases due to their unique characteristics derived from nanoscale and their ease of functionalization using various organic / inorganic compounds. have.

Publication No. 10-2008-0011856

SUMMARY OF THE INVENTION An object of the present invention is to provide a Janus particle and a method for preparing the same, which can fix DNA and protein, and thus can be used for DNA screening.

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

Janus particles according to an embodiment of the present invention for achieving the problem to be solved, the body particles, a self-assembled layer formed on the surface of the body particles, an inorganic metal layer formed on one surface of the self-assembled layer, It may include a target material linker portion coupled to the other surface other than the one surface.

The self-assembled layer includes any one of a thiol group, a carboxyl group, an amine group and an epoxy group.

The body particles are formed of any one selected from the group consisting of silica, glass, silicone, quartz, poly (meth) acrylate, polystyrene, polyethylene, polypropylene, polyester, polyamino acid and polyethyleneimine.

The inorganic metal layer may include an inorganic metal, and the inorganic metal may include gold (Ag), platinum (Pt), silver (Ag), copper (Cu), cobalt (Co), iron (Fe), and nickel (Ni). ), Manganese (Mn), zinc (Zn), molybdenum (Mo) and chromium (Cr) any one selected from the group consisting of.

The inorganic metal is laminated on the one surface by a sputtering method.

The target linker portion is any one selected from the group consisting of avidin (avidin), streptavidin (streptavidin), neutravidin, protein A, protein G, lectin and selectin.

According to another aspect of the present invention, there is provided a method of manufacturing Janus particles, the method comprising: providing body particles, forming a self-assembled layer on the surface of the body particles, and Stacking an inorganic metal on one surface of the layer to form an inorganic metal layer, and bonding the target material linker to the other surface other than the one surface.

The self-assembled layer includes any one of a thiol group, a carboxyl group, an amine group and an epoxy group.

The body particles are formed of any one selected from the group consisting of silica, glass, silicone, quartz, poly (meth) acrylate, polystyrene, polyethylene, polypropylene, polyester, polyamino acid and polyethyleneimine.

The inorganic metal is laminated on the one surface by a sputtering method.

The inorganic metal is gold (Ag), platinum (Pt), silver (Ag), copper (Cu), cobalt (Co), iron (Fe), nickel (Ni), manganese (Mn), zinc (Zn) , Molybdenum (Mo) and chromium (Cr) is any one selected from the group consisting of.

The target linker portion is any one selected from the group consisting of avidin (avidin), streptavidin (streptavidin), neutravidin, protein A, protein G, lectin and selectin.

The target material linker portion is covalently or hydrogen bonded to the self-assembled layer.

According to another aspect of the present invention, there is provided a DNA screening method comprising the steps of immobilizing a target DNA on the Janus particles, and fixing the target DNA on the Janus particles. Immersing in the expressed emulsion, synthesizing a target protein by the target DNA in the emulsion, and fixing the target protein to the Janus particles.

The Janus particles include body particles, a self-assembled layer formed on the surface of the body particles, an inorganic metal layer formed on one surface of the self-assembled layer, and a target material linker portion coupled to the other surface other than the one surface.

The target DNA is fixed to the target material linker portion.

The target protein is fixed to the inorganic metal layer.

And after the target protein is immobilized to the Janus particles, testing the activity of the target protein.

After testing the activity of the target protein, the method further comprises the step of conducting a PCR reaction of the target DNA matching the target protein to analyze the base sequence of the target DNA.

According to the present invention, Janus particles and methods for preparing the same are provided for DNA screening through Janus particles to which DNA and protein can be immobilized.

1A shows a conceptual diagram of Janus particles.
FIG. 1B is a cross-sectional view of Janus particles taken along line II ′ of FIG. 1A.
Figure 2 is a flow chart showing a manufacturing method of Janus particles according to an embodiment of the present invention.
3 is a view sequentially showing a manufacturing method of Janus particles.
Figure 4 shows the DNA screening (screening) process using the Janus particles of the invention.
5 and 6 are SEM images of the produced Janus particles.
7 to 9 are photographs showing that DNA is fixed and protein is expressed using Janus particles.

Advantages and features of the present invention and methods for achieving them will be apparent with reference to the embodiments described below in detail with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below, but will be implemented in various forms, and only the present embodiments are intended to complete the disclosure of the present invention, and the general knowledge in the art to which the present invention pertains. It is provided to fully convey the scope of the invention to those skilled in the art, and the present invention is defined only by the scope of the claims. DETAILED DESCRIPTION Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings. Regardless of the drawings, the same reference numbers refer to the same components, and “and / or” includes each and every combination of one or more of the items mentioned.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In this specification, the singular also includes the plural unless specifically stated otherwise in the phrase. As used herein, "comprises" and / or "comprising" does not exclude the presence or addition of one or more other components in addition to the mentioned components.

Unless otherwise defined, all terms (including technical and scientific terms) used in the present specification may be used in a sense that can be commonly understood by those skilled in the art. In addition, the terms defined in the commonly used dictionaries are not ideally or excessively interpreted unless they are specifically defined clearly.

Below look at the Janus particles and Janus particle production method according to the embodiments of the present invention.

Janus particles according to an embodiment of the present invention includes a surface of different chemical properties on the surface of the particles, thereby inducing protein expression through DNA. In addition, since the expressed protein can be immobilized on the surface, it can be usefully used for DNA screening.

First, referring to FIGS. 1A and 1B, a Janus particle according to an exemplary embodiment of the present invention will be described. FIG. 1A illustrates a conceptual diagram of Janus particles, and FIG. 1B is a cross-sectional view taken along line II ′ of FIG. 1A. 1A and 1B, a Janus particle 10 according to an embodiment of the present invention includes a main body particle 101, a self-assembled layer 102 formed on a surface of the main body particle 101, and the magnetic The inorganic metal layer 103 formed on one surface of the assembly layer 102 and a target material linker 104 coupled to the other surface other than the one surface may be included.

The main body particle 101 is a part which comprises the main body of the Janus particle 10. The self-assembled layer 102, the inorganic metal layer 103, and the linker portion 104 will be formed on the body particle 101, and the body particle 101 may be formed of a material capable of supporting the layer. . The body particle 101 may be formed of any one selected from the group consisting of silica, glass, silicon, quartz, poly (meth) acrylate, polystyrene, polyethylene, polypropylene, polyester, polyamino acid and polyethyleneimine. Among these, preferably silica may be used.

Subsequently, the self-assembly layer 102 is located on the surface of the main body particle 101. The self-assembled layer 102 may be formed to cover all of the surfaces of the body particles 101.

The self-assembled layer 102 may be formed using a monomolecular compound for forming the self-assembled layer 102 including at least one functional group selected from the group consisting of a thiol group, a carboxyl group, an amine group, an epoxy group, and -OH. However, the present invention is not limited thereto. For example, the monomolecular compound for forming the self-assembly layer 102 includes a hydrocarbon chain, a benzene ring or an alkylbenzene, including one or more functional groups selected from the group consisting of a thiol group, a carboxyl group, an amine group, an epoxy group, and -OH. It may be a compound including a ring, but is not limited thereto.

For example, the monomolecular compound for forming the self-assembled layer may include at least one thiol group, and may further include one or more functional groups selected from the group consisting of a carboxyl group, an amine group, an epoxy group, and -OH, but is not limited thereto. It is not. For example, the monomolecular compound for forming the self-assembled layer may be a compound including a hydrocarbon chain, a benzene ring, or an alkylbenzene ring, further comprising one thiol group, a carboxyl group, an amine group, an epoxy group, or -OH. It is not limited.

For example, carbon number of the hydrocarbon chain included in the self-assembly layer 102 may be 1, 3, or 6 or more, but is not limited thereto. For example, carbon number of the hydrocarbon chain included in the self-assembly layer 102 is 1 to 20, 1 to 18, 1 to 16, 1 to 14, 1 to 12, 1 to 10, 1 to 8, 1 to 6 , 1 to 4, 3 to 20, 3 to 18, 3 to 14, 3 to 12, 3 to 10, 3 to 8, 3 to 6, 6 to 20, 6 to 18, 6 to 16, 6 to 14, 6 to 12 or 6 to 10, but is not limited thereto.

The alkylbenzene ring included in the self-assembled layer 102 may include a benzene ring having an alkyl group having 1 to 6 carbon atoms, but is not limited thereto. For example, the alkylbenzene ring may include methyl benzene ring, ethylbenzene ring, propylbenzene ring, butylbenzene ring, pentylbenzene ring, hexylbenzene ring or isomer thereof, but is not limited thereto.

The self-assembly layer 102 is a target material linker portion 104 to be described later, where the biotin is fixed to form a biotin layer in the step of introducing a reactor suitable for causing biotin to react for immobilization This can also be useful.

An inorganic metal layer 103 is formed on one surface of the self-assembly layer 102. The inorganic metal layer 103 is a protein to be expressed by the DNA is fixed. To this end, a linker to be bonded to the inorganic metal layer 103 may be coupled to the terminal of the synthesized protein. An example of such a linker is GBP (Gold Binding Protein), but a linker capable of binding to the inorganic metal layer 103 may exist in various ways, but is not limited thereto.

The inorganic metal layer 103 may include an inorganic metal. These inorganic metals include gold (Ag), platinum (Pt), silver (Ag), copper (Cu), cobalt (Co), iron (Fe), nickel (Ni), manganese (Mn), zinc (Zn), It may be any one selected from the group consisting of molybdenum (Mo) and chromium (Cr).

The inorganic metal layer 103 may be formed on one surface of the self-assembled layer 102 by, for example, a sputtering method. That is, when one of the inorganic metals listed above is introduced into the sputtering device and the introduced inorganic metal collides with the charged ion particles, a part of the inorganic metal is directed onto the self-assembled layer 102 by the collision, and self-assembled Some particles of the inorganic metal directed onto the layer 102 will be deposited on the self-assembled layer 102. As a result, the inorganic metal layer 103 is formed on one surface of the self-assembly layer 102.

On the other hand, the target material linker 104 is coupled to the other surface other than the one surface of the self-assembly layer 102. The target material linker unit 104 is where DNA binds, and may form specific binding with biotin that binds to DNA.

The target linker portion 104 may be any one selected from the group consisting of avidin, streptavidin, neutravidin, protein A, protein G, lectin, and selectin. Can be. The interaction between the functional group of the target material ring cup portion 104 and the functional group of the self-assembly layer 102 may be combined and fixed. On the other hand, the target material linker 104 and the DNA can be cross-specific binding by biotin, thereby fixing the DNA to the target material linker 104.

Next, referring to Figures 1a to 3, it will be described a manufacturing method of Janus particles according to another embodiment of the present invention. Figure 2 is a flow chart showing a manufacturing method of Janus particles according to an embodiment of the present invention, Figure 3 is a view showing a manufacturing method of the Janus particles sequentially. Meanwhile, the same matters as the terms described in the above-described exemplary embodiment are substantially the same in the present exemplary embodiment, and thus repeated descriptions thereof will be omitted.

First, in order to manufacture Janus particles, the body particles 101 are provided (S10). The main body particle 101 forms the main body of the Janus particle 10, and any one formed of the above-described material may be selected. The size of the body particles 101 may have a particle diameter of 5 μm to 10 μm. For example, silica may be selected as the body particle 101, but is not limited thereto.

The provided body particles 101 are immersed in a solution containing any one of a thiol group, a carboxyl group, an amine group, and an epoxy group, and the self-assembled layer 102 having any one of the functional groups coated on the surface of the body particles 101 is coated. It forms (S20).

The self-assembled layer 102 includes octanethiol (OT), mercaptoundecanoic acid (MUA), cysteamine, 4-methylbenzene thiol (MBT), 4- Ethylbenzenethiol, mercaptopropionic acid (Mercaptopropionic acid: MPA), 4-mercaptophenol (mercaptophenol) or may be formed of a solution containing a combination thereof, but is not limited thereto. In addition, the self-assembly layer 102 may be formed by reacting 3-aminopropyltrimethoxysilane with ethanol and water (H ₂ O). That is, the material may be reacted to form the self-assembled layer 102 on the surface of the body particle 101.

For example, the self-assembled layer 102 may be a mixed solution including OT and MUA in a volume ratio of 3: 1 and ethanol as a solvent, but is not limited thereto. The self-assembled layer 102 may be formed by immersing the body particles 101 in the solution containing the material for 6 hours or more. On the other hand, when cysteamine is used, an amine group may be introduced to the surface of the body particle 101. The streptavidin, which is the target linker 104, may be specifically bound by the amine group.

Subsequently, the body particles 101 on which the self-assembly layer 102 is formed are arranged on the substrate. The substrate on which the body particles 101 are arranged is introduced into a chamber in a vacuum state. The substrate is disposed on one side of the chamber and an inorganic metal is disposed on the other side. When a negative voltage is applied to the target inorganic metal, gas molecules ionized by the discharge collide with the inorganic metal, and a part of the inorganic metal is sputtered to face the substrate. A portion of the inorganic metal facing in the substrate direction is deposited on the body particles 101 on the substrate. As a result, the inorganic metal layer 103 is laminated on one surface of the self-assembly layer 102 to form the inorganic metal layer 103 (S30).

Subsequently, the target material linker unit 104 is coupled to the other surface other than the one surface on which the inorganic metal layer 103 is formed (S40).

According to the exemplary embodiment of the present invention, the coupling of the target material linker unit 104 to the self-assembled layer 102 may include a functional group included in the self-assembled layer 102 and the target material linker unit 104. The target material linker 104 may be immobilized on the self-assembled layer 102 by forming a covalent bond through a reaction between functional groups. If necessary, a coupling agent may be further used to promote covalent bond formation between a functional group included in the compound for forming the self-assembled layer 102 and a functional group included in the target material linker unit 104. For example, the coupling agent may include, but is not limited to, EDC [1-ethy-3- (dimethylaminopropyl) carbodiimide], sulfo-NHS (N-hydroxysulfosuccinimide), or a combination thereof.

By the above-described process, the self-assembled layer 102 and the inorganic metal layer formed on one surface of the self-assembled layer 102 and the body particles 101 and the body particles 101 according to an embodiment of the present invention ( Janus particles comprising a 103, and the target material linker 104 coupled to the other surface other than the one surface can be produced.

On the other hand, Janus particles produced by the present invention can be used in the method for screening (DNA) as follows.

Figure 4 shows the DNA screening (screening) process using the Janus particles of the invention.

For DNA screening, the target DNA 201 is immobilized on Janus particles 10. The target DNA 201 may be fixed to the target material linker 104 of the Janus particle 10 described above. The target DNA 201 may proceed as follows. For example, when the target linker portion 104 is formed of streptavidin, streptavidin may not form a specific bond with the target DNA 201. To bind streptavidin and target DNA 201, biotin may be introduced as a linker between streptavidin and target DNA 201. For example, the 5 'end of the target DNA 201 may be modified with biotin. Through the above modification, the target DNA 201 can be immobilized to streptavidin using specific binding force of streptavidin and biotin.

Subsequently, the Janus particles to which the target DNA 201 is immobilized are immersed in an emulsion in which the protein is expressed. The emulsion is a single emulsion that can express the protein in response to the target DNA. That is, the target protein 202 is synthesized by the target DNA 201 in the emulsion by immersing the Janus particles to which the target DNA 201 is immobilized.

The synthesized target protein 202 may be immobilized on the inorganic metal layer 103 of the Janus particle 10. When the synthesis of the target protein 202 and the immobilization on the Janus particles 10 are terminated, the emulsion is broken and no further protein synthesis occurs.

Subsequently, the activity of the target protein 202 fixed to the Janus particle 10 is investigated. Among the synthesized proteins, if the protein having the highest activity is irradiated, the emulsion may be designated as a medium for synthesizing a protein having excellent chemical properties.

The target DNA is matched with a protein having excellent activity, and a polymerase chain reaction (PCR) reaction is performed on the target DNA. PCR reaction can be performed using a well-known PCR apparatus. After the PCR reaction, by analyzing the smoke sequence of the target DNA, it is possible to analyze the base sequence of the protein having excellent activity. Such DNA screening enables DNA sequencing of antigen antibodies through proteins, and thus, the present invention can be said to have many applications in the field of biotechnology.

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

Preparation Example: Preparation of Janus Particles

To prepare Janus particles, silica was used as the body particles. The particle diameter of silica was 6 micrometers-9 micrometers. For the amine group treatment on the surface of silica, 0.5 g of cysteamine was dissolved in 5 g of methanol. After immersing silica in the finished cysteamine solution, an amine group was introduced into the surface of silica.

Silica into which the amine group was introduced on the surface was aligned with a longitudinal 5 cm long substrate, and then introduced into the chamber for sputtering. After setting the pressure in the chamber to 10 ⁻² Pa, 1 g of platinum (Pt) was used as the target material. After applying a negative voltage to platinum (Pt), sputtering is performed for about 10 minutes. By the sputtering process, a platinum layer was successfully formed on one surface of the silica. Referring to FIG. 5, platinum may be laminated on the surface of silica coated with an amine group by a sputtering method.

Subsequently, streptavidin was formed on the surface of the silica by applying the EDC / NHs coupling technique on the amine-coated surface not coated with platinum. Referring to FIG. 6, the FITC fluorescent material was attached to the formed streptavidin, indicating that streptavidin was successfully introduced into the silica particles. This completed the Janus particle of the present invention.

Experimental Example: DNA Screening Using Janus Particles

DNA screening was performed using the Janus particles prepared by the preparation example.

Referring to FIG. 7, the target DNA was fixed to streptavidin using streptavidin-biotin on the surface of Janus stent where streptavidin was immobilized. It was confirmed using a Cy3 fluorescent material fixed to the DNA.

Janus particles immobilized with DNA were introduced as a single particle into an emulsion consisting of a cell-free expression solution / Oil and expression was performed using the single particles. Referring to Figure 8, it can be seen that the protein was expressed by Janus particles.

Subsequently, after the emulsion was broken, Janus particles were recovered and subjected to surface analysis. Referring to Figure 9, it was confirmed that the DNA and the protein (protein) expressed by the DNA to Janus particles are well fixed.

After protein expression, matching DNA was extracted and amplified by PCR reaction, through which DNA sequencing of the protein well expressed in the emulsion could be analyzed.

According to the present invention, Janus particles could be formed in a very simple manner using a vertical plasma sputter technique. Conventional methods for forming Janus particles have been mainly used by a chemical method of various chemicals, according to the present invention, it is possible to form Janus particles through a simpler sputtering method. In addition, by screening the chemical functional groups on the surface of Janus particles to fix the protein and DNA, DNA screening can be performed with the Janus particles of the present invention. On the other hand, it is possible to try a variety of applications by introducing a variety of functional groups on the surface of the Janus particles of the present invention.

Although the embodiments of the present invention have been described above, the present invention is not limited to the above embodiments and can be manufactured in various forms, and a person of ordinary skill in the art to which the present invention pertains has the technical idea of the present invention. However, it will be understood that other specific forms may be practiced without changing the essential features. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

Claims (19)

Body particles;
A self-assembled layer formed on the surface of the body particles;
An inorganic metal layer formed on one surface of the self-assembled layer; And
Janus particles comprising; a target material linker portion coupled to the other surface other than the one surface. According to claim 1,
Janus particles, characterized in that the self-assembled layer comprises any one of a thiol group, a carboxyl group, an amine group and an epoxy group. According to claim 1,
Janus particles, characterized in that the body particles are formed of any one selected from the group consisting of silica, glass, silicon, quartz, poly (meth) acrylate, polystyrene, polyethylene, polypropylene, polyester, polyamino acid and polyethyleneimine. . According to claim 1,
The inorganic metal layer may include an inorganic metal, and the inorganic metal may include gold (Ag), platinum (Pt), silver (Ag), copper (Cu), cobalt (Co), iron (Fe), and nickel (Ni). Janus particles, characterized in that any one selected from the group consisting of, (Mn), zinc (Zn), molybdenum (Mo) and chromium (Cr). The method of claim 4, wherein
Janus particles, the inorganic metal is laminated on the one surface by a sputtering method. According to claim 1,
The target material linker portion, characterized in that any one selected from the group consisting of avidin (avidin), streptavidin (streptavidin), neutravidin, protein A, protein G, lectin (selectin) and selectin (selectin) Janus particles. Providing body particles;
Forming a self-assembled layer on the surface of the body particles;
Forming an inorganic metal layer by laminating an inorganic metal on one surface of the self-assembling layer; And
Janus particle manufacturing method comprising a; linking the target material linker portion on the other surface other than the one surface. The method of claim 7, wherein
The self-assembled layer is a manufacturing method of Janus particles, characterized in that it comprises any one of a thiol group, a carboxyl group, an amine group and an epoxy group. The method of claim 7, wherein
Janus particles, characterized in that the body particles are formed of any one selected from the group consisting of silica, glass, silicon, quartz, poly (meth) acrylate, polystyrene, polyethylene, polypropylene, polyester, polyamino acid and polyethyleneimine. Manufacturing method. The method of claim 7, wherein
The inorganic metal is a manufacturing method of Janus particles, characterized in that the laminated on the one surface by a sputtering (sputtering) method. The method of claim 10,
The inorganic metal is gold (Ag), platinum (Pt), silver (Ag), copper (Cu), cobalt (Co), iron (Fe), nickel (Ni), manganese (Mn), zinc (Zn) , Molybdenum (Mo) and chromium (Cr) any one selected from the group consisting of Janus particle manufacturing method. The method of claim 7, wherein
The target material linker portion, characterized in that any one selected from the group consisting of avidin (avidin), streptavidin (streptavidin), neutravidin, protein A, protein G, lectin (selectin) and selectin (selectin) Janus particle production method. The method of claim 12,
The target material linker unit is a Janus particle manufacturing method, characterized in that coupled to the self-assembled layer by a covalent bond or a hydrogen bond. In the method of screening DNA using Janus particles,
Immobilizing a target DNA on the Janus particles;
Immersing the Janus particles in which the target DNA is immobilized in an emulsion in which a protein is expressed;
Synthesizing a target protein by the target DNA in the emulsion; And
DNA screening method comprising the step of immobilizing the target protein to the Janus particles. The method of claim 14,
The Janus particles include DNA including a body particle, a self-assembled layer formed on the surface of the body particle, an inorganic metal layer formed on one surface of the self-assembled layer, and a target material linker portion bonded to the other surface other than the one surface. Screening method. The method of claim 15,
DNA targeting method, characterized in that the target DNA is fixed to the target material linker. The method of claim 16,
The target protein is a DNA screening method, characterized in that fixed to the inorganic metal layer. The method of claim 14,
And after the target protein is immobilized to the Janus particles, examining the activity of the target protein. The method of claim 18,
And analyzing the base sequence of the target DNA by conducting a PCR reaction of the target DNA matching the target protein after examining the activity of the target protein.

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