KR102561465B1 - Water-soluble Carbon-coated magnetic nanoparticle by pyrene derivatives, its preparation method and its applications for purification of biological samples - Google Patents

Abstract

The present invention is a surface treatment method of carbon-coated magnetic nanoparticles for biological applications, more specifically, by adsorbing a hydrophilic pyrene derivative on the surface of hydrophobic carbon-coated magnetic nanoparticles, characterized by excellent water dispersibility and chemical stability. It relates to a water-soluble magnetic nanoparticle composite and a manufacturing method thereof.
According to the present invention, after the π-π stacking of the pyrene derivative on the coated carbon, the presence of a hydrophilic functional group (carboxyl group, amine group, etc.) in the pyrene derivative makes it easy to disperse in water on the outside of the surface. The existence of this functional group can be used effectively in fields such as life sciences, medicine, and diagnostics because it is used not only for water affinity but also for conjugation with biochemical substances, which is the next step. As a specific example, magnetic particles for antibody purification may be prepared by attaching Protein A protein, or magnetic particles for His-tag protein purification may be prepared by attaching Ni-NTA (Nickel-Nitrilo acetic acid) material.

Description

Water-soluble carbon-coated magnetic nanoparticle by pyrene derivatives, its preparation method and its applications for purification of biological samples}

The present invention is hydrophilicization through surface treatment of carbon-coated magnetic nanoparticles for biological applications, more specifically, pyrene derivatives containing hydrophilic functional groups (carboxyl groups, amine groups, etc.) are stacked with carbon surfaces by π-π After this is made, the presence of a hydrophilic functional group (carboxyl group, amine group, etc.) makes the surface outline easy to disperse in water, and this complex is the final product through covalent bonding with proteins and various biochemical substances leads to anger As a specific example, magnetic particles for antibody purification may be prepared by attaching Protein A protein, or magnetic particles for His-tag protein purification may be prepared by attaching Ni-NTA (Nickel-Nitrilo acetic acid) material.

The first life science application of magnetic particles was introduced by John Ugelstad (Norway) in the 1970s, and the main purpose was to separate related substances in samples. The main material is almost always iron oxide, and the main product is a polymer coating material containing a hydrophilic group to have water dispersibility. It is commercially used in the form of attaching various biochemical substances by using a polymer attached with a functional group. John Ugelstad's DynaBead, which was introduced for the first time, is a representative product. These products are mainly used in areas related to separation and purification, as well as in immunodiagnostic techniques.

What appeared as a next-generation product was a carbon-coated magnetic material. This product called Turbo Bead not only shows strong magnetism by carbon coating but also has good chemical stability. There are examples of use in environmental aspects such as heavy metal removal, but a wide range of uses such as DynaBead have not been reported yet. There is a magnetic material called CardioGenics as a multifunctional material in which a measurement method using light is added. This is a form in which the sensitivity to light is remarkably increased by coating the surface of the magnetic particle with silver.

In the case of magnetic iron oxide nanoparticles, a technology that can mass-produce high-quality iron oxide nanomaterials at low cost using a manufacturing technique using ultrasonic irradiation developed by the Korea Research Institute of Chemical Technology (Korean Patent Registration: 10-1350400) is an important advantage. Furthermore, the addition of carbon coating technique (domestic patent registration: 10-1335520), which is relatively inexpensive and easy to use compared to silica, which is a coating material of existing products, is a far more advanced technology in terms of performance and production cost reduction.

In the case of iron-cobalt magnetic particles, they are superior to existing iron oxide products in terms of performance because of the excellent magnetic force, which is a characteristic of the material. As mentioned above, it can be manufactured using ultrasonic irradiation and carbon coating technique (domestic patent registration: 10-1335520).

Nevertheless, the development of surface treatment methods for biological applications of these carbon-coated magnetic nanoparticles is insufficient, and the direct use of biosamples in an aqueous solution is not achieved due to the hydrophobic aspect of the carbon coating itself.

Accordingly, as a result of intensive research efforts to overcome the above problems, the inventors of the present invention reacted the pyrene derivative with the carbon-coated magnetic nanomaterial, thereby improving the stability of the aqueous phase by the hydrophilic functional group connected from the pyrene to the strong bonding force between the pyrene and the coated carbon. By imparting, it was confirmed that a complex having strong magnetism, chemical stability, and bioapplication ability was formed, and the present invention was completed.

US 20120208026 A1 KR 10-1335520 B

A method for producing carbon-coated magnetic nanoparticles uses a metal precursor and a carbon-containing solvent to form a carbon coating layer on the surface of chemically unstable magnetic nanoparticles, without reducing the magnetic properties of individual magnetic nanoparticles. The final result is the mass production of magnetic nanoparticles having a uniform size in the nanodomain with improved chemical and thermal stability. In this case, by using a solvent containing carbon when preparing the metal precursor solution, it serves not only for preparing the metal precursor solution but also as a raw material for coating carbon. Therefore, it has the advantage that there is no need to disperse or add a separate material for carbon coating. After that, the final product is obtained through the process of ultrasonic irradiation, precipitation, and heat treatment.

In order for the prepared carbon-coated nanomaterial to be used in the bio field, hydrophilic surface modification is required on the carbon surface, and proteins or various biochemical substances are attached by attaching functional groups. In addition, it is also a major purpose that there is no toxicity problem, manufacturing is simple, and mass production is possible.

According to one aspect of the present invention, the present invention uses a pyrene derivative containing a hydrophilic functional group to form a strong bond between pyrene and the carbon layer on the surface of the magnetic nanoparticles, thereby forming the pyrene derivative-carbon-coated magnetic nanoparticles. provides a complex.

In the present invention, the pyrene derivative containing a hydrophilic functional group can be any compound exhibiting hydrophilicity, but is preferably pyrene butylic acid or aminopyrene having a carboxyl group or an amine group as a functional group. A pyrene derivative-carbon-coated magnetic nanoparticle complex is provided. These hydrophilic functional groups are converted into water-soluble (biocompatible) nanoparticles.

In the present invention, rather than directly attaching biochemical substances such as antibodies or proteins to carbon-coated nanoparticles, an intermediate complex using a pyrene derivative that imparts water solubility on the carbon coating and induces covalent bond reactivity with these substances After making, a biochemical material-pyrene derivative-carbon-coated magnetic nanoparticle complex is formed, characterized in that the biochemical material is attached using a crosslinker.

In the present invention, preferably, protein A is covalently linked to a hydrophilic chemical functional group on the surface of the carbon-coated magnetic nanoparticles coated with the hydrophilic pyrene derivative, and is used to separate and purify antibodies. Provided is a carbon-coated magnetic nanoparticle composite.

Specifically, for the separation and purification of antibodies, the production of magnetic particles to which Protein A is attached is performed by using 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide (EDC) crosslinker on magnetic particles surface-treated with 1-pyrenebutylic acid. It is prepared by inducing a covalent bond between the primary amine group of A and the carboxyl group on the surface of magnetic particles.

In the present invention, NTA (Nickel-Nitrilo acetic acid) is covalently linked to the hydrophilic chemical functional group on the surface of the carbon-coated magnetic nanoparticles coated with the hydrophilic pyrene derivative to separate His-tag proteins through Ni ions. It provides a pyrene-carbon coated magnetic nanoparticle complex, characterized in that it is used for purification.

Specifically, the attachment of Ni-NTA (nitriloacetic acid) for separation and purification of His-tag protein is prepared by inducing covalent bonding between the primary amine value of amino-NTA and the carboxyl group on the surface of magnetic particles using EDC, and then nickel ions are added. Fill up and use.

According to another aspect of the present invention, there is provided a method for preparing the hydrophilic pyrene-carbon-coated magnetic nanoparticle composite of claim 1, comprising the following steps:

(a) coating hydrophilic pyrene on carbon-coated magnetic nanoparticles;

(b) binding protein A or nickel-nitrilo acetic acid (NTA) to the carbon-coated magnetic nanoparticles coated with hydrophilic pyrene.

According to the present invention, carbon-coated magnetic nanoparticles coated with pyrene derivatives are water-soluble, and in order to use the nanoparticles for bio-use, they are stored in physiological conditions (phosphate-buffered saline, PBS, pH 7.2-7.4) for several months. It is very stable in the refrigerator without losing any physicochemical properties. Additionally, the nanoparticles can be lyophilized and rehydrated upon addition of a physiological buffer without significant loss of any properties.

The method for producing pyrene derivative-carbon-coated magnetic nanoparticles according to the present invention is applied to biological applications after preparing carbon-coated magnetic nanoparticles having a stable carbon coating layer formed on the surface of unstable magnetic nanoparticles through the use of a non-toxic metal precursor and a carbon solvent. To this end, a new hydrophilic coating is formed on the carbon coating layer.

These hydrophilic pyrene derivative-carbon coated magnetic nanoparticles maintain the magnetic properties of individual magnetic nanoparticles while maintaining chemical and physical stability and suitability for use in aqueous solution. In addition, this hydrophilic functional group is a chemical functional group capable of bonding with biochemical substances such as bioactive substances in the carbon coating layer, and various types of surface modification are possible, so it can be applied and used in many areas such as biochemistry, thermotherapy and diagnosis. .

1 is a graph showing the formation of a hydrophilic pyrene derivative-carbon-coated magnetic nanoparticle complex according to an embodiment of the present invention, NTA binding through EDC, a binding derivative, and separation of his-tag protein through filling of nickel ions, and preparation of final products for purification. It is a schematic diagram showing the method.
2 is a transmission electron micrograph of carbon-coated magnetic nanoparticles used in preparing the hydrophilic pyrene derivative-carbon-coated magnetic nanoparticle composite in Example 1.
3 is a result of X-ray diffraction analysis of carbon-coated magnetic nanoparticles used in preparing the hydrophilic pyrene derivative-carbon-coated magnetic nanoparticle complex in Example 1.
4 is a result of magnetic hysteresis curves for the carbon-coated magnetic nanoparticles used in the preparation of the hydrophilic pyrene derivative-carbon-coated magnetic nanoparticle composite in Example 1 and Fe2O3dp, which is a general magnetic particle.
5 is a result of the manufacturing process of the hydrophilic pyrene derivative-carbon-coated magnetic nanoparticle composite in Example 1.
6 is a photograph of carbon-coated magnetic nanoparticles (left) in a chloroform solvent and a photograph of a hydrophilic pyrene derivative-carbon-coated magnetic nanoparticle complex (right) obtained through a coating reaction with pyrene butylic acid dissolved in an aqueous solution.
7 is an enzyme activity test (TMB Test) of the final HRP-hydrophilic pyrene derivative-carbon coated magnetic nanoparticle complex by reacting the hydrophilic pyrene derivative-carbon coated magnetic nanoparticle complex with the Horse Radish Peroxidase (HRP) enzyme among physiologically active substances. ) is the result of As a color change test according to enzyme activity after adding TMB, these are the reaction pictures of the hydrophilic pyrene derivative-carbon-coated magnetic nanoparticle complex (left) and the final HRP-hydrophilic pyrene derivative-carbon-coated magnetic nanoparticle complex (right) reaction picture.
Figure 8 shows the E. Coli RNAP alpha subunit remaining in the supernatant after the E. Coli RNAP alpha subunit separation experiment at 280 nm by coordinating Ni to the nitrilotriacetic acid (NTA)-coupled carbon-coated magnetic nanoparticle complex in Example 2. It is an absorption measurement table and graph. For the reproducibility test, the experiment was repeated 7 times.
9 is an SDS-PAGE image used to confirm the E. Coli RNAP alpha subunit by eluting the E. Coli RNAP alpha subunit separated from the Ni-coordinated carbon-coated magnetic nanoparticles with a 250 mM Imidazole solution.
Figure 10 shows the final complex of human Immunoglobulin G-ProteinA-hydrophilic pyrene derivative-carbon-coated magnetic nanoparticles by reacting ProteinA-hydrophilic pyrene derivative-carbon-coated magnetic nanoparticle complex with human Immunoglobulin G (antibody) for separation and purification of antibodies It is a diagram related to the amount of production of This is the result of measuring the absorbance at 280 nm by adding 200 μl of human Immunoglobulin G at a concentration of 1 mg/ml to the ProteinA-hydrophilic pyrene derivative-carbon-coated nanoparticle complex, and recovering the added solution after the reaction was finished. The absorbance at 280nm measured on the left is the absorbance of the first added solution and the recovered solution, and the absorbance at 280nm measured on the right is based on this to form the final complex of human Immunoglobulin G-ProteinA-hydrophilic pyrene derivative-carbon-coated magnetic nanoparticles. It is the expected absorbance of one human Immunoglobulin G. The concentration and weight values on the right side were derived using the Beer-Lambert law.

Hereinafter, the present invention will be described in more detail through examples. Since these examples are intended to illustrate the present invention only, the scope of the present invention is not to be construed as being limited by these examples.

<Example 1> Preparation of a hydrophilic pyrene derivative-carbon-coated magnetic nanoparticle composite

Step 1: Mix 20mg of carbon-coated magnetic nanoparticles with 1ml of chloroform organic solvent.

Step 2: After adding 3ml of 50mM pyrene butyric acid dissolved in 1M NaOH in the same volume, the carbon-coated magnetic nanoparticles and the reaction were mixed at room temperature using a shaker for 1 hour.

Step 3: After the reaction is completed, the pyrene derivative-carbon-coated magnetic nanoparticle complex is separated using a magnet and washed with distilled water.

Step 4: After washing, the pyrene derivative-carbon-coated magnetic nanoparticle complex is refrigerated after adding distilled water.

In step 2, the carbon-coated magnetic nanoparticles present in the organic solvent layer are separated into the aqueous solution layer, which means that the pyrene derivative-carbon-coated magnetic nanoparticle composite has hydrophilicity.

<Example 2> Preparation of HRP (Horse Radish Peroxidase)-hydrophilic pyrene derivative-carbon-coated magnetic nanoparticle complex

Step 1: Prepare a slurry of 0.5 ml from the hydrophilic pyrene derivative-carbon coated magnetic nanoparticle complex (2mg magnetic particles/ml phosphate buffered saline) stored in phosphate buffered saline.

Step 2: Hydrophilic pyrene derivative obtained by dissolving 140 ug of 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide and 300 ug of Sulfo NHS in 100 ul of phosphate buffered saline and removing the supernatant from the slurry of step 1- The carbon-coated magnetic nanoparticle composite was reacted with a vortex mixer for 10 minutes.

Step 3: After removing the supernatant, wash twice with phosphate buffered saline.

Step 4: Put in 100 ul of phosphate buffered saline and store in the refrigerator.

Step 5: To test HRP bonding, 100 ul of Tetramethylbenzidine (TMB) solution was reacted with 1 ul slurry of samples before and after HRP bonding.

<Example 3> Preparation of Nitrilotriacetic Acid (NTA) Coupled Magnetic Nanoparticle Complex and Protein Separation and Purification Test

The pyrene derivative-carbon-coated magnetic nanoparticle composite prepared in Example 1 was used.

Step 1: Put 2 mg of the pyrene derivative-carbon-coated magnetic nanoparticle complex into a tube and wash it three times using a magnet and 200 μl of 0.1M MES buffer (pH 4.6).

Step 2: Prepare by dissolving 15.3mg of 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide (EDC) in 200μl 0.1M MES buffer (pH 4.6) in a tube.

Step 3: Prepare a 50 mM Na,Na-Bis(carboxymethyl)-L-lysine hydrate (NTA) solution using 0.1M MES buffer (pH 4.6).

Step 4: After removing the solution of the pyrene derivative-carbon-coated magnetic nanoparticle complex that has been washed using a magnet, 200 μl of 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide (EDC) is added and left at room temperature for 10 minutes. Mix using a shaker.

Step 5: After the reaction is complete, wash 3 times with 200μl 0.1M MES buffer (pH 4.6) and remove the solution (remove residual EDC).

Step 6: Add 100μl 50mM a,Na-Bis(carboxymethyl)-L-lysine hydrate (NTA) solution to the washed EDC-pyrene derivative-carbon-coated magnetic nanoparticle complex and shaker at room temperature for 1 hour. Mix using

Step 7: After completion of the reaction, prepare by washing with distilled water three times.

Step 8: After adding 500 μl 0.1M NiSO ₄ solution to the washed magnetic nanoparticle composite, it was mixed for 10 minutes using a shaker at room temperature.

Step 9: After mixing, prepare by washing three times with 500 μl Ni binding buffer.

Step 10: After adding 100 μl E. Coli RNAP alpha subunit solution, mix using a shaker at room temperature for 30 to 60 minutes.

Step 11: After completion of the reaction, the supernatant is removed using a magnet and washed three times using Ni binding buffer. The supernatant is measured for absorption at 280 nm to determine the amount of remaining protein.

Step 12: Use Ni binding buffer with 100μl 250mM imidazole to separate the E. coli RNAP alpha subunit attached to Ni.

Step 13: Confirm the isolated E. Coli RNAP alpha subunit by SDS-PAGE.

<Example 4> Preparation of ProteinA-hydrophilic pyrene derivative-carbon-coated magnetic nanoparticle complex for antibody purification

Step 1: Prepare a slurry of 40 μl from the hydrophilic pyrene derivative-carbon-coated magnetic nanoparticle complex (10 mg magnetic particles/200 μl phosphoric acid-buffered saline) stored in phosphoric acid-buffered saline (PBS, pH 7.2-7.4).

Step 2: Remove the phosphoric acid-buffer brine from the hydrophilic pyrene derivative-carbon coated magnetic nanoparticle complex using a tube holder with a magnet, and add 0.1M 2-(N-morpholino)ethanesulfonic acid (pH:4.6), 0.5M Wash twice with 200 μl of NaCl.

Step 3: After dissolving 15.3 ug of 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide in 200 ul of 0.1M 2-(N-morpholino)ethanesulfonic acid (pH:4.6), 0.5M NaCl, The supernatant from the washed slurry is reacted with the hydrophilic pyrene derivative-carbon-coated magnetic nanoparticle complex at room temperature for 10 minutes with a vortex mixer.

Step 4: After removing the supernatant, wash twice with phosphoric acid-buffered saline.

Step 5: Prepare ProteinA-hydrophilic pyrene derivative-carbon-coated magnetic nanoparticle complex (10mg magnetic particles/200μl phosphate buffered saline) slurry by adding 200μl phosphate buffer saline.

Step 6: The supernatant of the slurry of step 5 is removed.

Step 7: 200 ul of phosphate buffered saline (pH: 7.4), 1mg/ml human Immunoglobulin G dissolved in 0.5M NaCl slurry from which the supernatant of step 6 was removed ProteinA-hydrophilic pyrene derivative-carbon coating magnetic React the nanoparticle complex with a vortex mixer for 10 minutes.

Step 8: After the reaction is over, the supernatant is removed, stored separately, and washed twice with phosphate-buffered saline.

Step 9: Absorbance at 280 nm wavelength of human Immunoglobulin G at a concentration of 1 mg/ml dissolved in the supernatant removed in step 8, 200 μl of phosphate buffered saline (pH: 7.4), and 0.5 M NaCl added in step 3 By comparing the amount of human Immunoglobulin G-ProteinA-hydrophilic pyrene derivative-carbon-coated magnetic nanoparticle composite, the amount of human Immunoglobulin G entered into the preparation was calculated by Beer-Lambert method (extinction coefficient of human Immunoglobulin G at 280 nm: 210,000 M ^-1 cm ^{- 1} , molecular weight of human Immunoglobulin G: 150,000).

As described above, according to the present invention, a pyrene derivative-carbon-coated magnetic nanoparticle complex is formed by forming a strong bond between pyrene and the carbon layer on the surface of magnetic nanoparticles using a pyrene derivative containing a hydrophilic functional group. By making the solubility and stability of the aqueous phase of magnetic nanoparticles, it is possible to attach them to various biological substances for separation, purification, concentration and detection by covalent bonds. Therefore, this magnetic nanoparticle complex can be effectively used in the fields of hematology such as biology, medicine and diagnostics.

Claims (5)

It consists of carbon-coated magnetic nanoparticles and a hydrophilic pyrene derivative coated on the outside, and the hydrophilic pyrene derivative is pyrene butyric acid in which a carboxyl group is bonded to a pyrene molecule, -Ethyl-3-[3-dimethylaminopropyl] carbodiimide (EDC) and hydrophilic pyrene-carbon coated magnetic nanoparticle complex.
delete The hydrophilic pyrene-carbon according to claim 1, wherein protein A is covalently linked to a hydrophilic chemical functional group on the surface of the carbon-coated magnetic nanoparticles coated with the hydrophilic pyrene derivative, and is used to separate and purify the antibody. Coated magnetic nanoparticle composite.
The method of claim 1, wherein NTA (Nickel-Nitrilo acetic acid) is covalently linked to the hydrophilic chemical functional group on the surface of the carbon-coated magnetic nanoparticles coated with the hydrophilic pyrene derivative, thereby forming His-tag proteins through Ni ions. A hydrophilic pyrene-carbon coated magnetic nanoparticle complex, characterized in that it is used for separation and purification. delete