KR20190082570A - Method for synthesis of monodisperse magnetic amylose microbeads and its applications - Google Patents

Abstract

Disclosed are a method for manufacturing magnetic amylose microbeads in the uniform size, and a method for purifying protein, and separating and concentrating target pathogens in a sample using the same. The method for manufacturing magnetic amylose microbeads comprises the following steps: coating dextran on the surface of magnetic nanoparticles and preparing a nucleation agent; and adding the nucleation agent to a substrate solution including short chain amylose (SCA) and synthesizing magnetic amylose microbeads.

Description

TECHNICAL FIELD [0001] The present invention relates to a method for producing magnetic amylose microbeads having uniform size,

More particularly, the present invention relates to a method for producing magnetic amylose microbeads having a uniform size and affinity with target materials such as separation and concentration of target pathogens, And a method for efficiently separating and purifying a desired substance from a sample using the magnetic properties of the magnetic substance.

A variety of ligands have been attracting attention in recent decades for applications in the separation and purification of proteins, which can bind proteins with high affinity and selectivity (Young et al. of affinity tags and microbial applications, Ha et al 2008 Purification of his-tagged proteins using Ni2 + -poly (2-acetamidoacrylic acid) hydrogel). Among them, selective separation and purification of proteins using complex matrices is an important technology for protein and enzyme engineering.

In addition, magnetic particles are widely used as a material for selectively purifying a protein and a target substance present in the sample. In particular, in order to detect specific pathogens present in small numbers in food, drinking water or the environment, it is necessary to effectively separate and concentrate them from the sample. In this case, when magnetic particles having receptors specifically binding to specific pathogens are used, The target pathogen is separated and concentrated from the sample to enable accurate detection. To meet this demand, several global companies, such as Pierce, Sigma, and Thermo Fisher, sell a wide variety of magnetic particles, and these magnetic particles are used in polymeric base materials such as polystyrene, polyacrylamide, polyvinyl alcohol, and polyethylene glycol It is manufactured by introducing nanoparticles, and its price is being sold at a high price of about USD100-300 per milliliter.

Therefore, a technique of synthesizing amylose from sucrose using the biological reaction of amylose synthase and producing the amylose in the form of particles through self-assembly reaction has been studied. When the magnetic nanoparticles are added during the self-assembly process of the amylose molecule, the amorphous nanoparticles form particles together with the amylose, and the amylose nanoparticles thus produced have superparamagnetic properties, It has high value as a material to be selectively separated from the sample. In particular, it uses low-cost sucrose as a substrate, it is produced through a simple biological reaction that does not require a complicated process and a self-assembly process at room temperature, so that the cost for production can not be compared with the commercial products described above It also has strengths.

However, this technique requires a heating process at a high temperature, and the amount of magnetic nanoparticles collected on the amylose particles is not so small, so that the magnetic sensitivity is low and the size of particles produced is not uniform. Therefore, in order to solve such a problem, there is a demand for a technique for producing particles having high magnetic sensitivity and uniform size. The high magnetic susceptibility of the magnetic particles is important for rapid separation and concentration, and the uniform particle size is important for the accurate estimation of the dispersibility and specific surface area of the magnetic particles in the solution. This is an essential factor in ensuring the reproducibility of the concentration process.

Korean Patent Publication No. 2009-0121313, "Production of a resistant starch product" Korean Patent Registration No. 1214572, "Method for producing cyclic amylose"

The present invention is to produce magnetic amylose microbeads of uniform size by promoting the crystallization of mono-chain amylose by using dextran-coated magnetic nanoparticles on the surface as a nucleating agent.

Disclosure of Invention Technical Problem [8] The present invention provides a magnetic nanoparticle having a magnetic nanoparticle-core structure having a core-shell structure of dextran-magnetic nanoparticles, improving the affinity between the magnetic nanoparticles and the mono-chain amylose to increase the amount of magnetic nanoparticles captured, To produce magnetic amylose microbeads.

The present invention relates to a method for preparing a magnetic amylose microbead by uniformly preparing magnetic amylose microbeads by controlling the concentration of the nucleating agent and determining the size and uniformity of the magnetic amylose microbeads to be produced, To produce particles.

The present invention relates to a method of separating an MBP-fusion protein using magnetic amylose microbeads prepared by using dextran-coated magnetic nanoparticles as a nucleating agent on a surface thereof and using magnetic amylose microbeads functionalized with a ligand-fused MBP protein Thereby improving the detection performance of the target molecule.

That is, the present invention is to utilize fused MBP as a crosslinked protein to effectively isolate and concentrate pathogenic microorganisms present in trace amounts in food, drinking water or the environment in order to modify an antibody specifically binding to a specific microorganism. In addition, the present invention can quantitatively detect the isolated and concentrated target pathogen through PCR or other analysis method.

The present invention relates to a method of separating an MBP-fusion protein using magnetic amylose microbeads prepared by using dextran-coated magnetic nanoparticles as a nucleating agent on a surface thereof and using magnetic amylose microbeads functionalized with a ligand-fused MBP protein To provide a means of separate enrichment required for accurate detection of specific target pathogens and target molecules.

A method for producing magnetic amylose microbeads according to an embodiment of the present invention includes the steps of: preparing a nucleation agent by coating dextran on the surface of magnetic nanoparticles; And adding the nucleating agent to a substrate solution containing short chain amylose (SCA) to synthesize magnetic amylose microbeads.

The nucleating agent may have a core-shell structure, the core portion may include the magnetic nanoparticles, and the shell portion may include the dextran.

The size of the magnetic amylose microbeads may be controlled depending on the content of the nucleating agent.

The size of the magnetic amylose microbeads can be controlled according to the synthesis time of the magnetic amylose microbeads.

The step of synthesizing the magnetic amylose microbeads by adding the nucleating agent to the substrate solution containing the short chain amylose (SCA) comprises: preparing an amylose synthase; Preparing a substrate solution comprising a substrate and a solvent; And adding the amylose synthase to the substrate solution and performing an enzymatic reaction to synthesize mono-chain amylose.

The amylose synthase may include at least one of amylosucrase, phosphorylase, starch synthase, amylase, and D-enzyme.

The magnetic nanoparticles may be iron oxide nanoparticles.

The magnetic amylose microbeads according to the embodiments of the present invention are produced by the method of producing magnetic amylose microbeads according to the embodiments of the present invention.

The magnetic amylose microbeads may be in the form of a rod.

The magnetic amylose microbeads can be used for therapeutic substances, diagnostic substances, packaging materials for functional health food materials, coating agents or chromatography resins.

A method for isolating and concentrating a target pathogen using magnetic amylose microbeads according to an embodiment of the present invention comprises: preparing a nucleating agent by coating dextran on the surface of magnetic nanoparticles; Synthesizing magnetic amylose microbeads by adding the nucleating agent to a substrate solution containing mono-chain amylose; Adding the magnetic amylose microbeads to a sample containing target pathogens and then binding target pathogens thereto; And separating and concentrating the magnetic amylose microbeads to which the target pathogen is bound.

The step of isolating and concentrating the magnetic amylose microbeads bound to the target pathogen may include a step of separating the target pathogen from the amylose magnetic beads by adding a competitive binder to the target pathogen on the surface of the magnetic amylose microbeads .

The surface of the magnetic amylose microbeads may be modified with an antibody specific to the target pathogen using MBP-SPG fusion protein as a cross-linking agent.

A method for purifying a target protein using magnetic amylose microbeads according to an embodiment of the present invention includes: preparing a nucleating agent by coating dextran on the surface of magnetic nanoparticles; Synthesizing magnetic amylose microbeads by adding the nucleating agent to a substrate solution containing mono-chain amylose; Binding the desired protein to the magnetic amylose microbeads; And separating and purifying magnetic amylose microbeads to which the target protein is bound.

The target protein may be a maltose binding protein (MBP) -fused protein (MBP-tagged protein).

The step of isolating and purifying the magnetic amylose microbeads to which the objective protein is bound may include separating the target protein from the amylose magnetic beads by adding a competitive binder to the target protein on the surface of the magnetic amylose microbeads have.

According to the embodiment of the present invention, magnetic nanoparticles coated with dextran on the surface can be used as a nucleating agent to promote crystallization of mono-chain amylose to produce magnetic amylose microbeads of uniform size.

According to the embodiment of the present invention, the nucleation agent of the dextran-magnetic nanoparticle core-shell structure is used to improve the affinity between the magnetic nanoparticles and the single-chain amylose to improve the collection efficiency of the magnetic nanoparticles, This improved magnetic amylose microbead can be produced.

According to an embodiment of the present invention, MBP-fusion protein is separated using magnetic amylose microbeads prepared by using dextran-coated magnetic nanoparticles as a nucleating agent on the surface, and ligand-fusion MBP protein is functionalized with magnetic Amylose microbeads can be used to enhance the detection performance of the target molecule.

According to an embodiment of the present invention, MBP-fusion protein is separated using magnetic amylose microbeads prepared by using dextran-coated magnetic nanoparticles as a nucleating agent on the surface, and ligand-fusion MBP protein is functionalized with magnetic Amylose microbeads can be used to reduce the detection cost of the target molecule.

According to an embodiment of the present invention, an MBP-SPG fusion protein is used as a cross-linking agent in magnetic amylose microbeads to modify an antibody specifically binding to a target pathogen, and by isolating and concentrating the target pathogen present in the sample, Separated and concentrated target pathogens can be quantitatively detected using PCR or other analytical methods.

According to an embodiment of the present invention, magnetic amylose microbeads functionalized with a ligand-fused MBP protein can be used in various fields such as small volume protein separation and purification, biosensors and actuators.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1A is a view showing a method for producing magnetic amylose microbeads according to an embodiment of the present invention. FIG.
FIG. 1B is a cross-sectional view illustrating the structure of a nucleating agent used in a method of producing magnetic amylose microbeads according to an embodiment of the present invention. FIG.
FIG. 2A is a transmission electron microscopy (TEM) image of a nucleating agent, and FIG. 2B is a graph showing a size distribution of a nucleating agent.
FIG. 2C is a graph showing FT-IR spectra of dextran and iron oxide nanoparticles constituting the nucleating agent. FIG.
Figure 2d is X- for the reference pattern (iv) of the _{γ-Fe 2 O 3 (i} ), nucleating agent (ii), Fe ₃ O ₄ reference pattern (reference patterns) (iii), and γ-Fe ₂ O ₃ of Ray diffraction spectroscopy (XRD).
FIG. 3A is an image showing a result of scanning electron microscopy of a magnetic amylose microbead produced without a nucleating agent, and FIG. 3B is an image showing a result of scanning electron microscopy of a magnetic amylose microbead according to an embodiment of the present invention.
4A is a graph showing the size (average diameter) of the magnetic amylose microbeads according to the embodiment of the present invention according to the concentration of the nucleating agent.
4B is a graph showing the size (average diameter) of the magnetic amylose microbeads according to the embodiment of the present invention with respect to the synthesis time.
5A is a graph showing a scanning electron microscopic result image and a size distribution of magnetic amylose microbeads according to an embodiment of the present invention.
5B is an image showing electron mapping and transmission electron microscopic results of magnetic amylose microbeads according to an embodiment of the present invention.
FIG. 5c is an image showing fluorescence microscopy image of magnetic amylose microbeads according to an embodiment of the present invention in which maltose binding protein-green fluorescent protein (MBP-GFP) fusion protein is functionalized.
5D is an image showing the reaction of magnetic amylose microbeads dispersed in a solution according to an embodiment of the present invention by external magnetic force.
6A is a graph showing the collection efficiency of magnetic amylose microbeads according to an embodiment of the present invention, depending on the number of bacteria in a phosphate buffer saline (PBS).
6B is a graph showing the collection efficiency of the magnetic amylose microbeads according to the embodiment of the present invention according to the number of bacteria in the milk.
FIG. 6c shows the efficiency of collecting magnetic amylose microbeads according to an embodiment of the present invention against E. coli, Bacillus cereus, Staphylococcus aureus and Vibrio cholera FIG.
Figure 6d is a graph showing the number of recycling of magnetic amylose microbeads according to an embodiment of the invention for bacterial isolation (left) and FITC-Ab-functionalized embodiments of the invention in each cycle (Right) image of the magnetic amylose microbeads according to the present invention.
FIGS. 7A to 7C are images showing rod-shaped magnetic amylose microbeads prepared by the method for producing magnetic amylose microbeads according to an embodiment of the present invention.
FIG. 7d is an image showing a scanning electron microscope result of a bar-shaped magnetic amylose microbead produced by the method of manufacturing magnetic amylose microbeads according to an embodiment of the present invention, FIG. 3 is an image showing a transmission electron microscope result of a bar-shaped magnetic amylose microbead produced by a method of producing a microbead. FIG.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings and accompanying drawings, but the present invention is not limited to or limited by the embodiments.

The terminology used herein is for the purpose of illustrating embodiments and is not intended to be limiting of the invention. In the present specification, the singular form includes plural forms unless otherwise specified in the specification. It is noted that the terms "comprises" and / or "comprising" used in the specification are intended to be inclusive in a manner similar to the components, steps, operations, and / Or additions.

As used herein, the terms "embodiment," "example," "side," "example," and the like should be construed as advantageous or advantageous over any other aspect or design It does not.

Also, the term 'or' implies an inclusive or 'inclusive' rather than an exclusive or 'exclusive'. That is, unless expressly stated otherwise or clear from the context, the expression 'x uses a or b' means any of the natural inclusive permutations.

Also, the phrase "a" or "an ", as used in the specification and claims, unless the context clearly dictates otherwise, or to the singular form, .

It will also be understood that when an element such as a film, layer, region, configuration request, etc. is referred to as being "on" or "on" another element, And the like are included.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1A is a view showing a method for producing magnetic amylose microbeads according to an embodiment of the present invention. FIG.

The method for producing magnetic amylose microbeads according to an embodiment of the present invention includes the steps of (S110) preparing a nucleation agent (Dex @ IONPs) by coating dextran on the surface of magnetic nanoparticles (S110) (S120) by adding a nucleating agent (Dex @ IONPs) to a substrate solution containing short chain amylose (SCA) to synthesize magnetic amylose microbeads (SAMBs).

In step S110, a method of producing magnetic amylose microbeads according to an embodiment of the present invention includes coating a surface of magnetic nanoparticles with dextran to prepare a nucleating agent (Dex @ IONPs).

More specifically, step S110 may comprise adding a magnetic nanoparticle precursor and dextran to the solvent to produce a nucleator solution and sonicating the nucleator solution.

First, a magnetic nanoparticle precursor and dextran are added into a solvent to prepare a nucleating agent solution.

Preferably, the magnetic nanoparticle precursor may include at least one of ferric chloride hexahydrate (FeCl _{3 ·} 6H ₂ O) and ferrous chloride tetrahydrate _{(FeCl 2 · 4H 2 O)} .

The solvent is not particularly limited, but deionized water may be preferably used as the solvent.

In addition, the nucleating agent solution may comprise a precipitation agent, a precipitating agent may comprise at least one of a sodium hydroxide (NaOH) and ammonium hydroxide (NH ₄ OH), the precipitating agent is the pH of the nucleating agent solution 9 - 12 Range.

In addition, the step of sonicating the nucleating agent solution can sonicate the nucleating agent solution for 1 to 20 minutes.

If the ultrasonic treatment time is less than 1 minute, the nucleating agent (Dex @ IONPs) is not completely formed, and if it exceeds 20 minutes, the size of the nucleating agent (Dex @ IONPs) becomes small.

Upon completion of the sonication, a nucleating agent (Dex @ IONPs) precipitates in the nucleating agent solution. Thus, the supernatant is discarded and the precipitated nucleating agent (Dex @ IONPs) is obtained as a tertiary distilled water by washing until the pH of the washings is 6-7. The reason for limiting the pH to 6-7 is to stabilize the nucleating agent (Dex @ IONPs) by neutral control.

Therefore, in the method of producing magnetic amylose microbeads according to the embodiment of the present invention, a safe colloidal solution can be prepared by rapidly dispersing the ultrasonic wave of the nucleating agent solution, and the nucleating agent (Dex @ IONPs) Dex @ IONPs) can be prevented.

The nucleating agent (Dex @ IONPs) prepared according to the method for producing magnetic amylose microbeads according to the embodiment of the present invention may have a core-shell structure, and the core-shell structure will be described in detail in FIG. 1B.

FIG. 1B is a cross-sectional view showing the structure of a nucleating agent prepared according to a method of producing magnetic amylose microbeads according to an embodiment of the present invention. FIG.

The core forming material (Dex @ IONPs) prepared according to the manufacturing method of the magnetic amylose microbeads according to the embodiment of the present invention may have a core-shell structure, the core portion 110 includes magnetic nanoparticles, 120) can include dextran.

The magnetic nanoparticles included in the core portion 110 may be used without limitation as long as the magnetic nanoparticles are composed of a magnetic metal material, a magnetic material, or a magnetic alloy, and the metal material includes at least one of Pt, Pd, Ag, Cu, may be, of the magnetic material is Co, Mn, Fe, Ni, Gd, Mo, MM _'2 O _4, and MpOq (M and M' are each independently selected from Co, Fe, Ni, Mn, Zn, Gd, and Cr And 0 < p < 3 and 0 &lt; q &lt; 5), and the magnetic alloy may include at least one of CoCu, CoPt, FePt, CoSm, NiFe, NiFeCo, and MnFe ₂ O ₄ .

Preferably, the magnetic nanoparticles include iron oxide nanoparticles. More preferably, the magnetic nanoparticles are selected from the group consisting of maghemite (Fe ₂ O ₃ ) and magnetite (Fe ₃ O ₄ ) having a diameter of 2 nm to 20 nm, (SPIO or USPIO) by including at least one of them.

It is preferable to use dextran as the shell 120 coated on the surface of the magnetic nanoparticles, but polysaccharides such as chitosan, starch or oligosaccharide can be used without limitation.

In the conventional case of producing magnetic amylose microbeads using magnetic nanoparticles, magnetic nanoparticles are formed on the surface of the magnetic amylose microbeads because the magnetic nanoparticles do not include the shell part 120, so that the cohesion between the magnetic amylose microbeads The magnetic amylose microbeads of uniform size can not be produced.

In addition, it is difficult to functionalize the surface of the magnetic amylose microbeads due to the magnetic nanoparticles formed on the surface of the magnetic amylose microbeads.

However, the nucleating agent (Dex @ IONPs) prepared according to the method of manufacturing magnetic amylose microbeads according to the embodiment of the present invention includes magnetic nanoparticles in the core portion 110 and dextrane in the shell portion 120 The magnetic nanoparticles are not exposed on the surface of the magnetic amylose microbeads (SAMBs) to reduce the cohesive force that may occur between the magnetic nanoparticles, and the magnetic amylose microbeads (SAMBs) Can be produced.

In addition, since the nucleating agent promotes nucleation reaction during the reaction of producing magnetic amylose microbeads (SAMBs) and induces nucleation in a short time, the size of magnetic amylose microbeads (SAMBs) can be uniformly formed.

However, in the case of the conventional reaction without nucleating agent, since the nucleation occurs throughout the reaction time, there is a problem that the size of magnetic amylose microbeads produced is not uniform.

Also, since the nucleating agent prepared according to the method of producing magnetic amylose microbeads according to the embodiment of the present invention includes dextran in the shell part 120, it is easy to functionalize the surface of the magnetic amylose microbeads.

1A, in step S120, a method for producing magnetic amylose microbeads according to an embodiment of the present invention comprises adding a nucleating agent (Dex @ IONPs) to a substrate solution containing short chain amylose (SCA) To thereby prepare a magnetic amylose microbead.

Preferably, the step of synthesizing magnetic amylose microbeads (SAMBs) by adding a nucleating agent to a substrate solution comprising short chain amylose (SCAs) comprises the steps of preparing a substrate solution and adding amylose synthesis Enzymes and nucleating agents (Dex @ IONPs) to synthesize magnetic amylose microbeads (SAMBs).

According to an embodiment, the step of synthesizing (SAMBs) magnetic amylose microbeads by adding a nucleating agent (Dex @ IONPs) to a substrate solution comprising mono-chain amylose (SCAs) comprises the steps of preparing an amylose synthase, Preparing a substrate solution containing a solvent, and adding an amylose synthase to the substrate solution and performing an enzyme reaction to synthesize mono-chain amylose. Then, the substrate solution containing single-chain amylose (SCAs) (Dex @ IONPs) can be added to synthesize magnetic amylose microbeads (SAMBs).

First, a method for producing magnetic amylose microbeads according to an embodiment of the present invention produces a substrate solution.

The substrate solution is a physiologically active solution containing a saccharide used as a substrate for the amylose synthase, and is prepared by adding a substrate to the solvent. The substrate serves as a monomer that is used by the amylose synthase in the process of synthesizing amylose to form a polymer. Such a substrate may include at least one of glucose, oligosaccharide, and sucrose.

As the solvent, Tris-HCl, PBS and the like can be used. In preparing the substrate solution, the substrate is preferably contained in an amount of 2 to 25 parts by weight based on 100 parts by weight of the solvent. When the substrate is contained in an amount of less than 2 parts by weight, an amylose chain is formed by the amylose synthase, but there arises a problem that magnetic amylose microbeads are not formed. If the substrate is more than 25 parts by weight, the amylose synthase consumes the substrate and the product produced is likely to form an isomer.

The pH value of the solvent is preferably 6 to 9. If the pH value of the solvent is less than 6, the activity of the amylose synthase is low in the enzyme reaction, resulting in a problem that the formation of the reaction product is not smooth. Even when the pH value of the solvent exceeds 9, there arises a problem that the activity of the amylose synthase decreases in the enzyme reaction.

Finally, in the method for producing magnetic amylose microbeads according to the embodiment of the present invention, amylose synthesizing enzyme and nucleating agent (Dex @ IONPs) are added to a substrate solution to synthesize magnetic amylose microbeads.

The amylose synthase may include at least one of amylosucrase, phosphorylase, starch synthase, amylase, and D-enzyme.

Thus, the substrate solution can produce mono-chain amylose by including amylose synthase and substrate in the solvent.

The nucleating agent (Dex @ IONPs) is preferably added in an amount of 0.1 to 30 parts by weight based on 100 parts by weight of the solvent. If the content of the nucleating agent (Dex @ IONPs) is less than 0.1 part by weight, the function of the magnetic nanoparticles may not be exhibited, which is undesirable. If the nucleating agent (Dex @ IONPs) exceeds 30 parts by weight, The activity of the magnetic amylose microbeads (SAMBs) may be inhibited and the reaction products may not be smoothly formed. The size of the produced magnetic amylose microbeads (SAMBs) is not uniform.

In the synthesis of a substrate solution by adding a nucleating agent (Dex @ IONPs) to a substrate solution and performing an enzymatic reaction, the enzyme reaction temperature is preferably within a temperature range of 20 ° C to 60 ° C. If the reaction temperature is less than 20 캜, the activity of the amylose synthase is low, and the formation of the reaction product is not smooth. If the reaction temperature exceeds 60 ° C, the amylose synthase loses its activity due to thermal stress, and the half life is also shortened.

Therefore, the method of producing magnetic amylose microbeads according to the embodiment of the present invention can be easily performed by using amylose synthesis enzymes and nucleating agents (Dex @ IONPs) without the complicated or high temperature process SAMBs) can be synthesized.

Also, the method for producing magnetic amylose microbeads according to an embodiment of the present invention comprises adding amylose synthesizing enzyme and a nucleating agent (Dex @ IONPs) to a substrate solution and conducting an enzyme reaction under a predetermined condition to produce magnetic amylose microbeads (SAMBs) Magnetic amylose microbeads (SAMBs) are spontaneously formed through self-assembly of single-chain amylose (SCAs) formed together with substrate consumption of amylose synthase.

Since the method of producing magnetic amylose microbeads according to the embodiment of the present invention forms magnetic amylose microbeads (SAMBs) by enzymatic synthesis, the manufacturing process is simple and high temperature heat treatment as in the prior art is not required. In addition, the magnetic amylose microbeads (SAMBs) prepared by the method for producing magnetic amylose microbeads according to the embodiments of the present invention have high purity of amylose.

The size (diameter) of the magnetic amylose microbeads (SAMBs) produced by the method for producing magnetic amylose microbeads according to the embodiment of the present invention can be controlled according to the content of the nucleating agent (Dex @ IONPs).

More specifically, since the amount of single-chain amylose (SCAs) contained in the substrate solution is limited, the size of the magnetic amylose microbeads (SAMBs) can be reduced as the concentration of the nucleating agent (Dex @ IONPs) is increased.

Accordingly, the method of producing magnetic amylose microbeads according to the embodiment of the present invention can easily control the size of magnetic amylose microbeads (SAMBs) according to the concentration of the nucleating agent (Dex @ IONPs).

The size of the magnetic amylose microbeads (SAMBs) produced by the method for producing magnetic amylose microbeads according to the embodiment of the present invention can be controlled according to the synthesis time of the magnetic amylose microbeads (SAMBs).

Since the amylose microbeads (SAMBs) are formed by the self-assembly reaction, the method of producing magnetic amylose microbeads according to the embodiment of the present invention includes reaction delay time for nucleation in the initial stage of the reaction, After this, nuclei are grown.

More specifically, in the method of producing magnetic amylose microbeads according to an embodiment of the present invention, a nucleating agent-short chain amylose complex (Dex @ IONPs) is reacted with a nucleating agent (Dex @ IONPs) and short chain amylose / SCAs complexes) and then self-assembly of mono-chain amylose (SCAs) formed by the starch-amylose complex (Dex @ IONPs / SCAs complexes) and substrate depletion of amylose synthase to form magnetic amylose microbeads SAMBs) can be spontaneously formed.

Also, since the size of the particles grows gradually according to the synthesis time, the magnetic amylose microbeads (SAMBs) can be increased in size as the synthesis time increases, have.

However, since the amount of the single-chain amylose (SCAs) contained in the substrate solution is limited, the size of the magnetic amylose microbeads (SAMBs) does not increase any more after a certain time.

According to the embodiment, the magnetic amylose microbeads (SAMBs) prepared by the method for producing magnetic amylose microbeads according to the embodiment of the present invention can be rearranged in the form of a rod.

After the step S120 of synthesizing the magnetic amylose microbeads by adding the nucleating agent to the substrate solution containing the single-chain amylose (SCAs), the magnetic amylose microbeads are arranged in a linear manner using the external magnetic force And the rod-like structure is stabilized through an additional self-assembly process of single-chain amylose (SCAs) on its surface.

Accordingly, the magnetic amylose microbeads (SAMBs) prepared by the method of the present invention can be used not only in spherical shapes but also in rod shapes, and thus can be utilized in various fields.

The magnetic amylose microbeads (SAMBs) prepared by the method for producing magnetic amylose microbeads according to the embodiments of the present invention can be used for therapeutic substances, diagnostic substances, functional health food packaging materials, coating agents or chromatography resins .

The magnetic amylose microbeads according to the embodiments of the present invention can be used for separation, concentration, or purification of a target pathogen.

In particular, the immunomagnetic particles using the magnetic amylose microbeads according to the embodiment of the present invention can be used for isolating and concentrating target pathogens for target pathogen detection.

A method for isolating and concentrating a target pathogen using magnetic amylose microbeads according to an embodiment of the present invention includes the steps of: preparing a nucleating agent by coating dextran on the surface of magnetic nanoparticles, adding a nucleating agent to a substrate solution containing mono-chain amylose, Adding magnetic amylose microbeads to a sample containing target pathogens to bind target pathogens and isolating and concentrating the magnetic amylose microbeads to which target pathogens are bound, .

A method for isolating and concentrating a target pathogen using magnetic amylose microbeads according to an embodiment of the present invention includes the steps of: preparing a nucleating agent by coating dextran on the surface of magnetic nanoparticles; and adding a nucleating agent to the substrate solution containing mono-chain amylose To synthesize magnetic amylose microbeads is the same as that described in Figs. 1A and 1B, and therefore, the same description will be omitted.

In the method of isolating and concentrating a target pathogen using magnetic amylose microbeads according to an embodiment of the present invention, magnetic amylose microbeads are added to a sample containing target pathogens to bind target pathogens.

The sample contains a pathogenic microorganism to be separated, microorganisms, other microorganisms, a food substrate, etc., and a targeting moyer that specifically binds to a target substance (for example, target pathogen) to be separated in order to isolate the microorganism to be separated Treatment of a magnetic amylose microbead according to an embodiment of the present invention in which the moiety is bonded to a surface to a sample containing the microorganism causes a targeting moiety that specifically binds to the target material To produce a target material-targeting moiety conjugate.

"Targeting moiety" means any substance capable of binding to an antigen or a protein or gene in a microorganism present on the surface of the target microorganism. The targeting moiety according to an embodiment of the present invention may be one or more selected from the group consisting of a protein, an assembling antibody using a fragment, a peptide, an antibody, an antibody fragment or a gene, preferably an antibody, It does not.

Preferably, the surface of the magnetic amylose microbeads can be modified by using an MBP-SPG fusion protein as a cross-linking agent to specifically target the pathogen.

Therefore, in the method of isolating and concentrating the target pathogen using the magnetic amylose microbeads according to the embodiment of the present invention, the MBP-SPG fusion protein is used as the cross-linking agent and the antibody specifically binding to the target pathogen is immobilized on the surface of the magnetic amylose microparticle, And can be used to isolate and concentrate the target pathogen present in the sample.

A method for isolating and concentrating a target pathogen using magnetic amylose microbeads according to an embodiment of the present invention includes separating and concentrating magnetic amylose microbeads to which a target pathogen is bound.

The step of isolating and concentrating the magnetic amylose microbeads bound to the target pathogen may comprise the step of isolating the target pathogen from the amylose magnetic beads by adding a competitive binder to the target pathogen on the surface of the magnetic amylose microbeads.

Preferably, the competitive binder for the target pathogen on the magnetic amylose microbead surface may be maltose.

The method of isolating and concentrating a target pathogen using the magnetic amylose microbeads according to an embodiment of the present invention may remove the magnetic amylose microbeads bound to the target pathogen to more easily detect the target pathogen. When the target substance is a microorganism, a method commonly used in the art may be used to detect microorganisms. If the target substance is a gene, the gene may be amplified and detected by a PCR method or the like.

More specifically, according to the method of isolating and concentrating the target pathogen using the magnetic amylose microbeads according to the embodiment of the present invention, the target pathogen isolated and concentrated can be quantitatively detected by PCR or other analysis method

Thus, magnetic amylose microbeads (SAMBs) can be used to isolate MBP-fusion proteins and detect target molecules and target microorganisms using magnetic amylose microbeads (SAMBs) functionalized with ligand-fused MBP proteins.

A method of purifying a target protein using magnetic amylose microbeads according to an embodiment of the present invention includes the steps of preparing a nucleating agent by coating dextran on the surface of magnetic nanoparticles, adding a nucleating agent to a substrate solution containing mono-chain amylose Thereby synthesizing magnetic amylose microbeads, binding the desired protein to the magnetic amylose microbeads, and separating and purifying magnetic amylose microbeads bound to the target protein.

A method for purifying a target protein using magnetic amylose microbeads according to an embodiment of the present invention comprises purifying a target protein using magnetic amylose microbeads (SAMBs) prepared by the method for producing magnetic amylose microbeads according to an embodiment of the present invention can do.

The target protein may be MBP (maltose binding protein) -fusion protein (MBP-tagged protein).

The step of isolating and purifying the magnetic amylose microbeads (SAMBs) to which the target protein is bound comprises the step of separating the target protein from the amylose magnetic beads by adding a competitive binder to the target protein on the surface of the magnetic amylose microbeads (SAMBs) can do.

The method of purifying a target protein using the magnetic amylose microbeads according to an embodiment of the present invention is a method of purifying magnetic amylose microbeads (SAMBs) composed of pure amylose molecules and nucleus forming agents (Dex @ IONPs) through amylose- And their application to magnetic separation and purification of MBP-fusion proteins.

The use of an enzyme catalyst based on a bottom-up approach allows nucleating agents (Dex @ IONPs) to react with the substrate to form magnetic amylose microbeads (SAMBs) complexes. Amylose molecules on the surface of magnetic amylose microbeads (SAMBs) can be used for affinity adsorption with MBP-fusion proteins.

The efficiency of magnetic amylose microbeads (SAMBs) as a template for magnetic separation and purification of proteins could be demonstrated by MBP-fusion green fluorescent protein (GFP) as a model protein. In order to study the application in biomolecule separation, the selectivity and recycling of amylose magnetic beads were investigated. Magnetic amylose microbeads can be recycled several times without loss effective in the purification capacity of the target protein.

Specifically, the enzymatic synthesis of magnetic amylose microbeads (SAMBs) in vitro is a powerful bottom-up approach for the production of amylose-nanomaterial hybrid microparticles.

Magnetic amylose microbeads (SAMBs) can form complexes with amylose molecules synthesized by the hydrophobic reaction of amylose and nucleating agents (Dex @ IONPs) during the enzymatic reaction. Because of its magnetic properties, amylose magnetic amylose microbeads (SAMBs) make it possible to use for magnetic separation applications of biomaterials. Magnetic separation power was measured by testing separation and purification of MBP-fused GFP from E. coli cell solution using magnetic amylose microbeads (SAMBs), and the purification capacity was 72 mu g of amylose magnetic bead mg protein.

Hereinafter, characteristics of the magnetic amylose microbeads produced by the method for producing magnetic amylose microbeads according to the embodiments of the present invention will be described.

Manufacturing example

Nucleating agents (Dex @ IONPs)

80mmol of the FeCl _{3 ·} 6H ₂ O, dextran (dextran) _{2 ·} 4H ₂ O and the 150mg FeCl 40mmol of deionized water of 20ml; was dissolved in (deionized water DW) was prepared in the nucleating agent solution. The nucleating agent solution was ultrasonicated using a Q500 sonicator (VC 750, Sonics & Materials Inc., Newtown, CT, USA) for 2 minutes while bubbling gently with gentle stream.

The ultrasonic treatment was performed by using a 6-mm ultrasound probe, an interval of 5 seconds and a disruption period of 5 seconds in an ice bath for 3 minutes at an amplitude of 25% .

Magnetic amylose microbeads (SAMBs)

Single-chain amylose (SCA) was prepared by enzymatic polymerization using amylosucrase derived from Deinococcus geothermalis (DGAS). A nucleating agent (Dex @ IONPs) was mixed in a 50 mM Tris-HCl solution (pH 8.0) in a substrate solution containing 500 units of Deanococcus zeussalmaris and 500 mM sucrose.

Magnetic amylose microbeads were mixed with 1 ml of deionized water (DW) and incubated at 40 ° C for 48 hours to synthesize magnetic amylose microbeads. The magnetic amylose microbeads were separated by a magnetic separation method, washed three times with deionized water and ethanol, and then stored at 4 ° C in 20% ethanol.

FIG. 2A is a transmission electron microscopy (TEM) image of a nucleating agent, and FIG. 2B is a graph showing a size distribution of a nucleating agent.

Referring to FIGS. 2A and 2B, it can be seen that a nucleation agent having a diameter of 127.4. + -. 35 nm is well formed.

2C is a graph showing FT-IR spectra of the nucleating agent, dextran and iron oxide nanoparticles.

In FIG. 2C, the nucleating agent shows a red line, the dextran shows a black line, and the iron oxide nanoparticles show a blue line.

Referring to FIG. 2C, the FT-IR spectrum of the nucleating agent shows absorption at 1150-1085 cm ^-1 and 3600-3200 cm ^-1 corresponding to the CCO and CH of dextran, and the absorption of iron-iron nanoparticles (magnetite) O bond at 538 cm &lt; ^{-1 &} gt ;.

Figure 2d is X- for the reference pattern (iv) of the _{γ-Fe 2 O 3 (i} ), nucleating agent (ii), Fe ₃ O ₄ reference pattern (reference patterns) (iii), and γ-Fe ₂ O ₃ of Ray diffraction spectroscopy (XRD).

Referring to FIG. 2D, the X-ray diffraction pattern of the nucleating agent shows a characteristic peak in the Fe ₂ O ₃ crystal, and the peak shifts slightly toward the lower peak as compared with the crystalline γ-Fe ₂ O ₃ .

FIG. 3A is an image showing a result of scanning electron microscopy of a magnetic amylose microbead produced without a nucleating agent, and FIG. 3B is an image showing a result of scanning electron microscopy of a magnetic amylose microbead according to an embodiment of the present invention.

Figure 3b shows the addition of 10 mg / ml of nucleating agent in the preparation of magnetic amylose microbeads according to an embodiment of the present invention.

3A and 3B, a magnetic amylose microbead according to an embodiment of the present invention uses magnetic nanoparticles coated with dextran on its surface as a nucleating agent, so that compared to magnetic amylose microbeads prepared without a nucleating agent, It can be seen that a magnetic amylose microbead of a size is formed.

In addition, the magnetic amylose microbeads produced without the nucleating agent cause coagulation between the magnetic amylose microbeads, but the magnetic amylose microbeads according to the embodiment of the present invention use the magnetic nanoparticles coated with dextran on the surface as nucleating agents , It can be seen that coagulation does not occur between the magnetic amylose microbeads.

4A is a graph showing the size (average diameter) of the magnetic amylose microbeads according to the embodiment of the present invention according to the concentration of the nucleating agent.

Figure 4a synthesized magnetic amylose microbeads for 24 hours.

Referring to FIG. 4A, as the concentration of the nucleating agent increases, the size of the magnetic amylose microbeads according to the embodiment of the present invention decreases.

More specifically, when preparing the magnetic amylose microbeads according to the embodiments of the present invention, since the amount of single-chain amylose is limited, as the concentration of the nucleating agent increases, the average of the magnetic amylose microbeads according to the embodiment of the present invention The diameter is reduced. The average diameter of the magnetic amylose microbeads can be controlled according to the concentration of the nucleating agent in the magnetic amylose microbeads according to the embodiment of the present invention.

FIG. 4B is a graph showing the size (average diameter) of the magnetic amylose microbeads according to the embodiment of the present invention according to the synthesis time. FIG.

FIG. 4b shows the preparation of magnetic amylose microbeads by using no nucleating agent (control), adding 6 mg / ml and 10 mg / ml nucleating agent.

Referring to FIG. 4B, the self-assembly reaction of the magnetic amylose microbeads according to the embodiment of the present invention shows a non-linear sigmoidal growth curve having a delay time at the beginning of the reaction , It can be seen that the self-assembly reaction of the magnetic amylose microbeads according to the embodiment of the present invention comprises the nucleation step and the growth step.

5A is a graph showing an electron scanning microscopic result image and a size distribution of magnetic amylose microbeads according to an embodiment of the present invention.

Referring to FIG. 5A, it can be seen that the magnetic amylose microbeads according to the embodiment of the present invention exhibit a uniform size distribution centered at 1.25 μm.

5B is an image showing electron mapping and transmission electron microscopic results of magnetic amylose microbeads according to an embodiment of the present invention.

FIG. 5B was a transmission electron microscope (TEM, JEM-2010F, JEOL, Tokyo, Japan) showing the morphology and electron mapping of the magnetic amylose microbeads according to the embodiment of the present invention. The chemical composition of magnetic amylose microbeads according to an embodiment of the present invention was measured by EDX spectrometer.

In Fig. 5B, iron, carbon and oxygen are indicated by dots of red, green and blue, respectively.

Referring to FIG. 5B, it can be seen that the magnetic amylose microbead according to the embodiment of the present invention has a spherical shape and has a diameter of about 1.25 mu m.

It can also be clearly observed from the transmission electron microscope and electron mapping that iron oxide and iron oxide are located in the magnetic amylose microbeads according to the embodiment of the present invention and that dextran and amylose molecules are located due to carbon and oxygen .

FIG. 5c is an image showing fluorescence microscopy image of magnetic amylose microbeads according to an embodiment of the present invention in which maltose binding protein-green fluorescent protein (MBP-GFP) fusion protein is functionalized.

Referring to FIG. 5C, it can be seen that the magnetic amylose microbeads according to the embodiment of the present invention specifically bind to the maltose binding protein through the green fluorescent protein.

Particularly, the right image shows the fluorescence microscopy of the magnetic amylose microbeads according to the embodiment of the present invention in which the maltose binding protein-green fluorescent protein (MBP-GFP) fusion protein is functionalized when the external magnetic field is applied ) Shows that the magnetic amylose microbeads according to the embodiment of the present invention having superparamagnetic characteristics are arranged in one line according to the direction of the magnetic force when an external magnetic field is applied thereto, .

5D is an image showing the reaction of magnetic amylose microbeads dispersed in a solution according to an embodiment of the present invention by external magnetic force.

Referring to FIG. 5D, it can be seen that the magnetic amylose microbeads according to the embodiment of the present invention can be separated from the solution within 5 seconds by bringing the magnets closer to the solution in which the magnetic amylose microbeads are dispersed there was.

Accordingly, the magnetic amylose microbeads according to the embodiments of the present invention can be used for separation, purification and concentration of maltose binding protein (MBP) fusion proteins to improve performance.

6A to 6D are graphs showing the relationship between the protein G of the recombinant MBP-target-streptococcal G group (recombinant) and the protein G of the recombinant MBP-target-streptococcal G group in order to measure the target pathogen capture efficiency of the magnetic amylose microbeads (immune-SAMBs) MBP-tagged Streptococcal protein G (MBP-SPG) fusion protein is bound on the surface of a magnetic amylose microbead according to an embodiment of the invention to bind an antibody specific for E. coli O157: H7 It was used as a cross-linker.

6A is a graph showing the collection efficiency of magnetic amylose microbeads according to the embodiment of the present invention in accordance with the number of bacteria dispersed in phosphate buffer saline (PBS).

6A, the target bacteria is E. coli.

6A, magnetic amylose microbeads (SAMBs) according to an embodiment of the present invention have a slightly higher collection efficiency than polystyrene based immunomagnetic beads (PSMBs) (commercial), which is the most widely used prototype. High.

6B is a graph showing the collection efficiency of the magnetic amylose microbeads according to the embodiment of the present invention according to the number of bacteria in the milk.

Figure 6b shows that the target bacteria was E. coli and proceeded at a bacterial concentration of 10 &lt; ² &gt; CFU / ml to 10 &lt; ⁶ &gt; CFU / ml.

Referring to FIG. 6B, the magnetic amylose microbeads (SAMBs) according to the embodiment of the present invention have a better collection efficiency than polystyrene based immunomagnetic beads (PSMBs) (commercial), which is the most widely used prototype &Lt; / RTI &gt;

FIG. 6C shows the collection efficiency of magnetic amylose microbeads according to an embodiment of the present invention against E. coli, Bacillus cereus, Staphylococcus aureus, and Vibrio cholera It is a graph.

6C, the target bacteria is E. coli.

Referring to FIG. 6C, it can be seen that the magnetic amylose microbeads (immune-SAMBs) according to the embodiment of the present invention are specifically bound to E. coli.

Figure 6d is a graph showing the number of recycling of magnetic amylose microbeads according to an embodiment of the invention for bacterial isolation (left) and FITC-Ab-functionalized embodiments of the invention in each cycle (Right) image of the magnetic amylose microbeads according to the present invention.

Figure 6d measures the relative tablet capacity of the magnetic amylose microbeads according to the FITC-Ab-functionalized inventive embodiments during three re-use.

Referring to FIG. 6D, the magnetic amylose microbeads according to the FITC-Ab-functionalized embodiments of the present invention have higher collection efficiency than polystyrene based immunomagnetic beads (PSMBs) (commercial), and have three regeneration and reuse Later, the affinity and specificity for the MBP-fusion protein is maintained.

Accordingly, the magnetic amylose microbeads according to the embodiments of the present invention are suitable for separation and purification of MBP-fusion proteins from cell lysates, and application to separation and concentration processes essential for the detection of specific microorganisms present in a sample.

FIGS. 7A to 7C are images showing rod-shaped magnetic amylose microbeads prepared by the method for producing magnetic amylose microbeads according to an embodiment of the present invention.

Referring to FIG. 7A, the method of manufacturing magnetic amylose microbeads according to an embodiment of the present invention is characterized in that the magnetic amylose microbeads can be easily aligned with an external magnetic field due to excellent sensitivity and stability of the nucleating agent added in the substrate solution And can be easily immobilized by the SCA-mediated precipitation process.

Accordingly, the method of producing magnetic amylose microbeads according to the embodiment of the present invention can control the shape of the magnetic amylose microbeads by controlling the external magnetic field.

FIG. 7d is an image showing a scanning electron microscope result of a bar-shaped magnetic amylose microbead produced by the method of manufacturing magnetic amylose microbeads according to an embodiment of the present invention, FIG. 3 is an image showing a transmission electron microscope result of a bar-shaped magnetic amylose microbead produced by a method of producing a microbead. FIG.

7D and 7E, it can be seen that the rod-shaped magnetic amylose microbeads prepared by the method of manufacturing magnetic amylose microbeads according to the embodiment of the present invention are linearly aligned and have a rod shape.

It should be noted that the embodiments of the present invention disclosed in the present specification and drawings are only illustrative of specific examples for the purpose of understanding and are not intended to limit the scope of the present invention. It will be apparent to those skilled in the art that other modifications based on the technical idea of the present invention are possible in addition to the embodiments disclosed herein.

110: core part 120: shell part

Claims (16)

Coating a surface of the magnetic nanoparticles with dextran to prepare a nucleation agent; And
Synthesizing magnetic amylose microbeads by adding the nucleating agent to a substrate solution containing short chain amylose (SCA)
Wherein the magnetic amylose microbeads (SAMBs) are formed on the substrate.
The method according to claim 1,
Wherein the nucleating agent has a core-shell structure, the core portion includes the magnetic nanoparticles, and the shell portion includes the dextran.
The method according to claim 1,
Wherein the size of the magnetic amylose microbeads is controlled according to the content of the nucleating agent.
The method according to claim 1,
Wherein the size of the magnetic amylose microbeads is controlled according to the synthesis time of the magnetic amylose microbeads.
The method according to claim 1,
The step of synthesizing the magnetic amylose microbeads by adding the nucleating agent to the substrate solution containing the short chain amylose (SCA)
Preparing an amylose synthase;
Preparing a substrate solution comprising a substrate and a solvent; And
Adding the amylose synthase to the substrate solution and performing an enzymatic reaction to synthesize mono-chain amylose
Wherein the magnetic amylose microbeads are prepared by a method comprising the steps of:
6. The method of claim 5,
Wherein the amylose synthase comprises at least one of amylosucrase, phosphorylase, starch synthase, amylase, and D-enzyme. (Method for producing magnetic amylose microbeads).
The method according to claim 1,
Wherein the magnetic nanoparticles are iron oxide nanoparticles.
A magnetic amylose microbead prepared by the process for producing magnetic amylose microbeads according to any one of claims 1 to 7.
9. The method of claim 8,
Wherein the magnetic amylose microbeads are rod-shaped.
9. The method of claim 8,
Wherein the magnetic amylose microbeads are used for a therapeutic substance, a diagnostic substance, a functional health food packaging agent, a coating agent or a chromatography resin.
Coating the surface of the magnetic nanoparticles with dextran to prepare a nucleating agent;
Synthesizing magnetic amylose microbeads by adding the nucleating agent to a substrate solution containing mono-chain amylose;
Adding the magnetic amylose microbeads to a sample containing target pathogens to bind target pathogens; And
Separating and concentrating the magnetic amylose microbeads to which the target pathogen is bound
And isolating and concentrating the target pathogen using the magnetic amylose microbeads.
12. The method of claim 11,
The step of isolating and concentrating the magnetic amylose microbeads to which the target pathogens are bound,
And adding a competitive binder to the target pathogen on the surface of the magnetic amylose microbead to isolate the target pathogen from the amylose magnetic bead. 2. A method for isolating and concentrating a target pathogen using magnetic amylose microbeads.
12. The method of claim 11,
Wherein the surface of the magnetic amylose microbeads is modified with an antibody specific to the target pathogen using a MBP-SPG fusion protein as a cross-linking agent, thereby isolating and concentrating the target pathogen using the magnetic amylose microbeads.
Coating the surface of the magnetic nanoparticles with dextran to prepare a nucleating agent;
Synthesizing magnetic amylose microbeads by adding the nucleating agent to a substrate solution containing mono-chain amylose;
Binding the desired protein to the magnetic amylose microbeads; And
Separating and purifying the magnetic amylose microbeads to which the target protein is bound
Wherein the magnetic amylose microbeads are used for purifying target proteins using magnetic amylose microbeads.
15. The method of claim 14,
Wherein the target protein is a maltose binding protein (MBP) -tagged protein. 2. A method for purifying a target protein using magnetic amylose microbeads,
15. The method of claim 14,
The step of separating and purifying magnetic amylose microbeads to which the target protein is bound comprises:
And separating the target protein from the amylose magnetic beads by adding a competitive binder to the target protein on the surface of the magnetic amylose microbeads, thereby purifying the target protein using the magnetic amylose microbeads.

Images