KR20110033575A - Mesoporous silica conjugate integrating magnetic nano particles having peroxidase activity and enzymes and method for manufacturing the same - Google Patents

Abstract

PURPOSE: A porous silica composite and a method for preparing the same are provided to sensitively diagnose pathogens, small molecular material, proteins, and DNA. CONSTITUTION: A porous silica composite comprises: magnetic nanoparticles with peroxidase activity; and an enzyme which provides a reactive substrate catalyzed by peroxidase or an enzyme using a reactant catalyzed by peroxidase. The substrate catalyzed by hydrogen peroxide is hydrogen peroxide. The reactant catalyzed by peroxidase is water(H_2O) or oxygen(O_2). The porous silica is MSU, SBA, MCM, or KIT. A method for preparing the porous silica composite comprises: a step of precipitating the magnetic particles inside a pore of the silica; and a step of fixing the enzyme into the pore.

Description

Mesoporous Silica Conjugate Integrating Magnetic Nano Particles Having Peroxidase Activity and Enzymes and Method for Manufacturing the Same}

The present invention relates to a porous silica composite in which magnetic nanoparticles having peroxidase activity and an enzyme are immobilized in pores of porous silica, and more particularly, to a porous silica composite having a large surface area and pore volume. The present invention relates to a porous silica composite in which magnetic nanoparticles having an activity as a peroxidase and an enzyme are simultaneously integrated in pores, and a method of manufacturing the same.

As society is advanced and modernized, human desire to live longer and healthier is increasing. In particular, the recent rapid development of medical technology is making disease diagnosis technology more important because it is making a society that can be cured if any disease is detected early. In addition, the development of diagnostic technology that can be easily and conveniently used by the general public is also very important in terms of economic feasibility, rather than disease diagnosis technology that requires expensive detection machines or skilled technicians. According to such necessity, the technology to detect and quantify small molecule substances such as glucose, galactose, and cholesterol, proteins, DNA, and pathogens, which are the causes of disease, more easily and with high sensitivity is very important economically and technically, and has a large ripple effect in industry. It is a field that is actively researched around the world. Another important flow of disease diagnosis is the field of diagnostic technology called Point of Care Testing (POCT), which includes technologies and devices that can provide immediate and easy diagnosis in the field without skilled analysts. do. The recently emerging global POCT market demands new technologies to diagnose diseases faster and easier.

Recently, as a strategy for developing high-sensitivity diagnostic technology, various nanoparticles are used for diagnosis, and recently, magnetic nanoparticles have been reported to have peroxidase activity.

However, in order to use these magnetic nanoparticles as more efficient and realistic peroxidase substitutes, it is important to solve problems such as more effective separation and reuse, increase activity and sensitivity per unit individual, and improve access to other enzymes.

Korean Patent Publication No. 2006-61494 discloses functional silica magnetic nanoparticles prepared by introducing an amine group and / or a chloro group on the surface of silica coated magnetic nanoparticles, and a method of manufacturing the same, and Korean Patent Registration No. 830,889 Nanoparticles consisting of a core compound made of a magnetic material and a silica compound shell containing an organic fluorescent substance, and a cetuximab antibody are introduced on a surface thereof, and methods for preparing the same are disclosed, but the patents are described as peroxidases of magnetic nanoparticles themselves. The activity of the nanoparticles is not limited to the use of the nanoparticles for nucleic acid isolation and purification or for diagnosing colorectal cancer, and organic functional groups or antibodies are attached to the surface of the particles.

Korean Patent Registration No. 821,192 discloses magnetic nanoparticles containing a magnetic material and an organic fluorescent material on the outside of the core and wrapped with surface-modified silica shells and a method of manufacturing the same. There is a need to surface modify the silica shell for the introduction of various chemical functional groups.

As a result of the present inventors' efforts to solve the above problems, the magnetic nanoparticles having peroxidase activity are first accumulated in the pores of the porous silica having a large surface area and pore volume, and then the enzyme is immobilized in the pores of the remaining porous silica. A porous silica composite was prepared, and it was confirmed that the substance serving as the target substrate of the enzyme can be easily diagnosed with high sensitivity, thereby completing the present invention.

It is an object of the present invention to provide a porous silica composite in which magnetic nanoparticles having peroxidase activity and an enzyme are immobilized in pores of porous silica, and a method of manufacturing the same.

Another object of the present invention is to provide a biomolecule detection method using the porous silica composite.

The present invention (a) a magnetic nanoparticle having peroxidase activity; And (b) one or more enzymes selected from the group consisting of (i) an enzyme that provides a substrate for the reaction catalyzed by a peroxidase and (ii) an enzyme that uses the product of the peroxidase-catalyzed reaction as a substrate. It provides a fixed porous silica composite.

The present invention also provides a method for preparing a porous silica-magnetic nanoparticle composite by (a) depositing magnetic nanoparticles having peroxidase activity inside pores in porous silica; And (b) at least one enzyme selected from the group consisting of (i) an enzyme that provides a substrate for the reaction catalyzed by a peroxidase and (ii) an enzyme that uses as a substrate the product of the peroxidase catalyzed reaction. It provides a method for producing a porous silica composite containing a magnetic nanoparticles having peroxidase activity and enzyme comprising the step of fixing to the nanoparticle complex.

The present invention also provides a biomolecule detection method using the porous silica composite. The detection method comprises the steps of: (a) reacting the porous silica composite, a sample containing a biomolecule to be detected and a color substrate; And (b) measuring the absorbance of the reaction solution to detect the biomolecule.

The porous silica composite according to the present invention can easily and sensitively diagnose color development of small molecules such as glucose, galactose and cholesterol, proteins, DNA and pathogens, which are markers of various diseases, and integrate magnetic nanoparticles and enzymes into porous silica. As a result, high sensitivity and quantitative analysis is possible, and the magnetic nanoparticles can be used for efficient separation and reuse.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein and the experimental methods described below are well known and commonly used in the art.

The present invention in one aspect, (a) magnetic nanoparticles having peroxidase activity; And (b) one or more enzymes selected from the group consisting of (i) an enzyme that provides a substrate for the reaction catalyzed by a peroxidase and (ii) an enzyme that uses the product of the peroxidase-catalyzed reaction as a substrate. It relates to a fixed porous silica composite.

The present invention can be easily quantified the presence and concentration of the target substrate of the enzyme through the H ₂ O ₂ detection reaction by incorporating a high density of magnetic nanoparticles having peroxidase activity in the pores of the porous silica, and then fixed the enzyme It can be.

In the present invention, the enzyme is a substance that catalyzes various chemical reactions including biological activities, and is recently used industrially for the diagnosis of diseases. In particular, peroxidase is commonly present in the biological system, Oxidation / reduction enzymes are used, using hydrogen peroxide or alkyl peroxide as oxidant.

In the present invention, "peroxidase-catalyzed reaction" means all redox reactions catalyzed by peroxidase. Specifically, as shown in FIG. 2, generally, the peroxidase catalyzed reaction includes oxidative dehydrogenation, oxidative halogenation, hydrogen peroxide decomposition, or H ₂ O ₂ dismutation. (oxygen-transfer reaction) (Stefano Colonna, et al ., Tibtech April 1999, vol. 17).

In the present invention, the term "oxidative dehydrogenation" is a reaction in which peroxidase oxidizes a substrate by electron transfer, which can be represented by the following Scheme 1.

Scheme 1

2SH + H ₂ O ₂ → 2S + H ₂ O

In the present invention, "oxidative halogenation" is a reaction in which the peroxidase halogenates the carbon atoms of the substrate that activates the benzyl / allyl C-H bond with hydrogen peroxide and halide ions, and can be represented by the following scheme 2:

Scheme 2

SH + H ₂ O ₂ + H (+) + X (-) → SX + 2H ₂ O (X = Cl, Br, I)

In the present invention, "H ₂ O ₂ dismutation" is a reaction in which the peroxidase catalyst decomposes hydrogen peroxide into water (H ₂ O) and oxygen (O ₂ ), which can be represented by the following reaction formula 3. :

Scheme 3

2H ₂ O ₂ → 2H ₂ O + O ₂

In the present invention, the oxygen-transfer reaction means that the oxygen atom is enantioselectively introduced into the organic substrate by peroxidase, and can be represented by the following Scheme 4.

Scheme 4

SH + H ₂ O ₂ → SOH + H ₂ O

In the present invention, the substrate of the reaction catalyzed by the peroxidase may be characterized in that the hydrogen peroxide, as shown in the reaction schemes.

In the present invention, the enzyme providing a substrate of the reaction catalyzed by the peroxidase may be characterized in that the oxidase.

In the present invention, oxidase refers to the enzyme that acts on the oxidation reaction, and includes glucose oxidase, xanthine oxidase, cytochrome oxidase, ascorbic acid oxidase, and the like. oxidoreductase) is a group of enzymes called, and the common name is oxidase. Oxygen is oxidized and the substrate is oxidized and oxygen is reduced. At that time, oxygen is divided into hydrogen peroxide and water. Examples of hydrogen peroxide include glucose oxidase present in blue molds, xanthine oxidase present in live milk and liver, and D-amino oxidase present in animal tissues.

In the present invention, the oxidase may be selected from the group consisting of glucose oxidase, cholesterol oxidase, galactose oxidase, pyranose oxidase, lactose oxidase, hexose oxidase and glutamate oxidase. have.

In the present invention, the product of the reaction catalyzed by the peroxidase may be characterized in that the water (H ₂ O) or oxygen (O ₂ ), as shown in the reaction schemes.

In the present invention, the enzyme that uses the product of the reaction catalyzed by the peroxidase as a substrate may be characterized in that the luciferase (Luciferase), the luciferase is an enzyme that shows a color signal in the presence of oxygen and luciferin (luciferin), The magnetic nanoparticle-luciferase porous silica composite can be used to prepare a system capable of developing hydrogen peroxide detection.

In the present invention, the porous silica refers to mesoporous silica or silicate having a pore of 2-100 nm in a regular arrangement of a straight line, a hexagon, a cube, and the like. Porous silica of the present invention is a silica in which pores are formed to fix the magnetic nanoparticles and oxidase, MSU (Mishigan State University), MCM, SBA and KIT series has been developed, more preferably, pores It is preferable to use MSU-F large enough in pore size for the accumulation of enzymes in the pores.

In the present invention, the magnetic nanoparticles having the peroxidase activity are ferromagnetic particles and generally have a size of about 10 nm, and types thereof include iron oxide (Fe ₂ O ₃ , Fe ₃ O ₄ ), and ferrite (Fe _3). Fe changed from O ₄ to another magnetically related atom, eg CoFe ₂ O ₄ , MnFe ₂ O ₄ ), alloys (oxidation problems caused by magnetic atoms, alloyed with precious metals to increase conductivity and stability , For example, FePt, CoPt, etc.), and the magnetic nanoparticles may be formed by a conventional particle manufacturing method such as a wet method, a dry method, or a vacuum method.

The magnetic nanoparticle having the peroxidase activity may be characterized in that it is selected from the group consisting of magnetite, hematite, maghemite and soft ferromagnetic, preferably magnetite is used.

In the present invention, the pore diameter of the porous silica may be characterized in that 10 ~ 30nm, if the diameter is less than 10nm in general compared to the size of the enzyme (6-7 nm), there is a problem in integration, the diameter is If it exceeds 30 nm, the increase in stability of enzymes characteristic of the porous silica is inhibited. Therefore, in the present invention, a porous silica having a pore diameter of 10 to 30 nm is preferable.

Specifically, in the present invention, it is possible to integrate magnetic nanoparticles having peroxidase activity in the pores of porous silica having pores having a diameter of 10 to 30 nm at high density, and to immobilize various enzymes in the remaining pores. Can be easily implemented. For example, the presence and concentration of the target substrate of the enzyme can be easily quantified through the color reaction through the linkage reaction between the immobilized enzyme and the magnetic nanoparticles (FIG. 3).

In the present invention, the protein, RNA, DNA, virus, pathogens, hormones, hapten, avidin (streptavidin), neutravidin, lectin (lectin) on the surface of the porous silica composite ), A selectin, a radioisotope labeled substance, and a substance selected from the group consisting of a substance capable of specifically binding to a tumor marker may be further attached. The materials may be introduced directly to the surface of the porous silica composite or bonded to the surface through surface modification of chemical functional groups (eg, -COOH, -NH ₂ , -SH, etc.). For example, Aminopropyl triethoxysilane (APTES) may be reacted by boiling at 80 ° C. on the surface of the porous silica to bind a -NH ₂ reactor, and to react the -SH reactor by reacting 3-mercaptopropyl trimethoxysilane. The -COOH reactor can be bonded by reacting succinic anhydride with -NH ₂ bonded porous silica. The functional groups can be bound to the surface of the biological material through chemical covalent bonds. For example, the combination of —NH ₂ and —COOH is coupled by an EDC-NHS reaction. In other words, EDC (1-Ethyl-3- [3-dimethylaminopropyl] carbodiimide Hydrochloride reacts with COOH to form amine-reactive intermediate when NHS (N-hydroxysulfosuccinimide) is present to form amide bond with NH ₂ . In the same manner as above, the biological material and the porous silica may be chemically combined.

The tumor marker may be a ligand, an antigen, a receptor or a nucleic acid encoding them.

When the tumor marker is "ligand", a substance capable of specifically binding to the ligand may be introduced to the surface of the porous silica composite according to the present invention, and a receptor or antibody capable of specifically binding to the ligand may be introduced. Will be appropriate. Examples of ligands and receptors that can specifically bind to the present invention include synaptotagmin C2 (synaptotagmin C2), phosphatidylserine, annexin V and phosphatidylserine, integrin and its Receptors, Vascular Endothelial Growth Factor (VEGF) and its receptors, angiopoietin and Tie2 receptors, somatostatin and its receptors, vasointestinal peptides and their receptors It is not limited.

When the tumor marker is an "antigen", a substance capable of specifically binding to the antigen can be introduced to the surface of the porous silica composite according to the present invention, and an antibody capable of specifically binding to the antigen will be suitable. Examples of antigens and antibodies that specifically bind to the present invention include carcinoembryonic antigens (colon cancer marker antigens), Herceptin (Genentech, USA), and HER2 / neu antigens (HER2 / neu antigens-breast cancer markers). Antigen) and Herceptin, prostate-specific membrane antigen (prostate cancer marker antigen) and rituxan (IDCE / Genentech, USA).

Representative examples in which the tumor marker is a "receptor" include folic acid receptors expressed in ovarian cancer cells. A substance capable of specifically binding to the receptor may be introduced to the surface of the porous silica composite according to the present invention, and ligands or antibodies capable of specifically binding to the receptor, preferably antibodies will be suitable.

Herein, the antibody has a property of selectively and stably binding only to a specific target, and -NH _{2 of} lysine, -SH of cysteine, -COOH of aspartic acid and glutamic acid in the Fc region of the antibody bind to the surface of the porous silica composite. It can be usefully used. For example, it can be introduced directly to the surface of the complex or by modifying the surface of the complex with a functional group that organically binds the functional group. Such antibodies can be obtained commercially or prepared according to methods known in the art. In general, a mammal (eg, mouse, rat, goat, rabbit, horse or sheep) is immunized one or more times with an appropriate amount of antigen. After a period of time when the titer reaches an appropriate level, it is recovered from the serum of the mammal. The recovered antibody can be purified using known processes if desired and stored in a frozen buffered solution until use. Details of this method are well known in the art.

On the other hand, "nucleic acid" includes RNA and DNA encoding the aforementioned ligand, antigen, receptor or part thereof. Nucleic acid having a specific base sequence can be detected using a nucleic acid having a base sequence complementary to the base sequence because the nucleic acid has a feature that forms a base pair between complementary sequences as known in the art . Nucleic acid having a base sequence complementary to the nucleic acid encoding the enzyme, ligand, antigen, receptor can be introduced to the surface of the porous silica composite according to the present invention through various methods known in the art.

In addition, the nucleic acid has functional groups such as -NH ₂ , -SH, and -COOH at 5'- and 3'-terminals, so that the nucleic acid is introduced directly to the surface of the silica composite or through surface modification to a functional group that organically binds the functional group. Can be. Such nucleic acids can be synthesized by standard methods known in the art, for example using automated DNA synthesizers (such as those available from BioSearch, Applied Biosystems, etc.). As an example, phosphorothioate oligonucleotides can be synthesized by the methods described in Stein et al . Nucl. Acids Res . 1988, vol. 16, p. 3209. Methylphosphonate oligonucleotides can be prepared using controlled free polymeric supports (Sarin et al . Proc. Natl. Acad. Sci. USA . 1988, vol. 85, p.7448).

That is, by further binding the above-described specific substances to the surface of the magnetic nanoparticle-porous silica-enzyme complex according to the present invention, the substance which provides specific information on the progress of the disease by the color development by their complementary binding It can be used for analysis.

The porous silica complex according to the present invention may additionally be used for detecting and diagnosing pathogen DNA, proteins and pathogens, and attaching DNA probes that specifically bind to the pathogen DNA to the surface of the complex or causing the disease. And attaching an antibody that binds specifically to the pathogen to the surface of the complex, and then binding the magnetic nanoparticle-porous silica to specific DNA, protein, pathogen through reaction with an unknown sample. Then, by adding a substrate causing a color reaction, it can be detected and diagnosed simply as a change in color.

In another aspect, (a) preparing a porous silica-magnetic nanoparticle composite by depositing a magnetic nanoparticle having a peroxidase activity inside the pores of the porous silica; And (b) at least one enzyme selected from the group consisting of (i) an enzyme that provides a substrate for the reaction catalyzed by a peroxidase and (ii) an enzyme that uses as a substrate the product of the peroxidase catalyzed reaction. It relates to a method for producing a porous silica composite containing magnetic nanoparticles and peroxidase activity having a step of immobilizing on the nanoparticle composite.

In the present invention, the porous silica is selected from the group consisting of MSU series, SBA series, MCM series and KIT series, the magnetic nanoparticles having the peroxidase activity is magnetite, hematite, maghemite and soft ferromagnetic Selected from the group consisting of.

In the present invention, the fixing in step (b) may be characterized by using a crosslinked aggregate aggregation (CLEA) method.

The magnetic nanoparticle-porous silica composite of the present invention is simply produced by first uniformly depositing magnetic nanoparticles in porous silica. Then, for efficient immobilization of enzymes, a CLEA (Crosslinked Enzyme Aggregate) type capture method is used. For the CLEA capture, oxidase is first adsorbed into the pores of silica through physical adsorption and then glutaraldehyde is used as a crosslinking agent to form enzyme molecules in the pores through crosslinking of enzymes. The formed enzyme molecule mass does not escape well in the pores of silica, thereby reducing the loss of the enzyme molecule, and can inhibit the three-dimensional structural modification of the enzyme to maintain the enzyme activity more stably (Kim et al., Biotechnol. Bioeng 96 (2): 210-218, 2007).

In another aspect, the present invention relates to a method for detecting a biomolecule, wherein the porous silica composite reacts with a sample containing a biomolecule to be detected and then detects H ₂ O ₂ generated by the reaction. will be. More specifically, reacting the porous silica composite, a sample containing a biomolecule to be detected, and a coloring substrate; And it relates to a biomolecule detection method comprising the step of detecting the biomolecule by measuring the absorbance of the reaction solution.

In the present invention, the detection of H ₂ O ₂ may be characterized by using a color reaction, a fluorescence reaction, a luminescence reaction or an electrochemical reaction.

In this case, the fluorescence reaction is a method of detecting H ₂ O ₂ by reacting with a sample and magnetic nanoparticles having peroxidase activity to fluoresce. For example, Amplex-Red may include Peroxidase and H ₂ O ₂ . By generating a fluorescent substrate called Resorufin, hydrogen peroxide can be detected.

In the case of the luminescence reaction, it can be detected by simply measuring the luminescence using a light emitting substrate called luminol.

In addition, electrochemistry is a method of detecting hydrogen peroxide by performing an electrochemical treatment on a sample, and magnetic nanoparticles having peroxidase activity react to the flow of electrons generated by reduction of H ₂ O ₂ . The electrical signal can be detected. For example, as a representative example, a method of detecting a flow due to electron transfer as an electrical signal through an enzyme reaction by HRP (horse radish peroxidase) and H ₂ O ₂ may be mentioned.

In the present invention, the color substrate is a substrate exhibiting a specific color reaction, such as peroxidase and hydrogen peroxide, TMB (3,3 ', 5,5'-tetramethyl benzidine), ABTS [2,2'-azino-bis ( 3-ethylbenzothiazoline-6-sulfonic acid)] and Amplex-Red (10-acetyl-3,7-dihydroxyphenoxazine), and the biomolecules may be characterized as nucleic acids, proteins, polysaccharides or pathogens. have.

In the present invention, as described above, the magnetic nanoparticles and the enzyme are simply immobilized on the porous silica. For performance analysis of the prepared porous silica composite, ABTS (2,2'-azino-bis (3-ethylbenzo-thiazoline-6-sulfonic acid), TMB (3,3), which is a coloring substrate in an embodiment of the present invention , 5,5-tetramethylbenzidine) and Amplex-Red (10-acetyl-3,7-dihydroxyphenoxazine) were used to determine the presence and quantitative analysis of the target substrate of hydrogen peroxide and the immobilized enzyme (Fig. 4). In another embodiment of the present invention, a color sensor of a target material is implemented by immobilizing glucose oxidase and cholesterol oxidase together on a magnetic nanoparticle-porous silica complex to develop a system for analyzing color of glucose and cholesterol, which are clinically important substrates. (FIG. 5 and FIG. 6).

When immobilization using the present invention is performed, high sensitivity quantitative analysis using color is possible without using peroxidase. Since the peroxidase used in the conventional diagnostic method is essentially a protein-based organic enzyme, there is a consequent deterioration of protein structure and activity over time, but the porous silica complex of the present invention uses magnetic nanoparticles, which are inorganic instead of peroxidase. Because it is a more stable system. In addition, by incorporating magnetic nanoparticles into the porous silica, it is possible to quantitatively analyze high sensitivity by increasing activity per unit, and more efficient separation and reuse using magnetic force (FIG. 7). It is very cost-effective because quantitative analysis of economically and industrially important substances through linkage reaction using oxidase with porous silica is easily possible due to color differences through the naked eye.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Example 1: Preparation of magnetic nanoparticle-porous silica composite

1-1: MSU-F Porous Silica Preparation

MSU-F porous silica was prepared through the following reaction. (Kim et al., Chem. Comm. 1661-1662, 2000) Dissolve 9.8 g triblock copolymer pluronic P123, 4.4 ml acetic acid, 5.9 g 1,3,5-trimethylbenzene in 200 mL water for 2 hours at 60 ° C. By reacting, an oil-in-water emulsion was produced that produced the desired nanostructures. 16 mL of silica sodium (sodium silica) was added to the reaction product and reacted at 60 ° C. for 20 hours, and then aged at 100 ° C. for one day. By removing the block copolymer through calcination at 550 ℃, a porous silica having a pore diameter of about 25nm was prepared.

1-2: Preparation of magnetic nanoparticle-porous silica composite

The immobilization of the magnetic nanoparticles on the porous silica was performed using the following method. First, 6.97 g of Fe (NO ₃ ) ₃ .6H ₂ O and ₂ g of the porous silica prepared in Example 1-1 were dissolved in 80 ml ethanol, and the solution was stirred until all of the ethanol evaporated. (stirring) Thereafter, the remaining powder was heat-treated at 400 ° C. for 6 hours on an argon (95%) / hydrogen (5%) mixture gas, and through the capillary phenomenon caused by the reaction, magnetic nanoparticles in pores of porous silica The formation of magnetic nanoparticle-porous silica composite in the form of magnetite was confirmed by TEM (bottom photo of FIG. 1), XRD (FIG. 8), and the like. Table 1 below shows the treatment conditions and magnetic nanoparticle size of magnetite in the XRD test sample.

TABLE 1

Kinds Magnetite Size (nm) 20% loading, 400 ℃ treatment 14 40% loading, 400 ℃ 16 20% loading, 330 ℃ treatment 10.4 40% loading, 330 ℃ 13.6

Example 2 color development sensor using magnetic nanoparticle-porous silica

ABTS, TMB and Amplex-Red prepared in advance in the magnetic nanoparticle-porous silica composite prepared in Example 1 were added under sodium acetate buffer (0.2 M, pH 4.0) at 45 ° C. to adjust the amount of H ₂ O _{2 in} the reaction. It was measured by the color reaction. Specific experimental conditions are as follows. In other words, 0.2 mg / mL of porous silica-magnetic nanoparticle composite loaded with magnetite by 20 wt% and 40 wt%, chromogenic substrate ABTS, TMB 1 mM, Amplex-Red 0.2 mM, H ₂ O ₂ 10 mM sodium acetate The color change was observed by incubation in buffer (0.2 M, pH 4.0) and reaction at 45 ° C. for 30 minutes.

As a result, in the case of ABTS compared to the control green (Fig. 4 (A)), in the case of the TMB blue (Fig. 4 (B)), and in the case of Amplex-Red Red (Fig. 4 (C)) The H ₂ O _The color was developed according to ₂ , and the results of absorbance and fluorescence scanning showed specific absorbances according to the presence or absence of H ₂ O ₂ , thereby confirming that the magnetic nanoparticles to which the enzyme was immobilized acted as peroxidase.

Example 3 Preparation of Magnetic Nanoparticle-porous Silica-enzyme Complex and Application to Color Sensor

3-1. Preparation of magnetic nanoparticle-porous silica-oxidase complex using glucose oxidase and its application as color sensor

By immobilizing a specific enzyme together with the magnetic nanoparticle-porous silica composite prepared in Example 1, the presence and concentration of a specific substance were measured by the color reaction according to H ₂ O ₂ , which occurs when the enzyme meets a substrate.

First, in order to measure glucose color development, glucose oxidase per 100 mg of the magnetic nanoparticle-porous silica was collected in a crosslinked enzyme aggregate (CLEA) method and immobilized as follows. First, 5 mg / mL glucose oxidase per 10 mg porous silica was adsorbed by incubation for 30 minutes using hydrophobic interaction, which is physical adsorption, and 0.1 wt% glutaraldehyde was treated for 30 minutes as a crosslinking agent after washing. After crosslinker treatment, the glucose oxidase was immobilized in the pore of porous silica in a CLEA manner by washing with Tris-HCl buffer to remove remaining aldehyde functional groups.

As a result, it was possible to visually confirm that the color is changed to green only when glucose is present when the ABTS is added as a color substrate, and the absorbance scanning according to it (Fig. 5 (A)).

Glucose was measured using TMB as a chromogenic substrate, and the color change to blue was observed only when glucose was present. Also, the absorbance change was observed and other sugars such as Lactose, arabinose, galactose, and fructose were added. When it was confirmed that the color and absorbance does not change as when the buffer was added (Fig. 5 (B)).

Accordingly, when glucose oxidase was immobilized on the magnetic nanoparticle-porous silica, glucose of a high sensitivity could be measured through color development.

3-2. Preparation of Magnetic Nanoparticle-porous Silica-oxidase Complex Using Cholesterol Oxidase and Its Application as Color Sensor

For cholesterol colorimetry, cholesterol oxidase was immobilized on the magnetic nanoparticle-porous silica as follows. First, 4 mg / mL cholesterol oxidase per 10 mg porous silica was adsorbed for 30 minutes by physical adsorption, and after washing, 0.1 wt% glutaraldehyde was treated for 30 minutes as a crosslinking agent. Washed with Tris-HCl buffer to remove, u-cholesterol oxidase was immobilized in a CLEA manner into the pores of porous silica.

As a result of the measurement of cholesterol coloration using ABTS, it was confirmed that the color of green color in the case of cholesterol-containing reaction tube was observed, and the increase in absorbance was observed at 417 nm. When glucose or glycerol was present in the reaction tube, it did not develop color (FIG. 6).

Therefore, by immobilizing the enzyme on the magnetic nanoparticle-porous silica as described above, the peroxidase activity of the magnetic nanoparticle and the specific enzyme activity are combined to make the presence and concentration of the target substrate of the enzyme such as glucose, galactose and cholesterol very sensitive. It was successful to detect.

In addition, as described above, by additionally binding a substance capable of specifically binding to proteins, DNA, pathogens, etc., which are marker substances capable of providing information of various diseases, on the surface of the complex to which the enzyme is immobilized, It will be apparent to those skilled in the art that, for identification of biomaterials, they can be applied to easy color detection.

Example 4 Separation and Reuse Evaluation of Magnetic Nanoparticle-Porous Silica-Enzyme Complex Using Magnet

In order to evaluate the possibility of simple and efficient separation and reuse using the magnet of the porous silica composite, as shown in FIG. 7 (B), how quickly the magnetic nanoparticle-porous silica-glucose oxidase complex was completely captured using a magnet. Was visually observed. As a result, it was confirmed that the porous silica composite was attracted to the magnet within 20-30 seconds after the magnet was applied. In addition, as a result of protein quantitative assay, oxidase was also collected in the porous silica complex in the form of CLEA, so there was no loss during reuse using magnets. Therefore, it was confirmed that the activity of the enzyme was almost maintained.

Using this property, the processes described above, i.e., color reaction using ABTS-collection using magnet-absorbance measurement of product-removal of reactant and product (removal of supernatant)-3 washings-cycle of ABTS reaction again The reuse efficiency was measured. As a result, as shown in FIG. 7 (A), when glucose oxidase was immobilized in a CLEA manner to porous silica immobilized with 40 wt% magnetic nanoparticles relative to the mass of porous silica, 90 times of original activity was used during reuse using 30 times magnetic force. It was confirmed that more than% is maintained.

Having described the specific part of the present invention in detail, to those skilled in the art, such a specific description is only a preferred embodiment, whereby the scope of the present invention is not limited, it will be apparent. will be. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Figure 1 (a) shows a schematic diagram of the present invention, by using a large pore volume and surface area of the porous silica to immobilize the magnetic nanoparticles and the enzyme, to prepare a porous silica composite as a nanoscale sensor, (b) Shows a TEM picture of the porous silica composite.

2 exemplarily shows reactions catalyzed by the peroxidase activity of magnetic nanoparticles according to the present invention (a: oxidative dehydrogenation; b: oxidative halogenation; c: H ₂ O ₂ decomposition; d: Oxygen transfer reaction).

Figure 3 shows a reaction scheme for easily measuring and quantifying the presence and concentration of the target substrate of the enzyme by immobilizing the enzyme together in the inorganic complex in which the magnetic nanoparticles according to the present invention is integrated in the porous silica.

Figure 4 is ABTS (2,2'-azino-bis (3-ethylbenzo-thiazoline-6-sulfonic acid) diammonium salt (A), TMB (3,3) by magnetic nanoparticle-porous silica inorganic composite according to the present invention Easily detect the presence or absence of hydrogen peroxide through color oxidation and absorbance scanning of red, 5,5-tetramethylbenzidine) (B), and Amplex-Red (10-acetyl-3,7-dihydroxyphenoxazine) (C) It is shown ((a): a) Buffer with 6 mM ABTS, b) 6 mM ABTS + porous silica complex, c) 6 mM ABTS +10 mM H 2 O 2, d) 6 mM ABTS + 10 mM H 2 O 2 + Porous silica composites; (B): a) 6 mM TMB + porous silica complex, b) 6 mM TMB +10 mM H 2 O 2, c) 6 mM TMB + 10 mM H 2 O 2 + porous silica complex; (C): a) 6 mM Amplex-Red + porous silica complex, b) 6 mM Amplex-Red + 10 mM H ₂ O ₂ , c) 6 mM Amplex-Red + 10 mM H ₂ O ₂ + porous silica complex) .

5 is a photograph of glucose measurement using magnetic nanoparticle-porous silica-glucose oxidase complex (ABTS (A), TMB (B)) and absorbance scanning results ((A): a) 0.5 mM glucose, b) 5 mM lactose, c) 5 mM arabinose, d) 5 mM galactose e) 5 mM fructose, f) buffer; (B): a) 0.5 mM glucose, b) 5 mM lactose, c) 5 mM arabinose, d) 5 mM galactose e) 5 mM fructose, f) buffer).

FIG. 6 shows a photograph (ABTS) and absorbance scanning results of cholesterol measurement using magnetic nanoparticle-porous silica-cholesterol oxidase complex (a) 0.5 mM cholesterol, b) buffer, c) 5 mM glycerol, d) 5 mM glucose).

Figure 7 (A) is a rapid separation of the complex using the magnetic force of the magnetic nanoparticles-porous silica-glucose oxidase complex according to the present invention (CLEA-GOx in MPS: porous silica complex fixed in the CLEA method, Ads-GOx in MPS: porous silica composite fixed by physical adsorption) and (B) are graphs and photographs showing reuse efficiency using the same.

Figure 8 shows the magnetite XRD analysis of the magnetic nanoparticle-porous silica composite prepared in Example 1 and the size of the analyzed magnetic nanoparticles immobilized.

Claims (22)

(a) magnetic nanoparticles having peroxidase activity; And (b) one or more enzymes selected from the group consisting of (i) an enzyme that provides a substrate for the reaction catalyzed by a peroxidase and (ii) an enzyme that uses the product of the peroxidase-catalyzed reaction as a substrate. Fixed porous silica composite. According to claim 1, wherein the reaction catalyzed by the peroxidase is oxidative dehydrogenation (oxidative dehydrogenation), oxidative halogenation (oxidative halogenation), hydrogen peroxide decomposition (H ₂ O ₂ dismutation) and oxygen-transfer reaction (oxygen-transfer reaction) porous silica composite, characterized in that any one selected from the group consisting of. The porous silica composite of claim 1, wherein the substrate of the reaction catalyzed by the peroxidase is hydrogen peroxide. The porous silica composite of claim 1, wherein the enzyme providing a substrate of the reaction catalyzed by the peroxidase is an oxidase. The method of claim 4, wherein the oxidase is selected from the group consisting of glucose oxidase, cholesterol oxidase, galactose oxidase, pyranose oxidase, lactose oxidase, hexose oxidase and glutamate oxidase Porous silica composite. The porous silica composite of claim 1, wherein the product of the peroxidase-catalyzed reaction is water (H ₂ O) or oxygen (O ₂ ). The porous silica composite according to claim 1, wherein the enzyme which uses the product of the reaction catalyzed by the peroxidase as a substrate is luciferase. The porous silica composite of claim 1, wherein the porous silica is selected from the group consisting of MSU series, SBA series, MCM series, and KIT series. The porous silica composite of claim 8, wherein the porous silica is MSU-F. The porous silica composite of claim 1, wherein the magnetic nanoparticles having peroxidase activity are selected from the group consisting of magnetite, hematite, maghemite, and soft ferromagnetic. The porous silica composite of claim 10, wherein the magnetic nanoparticle having the peroxidase activity is magnetite. The porous silica composite of claim 1, wherein the pore diameter of the porous silica is 10 to 30 nm. According to claim 1, wherein the surface of the porous silica complex protein, RNA, DNA, virus, pathogens, hormones, hapten (hapten), avidin (avidin), streptavidin (streptavidin), neutravidin, lectin ( lectin, a selectin, a radioisotope-labeled substance, and a substance selected from the group consisting of a substance capable of specifically binding to a tumor marker, wherein the porous silica composite is further attached. A method for preparing the porous silica composite of claim 1, wherein the magnetic nanoparticles having peroxidase activity and the enzyme are immobilized in the pores of the porous silica, comprising the following steps: (a) preparing a porous silica-magnetic nanoparticle composite by depositing magnetic nanoparticles having peroxidase activity inside pores of the porous silica; And at least one enzyme selected from the group consisting of (i) an enzyme providing a substrate of a reaction catalyzed by a peroxidase and (ii) an enzyme using as a substrate a product of a reaction catalyzed by a peroxidase. Fixing in the pores of the particle composite. The method of claim 14, wherein the porous silica is selected from the group consisting of MSU series, SBA series, MCM series and KIT series. 15. The method of claim 14, wherein the magnetic nanoparticles having peroxidase activity are selected from the group consisting of magnetite, hematite, maghemite and softferromagnetic. 15. The method of claim 14, wherein the fixing in step (b) uses a crosslinked aggregate aggregation (CLEA) method. The method of claim 1, wherein the porous silica composite of claim 1 is reacted with a sample containing a biomolecule to be detected, and then H ₂ O ₂ generated by the reaction is detected. The method of claim 18, wherein the detection of H ₂ O ₂ is characterized by using a color reaction, a fluorescence reaction, a luminescence reaction or an electrochemical reaction. 19. The method of claim 18, wherein the method comprises the steps of: (a) reacting the porous silica complex of claim 1, a sample containing a biomolecule to be detected, and a coloring substrate; And (b) measuring the absorbance of the reaction solution to detect biomolecules. The method of claim 20, wherein the chromogenic substrate is TMB (3,3 ', 5,5'-tetramethyl benzidine), ABTS [2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)] or Amplex- Red (10-acetyl-3,7-dihydroxyphenoxazine). The method of claim 18, wherein the biomolecule is a nucleic acid, a protein, a polysaccharide or a pathogen.

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