KR20130081429A - Synthesis method of enzyme-mimic magnetic nanocatalysts, and enzyme-mimic magnetic nanocatalysts thereby - Google Patents

Abstract

PURPOSE: A method for synthesizing an enzyme-mimic magnetic nanocatalyst and the synthesized enzyme-mimic magnetic nanocatalyst synthesized thereby are provided to obtain the same effect of an enzyme and to reduce the coagulation, toxicity, and oxidation of particles. CONSTITUTION: A method for synthesizing an enzyme-mimic magnetic nanocatalyst comprises the steps of: synthesizing magnetite magnetic nanoparticles by adding an alkaline aqueous solution into a mixed aqueous solution of Fe^2+ and Fe^3+ to be simultaneously precipitated; coating the surfaces of the magnetite magnetic nanoparticles with silica based on a silica precursor using a sol-gel method under a basic atmosphere; and reforming the surfaces of the silica coated magnetic nanoparticles using a functional silane cross-linking agent.

Description

Synthesis method of enzyme-mimic magnetic nanocatalysts, and enzyme-mimic magnetic nanocatalysts Thus

The present invention relates to a method for synthesizing an enzyme-mimicking magnetic nanocatalyst and to an enzyme-mimicking magnetic nanocatalyst synthesized by the present invention. The present invention relates to a method for synthesizing an enzyme-mimicking magnetic nanocatalyst that mimics the active site of the enzyme chymotrypsin, which is well known in terms of catalytic reaction, and to an enzyme-mimicking magnetic nanocatalyst synthesized thereby.

Even in vivo, biopolymers such as antibodies and enzymes selectively recognize substrates according to their specific uses, thereby transmitting or catalyzing signals to maintain unique biological functions. Biopolymers, with few exceptions, are very difficult to synthesize artificially and have limitations in producing the quantities needed for research and applications. In addition, it is inferior to artificial enzymes that mimic the functions of biopolymers such as antibodies and enzymes in stability or long-term storage, and because the reaction is performed almost in aqueous solution, other solvents such as organic solvents cannot be used and recovery after use is also possible. It has the disadvantage of being difficult. In order to solve these drawbacks, research on the development of artificial enzymes that mimic the functions of biological polymers such as antibodies and enzymes has been increasing.

Magnetite (magnetite, Fe ₃ O ₄ ) magnetic nanoparticles are a type of iron oxide nanoparticles (iron oxide nanoparticles) having a size of 1 ~ 100nm and has a superparamagnetic characteristic. When the silica coating on the magnetic nanoparticles is easy to modify the surface of the particles by using a functional silane coupling agent (silane coupling agent), the chemical stability is excellent. Magnetic nanoparticles can be synthesized at low cost and have advantages such as separation and reuse of materials, mass production, efficiency and simplicity.

Meanwhile, the enzyme chymotrypsin is a protease that catalyzes the hydrolysis of peptide bonds. The active site is composed of a hydroxyl group of Serin (195), a carboxyl group of Aspartic acid (102) and an imidazole group of Histidine (57). This enzyme specifically acts on peptide bonds adjacent to aromatic amino acid residues (Trp, Phe, Tyr, etc.) and catalyzes the hydrolysis of small ester and amide compounds.

In the present invention, by applying the silica coating on the magnetite magnetic nanoparticles described above, it is not only easy to separate and reuse materials, but also reduce the aggregation, toxicity and oxidation characteristics of the particles, and increase the economic efficiency while implementing a catalytic effect such as the enzyme chymotrypsin. A method of synthesizing a magnetic nanocatalyst that mimics an enzyme can be developed.

The object of the present invention is superparamagnetic, so it is easy to separate and reuse materials, and can reduce particle aggregation, toxicity and oxidation, and can realize the same catalytic effect of enzymes, which is widely used in biosensors, biocatalysts, and proteome research. The present invention provides a method for synthesizing an enzyme-mimicking magnetic nanocatalyst and an enzyme-mimicking magnetic nanocatalyst synthesized thereby.

In order to achieve the object of the present invention as described above, the present invention i) by adding an aqueous alkali solution to the mixed aqueous solution of Fe ²⁺ and Fe ³⁺ to simultaneously precipitate the ions magnetite (magnetite, Fe ₃ O ₄ ) magnetic Synthesizing nanoparticles; ii) silica coating the magnetite magnetic nanoparticle surface with a sol-gel process using a silica precursor under a base atmosphere; And iii) modifying the surface of the magnetic nanoparticles coated with silica using a functional silane couplig agent.

At this time, the functional silane crosslinking agent to mimic the active sites (Ser-195, His-57, Asp-102) of the chymotrypsin (Chymotrypsin), 3-aminopropyl trimethoxysilane, carboxy ethylsilane tree Carboxy ethylsilanetriol, triethoxy-3- (2-imidazolin-1-yl) propylsilane (Triethoxy-3- (2-imidazolin-1-yl) propylsilane) or mixtures thereof, In this case, the prepared enzyme-mimicking magnetic nanocatalyst is characterized by hydrolyzing a peptide bond, an ester compound and an amide compound.

It is also preferred that the functional silane crosslinker has at least one functional group selected from amine groups, imidazole groups and carboxyl groups in order to constitute amino acid residues which are active sites of the enzyme.

On the other hand, the present invention provides an enzyme-mimetic magnetic nanocatalyst synthesized by the above method, wherein the particle size of the magnetized magnetic nanoparticles is 7 ~ 10nm, the particle size of the magnetic nanoparticles coated with silica is 20 ~ It is preferable that it is 100 nm. The enzyme mimic magnetic nanocatalyst synthesized as described above may be applied to various fields such as biosensor, biocatalyst, proteome research, and the like.

Enzyme-mimicking magnetic nanocatalyst synthesized according to the present invention has superparamagnetism, which is easy to separate and reuse materials, reduce aggregation, toxicity and oxidation of particles, and can implement the catalytic effect of enzymes in biosensor, It can be widely applied to biocatalyst, proteome research and the like.

1 is a schematic view showing the hydrolysis pathway of p -nitrophenyl alkyl esters.
2 is a synthetic process chart of magnetic nanoparticles.
3 is a synthetic process chart of silica coated magnetic nanoparticles.
4 is a synthetic process diagram of an enzyme-mimicking magnetic nanocatalyst.
5 is a schematic diagram showing the synthesis of an enzyme-mimicking magnetic nanocatalyst.
6 is a transmission electron microscopy (TEM) photograph of magnetic nanoparticles.
7 is a transmission electron microscopy (TEM) photograph showing the outer skin / internal structure of silica coated magnetic nanoparticles.
8 is a graph of four-ier transform infrared (FT-IR) spectroscopy of magnetic nanoparticles and silica coated magnetic nanoparticles.
9 is a graph of four-ier transform infrared (FT-IR) spectroscopy of an enzyme-mimicking magnetic nanocatalyst.
Figure 10 (a) ~ (d) is a graph quantifying the free p -Nitrophenol over time, (a) is a graph quantifying the p -Nitrophenol liberated by chymotrypsin by substrate, (b) ~ ( d) is a graph of quantifying p -Nitrophenol in which substrates p -Nitrophenyl butyrate (PNPB), p -Nitrophenyl octanoate (PNPO), and p -Nitrophenyl dodecanoate (PNPD) are liberated by an enzyme mimicking magnetic nanocatalyst, respectively.
Figure 11 is a graph of the reaction rate of chymotrypsin and enzyme mimic magnetic nanocatalyst by substrate.
12 is a graph showing the maximum rate of enzyme-mimicking magnetic nanocatalyst by substrate.
13 is a graph of the number of conversion of enzyme-mimicking magnetic nanocatalyst by substrate.
14 is a graph of catalytic efficiency constants of enzyme-mimicking magnetic nanocatalysts by substrate.
15 is a graph of specificity constants of enzyme-mimicking magnetic nanocatalysts by substrate.

A method of synthesizing an enzyme-mimicking magnetic nanocatalyst according to an aspect of the present invention and an enzyme-mimicking magnetic nanocatalyst synthesized thereby are described in detail with reference to the accompanying drawings.

Magnetic nanoparticles of the present invention comprises the steps of i) adding an aqueous alkali solution to a mixed aqueous solution of Fe ²⁺ and Fe ³⁺ to precipitate the ions simultaneously to synthesize magnetite (magnetite, Fe ₃ O ₄ ) magnetic nanoparticles, ii) Silica coating the surface of the magnetite magnetic nanoparticles by a sol-gel process using a silica precursor under a base atmosphere, and iii) a functional silane crosslinker is formed on the surface of the magnetic nanoparticles coated with silica. It is prepared through the step of modifying using a couplig agent.

Magnetic nanoparticles of the present invention synthesized by the co-precipitation method has a size of 7 ~ 10nm and the surface is composed of hydroxyl groups (OH) is hydrophilic (hydrophilic). The general scheme of the magnetite magnetic nanoparticle synthesis is shown in the following scheme (1), and in the present invention, magnetic nanoparticles were synthesized based on the following formula.

FeCl ₂ + 2FeCl ₃ + 8 NH ₄ OH-> Fe (OH) ₂ + 2Fe (OH) ₃ + 8 NH ₄ Cl (1)

-> Fe ₃ O ₄ + 8 NH ₄ Cl + 4H ₂ O

The silica coating of the magnetic nanoparticles is synthesized in a base atmosphere by a sol-gel process for producing a metal oxide through hydrolysis and condensation reaction of a metal alkoxide using a silica precursor such as tetraethyl orthosilicate (TEOS). . It is briefly shown in the following scheme (2).

Hydrolysis: M-OR + H ₂ O-> M-OH + RO H (2)

Condensation: M-OR + M-OH-> M-O-M + R-OH

M-OR + M-OH-> MOM + H ₂ O

(M: Metal or Silicon, R: Alkyl)

The hydrolysis reaction under basic atmospheric conditions is further accelerated as the R group of the alkoxide precursor is substituted with the hydroxy group (OH), and the hydrolysis proceeds in a state where the substitution is completed. The high rate of polymerization results in the formation of cross-linked sol first, resulting in an amorphous silica shell.

Silica coating prevents agglomeration of the magnetic nanoparticles, and the hydroxy group on the surface of the particle covalently bonds with various coupling agents to facilitate synthesis of an enzyme-mimicking magnetic nanocatalyst and improve chemical stability. It has the advantage that it can be applied to various bio fields. Magnetic nanoparticles coated with silica have a particle size of 20 ~ 100nm.

On the other hand, the enzyme mimetic magnetic nanocatalyst of the present invention was prepared by modifying the surface of the magnetic nanocatalyst coated with silica using a functional silane couplig agent to form an amino acid residue which is an active site of the enzyme.

The functional silane crosslinking agent may have any one or more functional groups selected from amine groups, imidazole groups, and carboxyl groups, and in one embodiment, active sites (Ser-195, His-57, Asp-102) of chymotrypsin To mimic the 3-aminopropyl trimethoxysilane, Carboxyethylsilanetriol, Triethoxy-3- (2-imidazolin-1-yl) propylsilane (Triethoxy-3 -(2-imidazolin-1-yl) propylsilane) or mixtures thereof.

The hydroxy group (OH) formed on the surface by the silica coating binds to the silane by a covalent bond type such as Si-O-Si. The silanization reaction occurs in two stages. Firstly, a silane coupling agent is condensed into a silane polymer. Second, the condensed polymers form the surface of the silica-coated magnetic nanoparticles by covalent bonding through hydroxylation with hydroxyl groups. A schematic diagram of the synthesis process is shown in FIG. 5.

Hereinafter, examples and experimental examples of the enzyme-mimicking magnetic nanocatalyst according to the present invention will be described in detail. However, the following examples are provided only to aid understanding of the present invention, and the scope of the present invention is not limited to the following examples.

[ Example  1] Synthesis of Magnetic Nanoparticles

₂ · 4H ₂ O with an FeCl ₃ · 6H ₂ O to 2M FeCl Fe ^{+ 2} solution and 1M Fe ^{+ 3} solution dissolved in H ₂ O each was prepared at room temperature. 2M Fe ^{+ 2} solution and 10mL 1M Fe ^{+ 3} solution was added dropwise to a solution mixture of 40mL 0.7M NH ₄ OH 500mL slowly dropwise and the mixture was stirred (stirring) for the time. The color of the solution changes from orange to black. After magnetic separation, washed with ethanol (ethanol) and vacuum dried for 24 hours to synthesize magnetic nanoparticles (magnetic nanoparticles, MNPs). A synthesis process of the magnetic nanoparticles of the above example is shown in FIG. 2.

Example 2 Synthesis of Silica Coated Magnetic Nanoparticles

100 mg of MNPs were added to 100 mL of cyclohexane and 4 mL of oleic acid was dispersed for 3 hours. Next, MNPs dispersion solution was added to a solution of 44 g of Igepal CO-520 dispersed in 900 mL of cyclohexane, and stirred, and ₄ mL of NH ₄ OH and 6 mL of tetraethyl orthosilicate (TEOS) were added and stirred at room temperature for 20 hours. After the reaction, methanol (methanol) was added and the precipitate obtained by centrifugation was washed with ethanol, and vacuum dried for 24 hours, and then coated with silica coated silica nanoparticles having a diameter of 20 to 100 nm. MNPs) were prepared. The synthesis process of the silica coated magnetic nanoparticles of the above example is shown in FIG. 3.

Example 3 Synthesis of Enzyme-mimicking Magnetic Nanocatalyst

250 mL of toluene was added to 2.0 g of the Si-MNPs prepared in each round flask, followed by stirring for about 2 hours. ) 8.0 g of) propylsilane is added and refluxed at a temperature of 110 ° C. for 6 hours. After the reaction was centrifuged (3200rpm, 5min) and washed with toluene, vacuum drying for 24 hours to synthesize enzyme-mimic magnetic nanocatalysts (enzyme-mimic magnetic nanocatalysts). The synthesis process of the enzyme-mimicking magnetic nanocatalyst of the above example is shown in FIG. 4.

Example 4 p Hydrolysis of Nitrophenyl Alkyl Esters (PNPE)

1) Preparation of 0.1M Tris-buffer

Add 21.876mL of hydrochloric acid (HCl) to 100mL of distilled water to make 6M HCl. Add 800 mL of distilled water to 12.114 g of Tris and adjust the pH to 7.6 with a pH meter using 6M HCl. Add distilled water to make 1000mL solution.

2) Preparation of PNPE Solution

P- Nitrophenyl butyrate (PNPB), p- Nitrophenyl octanoate (PNPO), and p- Nitrophenyl dodecanoate (PNPD) were used as substrates. It is dissolved in tetrahydrofuran (THF) and diluted in distilled water. Dilute to 0.1M Tris-HCl buffer according to the concentration to be used.

3) PNPE hydrolysis of chymotrypsin

Was dissolved in 0.1M Tris buffer 1mL chymotrypsin 1mg PNPE put in solution is quantified by a p -Nitrophenol (PNP) is glass and by time shaking in 25 measures the UV-Vis spectrophotometer.

4) PNPE Hydrolysis of Enzyme-mimicking Magnetic Nanocatalysts

150mg of each catalyst is added to 1mL 0.1M Tris buffer and 1mL PNPE solution in a 15mL centrifuge tube. At this time, the temperature is maintained at 25 ℃. Free PNP was quantified by UV-Vis spectrophotometer.

[ Experimental Example  1] of magnetic nanoparticles and silica coated magnetic nanoparticles TEM  Analysis

6 and 7 are images taken with a transmission electron microscope (TEM) to observe the microstructure of the prepared magnetite magnetic nanoparticles and silica coated magnetic nanoparticles. The particle size of the magnetic nanoparticles was not nearly spherical but about 7-10 nm, but almost spherical. Silica-coated magnetic nanoparticles were spherical silica coated around magnetic nanoparticles such as dots. The size of the particles was increased to 20-100 nm by the formation of the silica coating film of the magnetic nanoparticles.

[ Experimental Example  2] of magnetic nanoparticles and silica coated magnetic nanoparticles FT - IR  Analysis

8 is a spectrum of magnetic nanoparticles and silica coated magnetic nanoparticles by KBr method using FT-IR. Was measured by the nature 650cm ^-1 ~ 4000cm ^-1 of the device, from 3428cm ^-1 hydroxy stretching vibration peak at the ^{2960cm -1 CH stretching vibration peak, Metal} oxide peak, Si-O-Si vibration at 1098cm ^-1 1630cm ^-1 in A Si-C vibration peak was observed at the peak, 860 cm ⁻¹ .

[ Experimental Example  3] Enzyme Mimicization of Magnetic Nanocatalysts FT - IR  Analysis

FIG. 9 is a spectrum of FT-IR of an enzyme-mimicking magnetic nanocatalyst prepared by modifying a silane on a silica coated magnetic nanoparticle surface by KBr method. Due to the nature of the device measured up to 650cm ^-1 ~ 4000cm ^{- 1} . At 3426cm ^-1 appear in the magnetic nanoparticles coated with silica hydroxy stretching vibration peak yi at ^{2944cm -1 CH stretching vibration peak, Metal} oxide peak, Si-O-Si vibration peak, 800 ~ 950cm at 1095cm ^-1 1664cm ^-1 in Si-C vibration peaks were observed at ^-1 . NH amine is a vibration peak imidazole is the C = C peak at 1450cm ^-1 aromatic ring, from 1488cm ^-1-carboxy was confirmed from 1571cm ^-1 C = O vibration peak. Multifunction is a modification of all of the amine, imidazole and carboxy groups on the particle surface. The analysis confirmed that all three groups were modified.

[ Experimental Example  5] Efficiency of Enzyme-mimicking Magnetic Nanocatalyst

Si-MNPs @ Amine, Si-MNPs @ Imidazole, Si-MNPs @ Carboxyl, Si-MNPs @ Multifunction and the substrates p -Nitrophenyl butyrate (PNPB), p -NItrophenyl octanoate PNPO), p -Nitrophenyl dodecanoate (PNPD) were used to compare the hydrolysis efficiency.

FIG. 10 is a graph quantifying PNP released over time using chymotrypsin and an enzyme mimicking magnetic nanocatalyst through UV-Vis analysis. The substrate concentration was 50 μM and the catalyst amount was 150 mg. Enzyme-mimicking magnetic nanocatalyst, as shown in the graph, embodies the same effect as chymotrypsin, hydrolyzing PNPE to PNP, and confirmed that the reaction was almost finished at 30 min.

FIG. 11 is a graph showing the reaction rate between chymotrypsin and enzyme-mimicking magnetic nanocatalyst obtained through FIG. 10. It was confirmed that the reaction rate of Si-MNPs @ Multifunction catalyst was better than chymotrypsin.

Tables 1, 2, 3, 4 and 12, 13, 14, and 15 show the efficiency of the respective enzyme-mimicking magnetic nanocatalysts. It was confirmed that the efficiency of the Si-MNPs @ Multifunction catalyst modified with three functional groups was higher than that of the magnetic nanocatalyst whose surface was modified with one functional group.

        <Michaelis-menten parameter of Si-MNPs @ Amine>

        <Michaelis-Menten Parameters of Si-MNPs @ Imidazole>

        <Michaelis-Menten Parameters of Si-MNPs @ Carboxyl>

<Michalis-Menten parameter of Si-MNPs @ Multifunction>

As can be seen in the above Examples and Experimental Examples, the present invention provides an enzyme-mimicking magnetic nanocatalyst that mimics the active site of chymotrypsin (Ser ¹⁹⁵ , His ⁵⁷ , Asp ¹⁰² ) to carry out the hydrolysis reaction of the ester compound. It has been shown that it can achieve the same effect as trypsin.

The present invention is not limited to the above-described specific embodiments and descriptions, and various modifications can be made to those skilled in the art without departing from the gist of the present invention claimed in the claims. And such modifications are within the scope of protection of the present invention.

Claims (8)

i) adding an aqueous alkali solution to the mixed aqueous solution of Fe ² + and Fe ^{3 +} to precipitate the ions simultaneously to synthesize magnetite (magnetite, Fe ₃ O ₄ ) magnetic nanoparticles;
Ii) silica coating the magnetite magnetic nanoparticle surface by a sol-gel process using a silica precursor under a base atmosphere; And
Iii) modifying the surface of the magnetic nanoparticles coated with silica using a functional silane couplig agent;
Synthesis method of enzyme-mimicking magnetic nanocatalyst comprising a. The method of claim 1,
The functional silane crosslinking agent has an at least one functional group selected from an amine group, an imidazole group and a carboxyl group to form an amino acid residue which is an active site of the enzyme. The method of claim 2,
The functional silane crosslinker is 3-aminopropyl trimethoxysilane, carboxyethyl silanetriol (Carboxyethyl) to mimic the active sites (Ser-195, His-57, Asp-102) of chymotrypsin silanetriol), triethoxy-3- (2-imidazolin-1-yl) propylsilane (Triethoxy-3- (2-imidazolin-1-yl) propylsilane) or mixtures thereof Synthesis method of magnetic nanocatalyst. The method of claim 3,
A method for synthesizing an enzyme-mimicking magnetic nanocatalyst comprising hydrolyzing a peptide bond, an ester compound, and an amide compound. An enzyme-mimicking magnetic nanocatalyst prepared by the method of any one of claims 1 to 4. The method of claim 5,
A method for synthesizing an enzyme-mimicking magnetic nanocatalyst, characterized in that the average particle size of the magnetic magnetic nanoparticles is 7-10 nm. The method of claim 5,
The method of synthesizing a magnetic nanocatalyst according to claim 2, wherein the average particle size of the magnetic nanoparticles coated with silica is 20 to 100 nm. The method of claim 5,
A method for synthesizing an enzyme-mimicking magnetic nanocatalyst, which is used for biosensor, biocatalyst, proteome research.

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