KR101068873B1 - Hydrogel entrapping biomarker-immobilized silica nanoparticles and method for preparing the same - Google Patents

Abstract

The present invention relates to a hydrogel containing silica nanoparticles to which the biomarker is immobilized, and to a method of manufacturing the same. More specifically, the biomarker is fixed to the surface-modified silica nanoparticles and encapsulated into a hydrogel having excellent biocompatibility. Hydrogels containing silica nanoparticles immobilized with biomarkers that can maintain biomarker activity and stably react with substrates by preventing biomarker denaturation and leakage due to environmental factors, and their preparation It is about a method.

Hydrogels, Silica Nanoparticles, Biomarkers

Description

Hydrogel containing silica nanoparticles immobilized with biomarker and method for preparing the same {Hydrogel entrapping biomarker-immobilized silica nanoparticles and method for preparing the same}

The present invention relates to a hydrogel containing silica nanoparticles to which the biomarker is immobilized, and to a method of manufacturing the same. More specifically, the biomarker is fixed to the surface-modified silica nanoparticles and encapsulated into a hydrogel having excellent biocompatibility. Hydrogels containing silica nanoparticles immobilized with biomarkers, which can maintain biomarker activity and stably react with substrates by preventing biomarker denaturation and biomarker leakage due to environmental factors. It relates to a manufacturing method.

Conventional protein biosensors use microarrays to fix biomarkers on glass plates, plastics or by using two-dimensional planes ( Analytical Biochemistry 368 (2007) 205-213). Microarray fabrication is diverse and complex due to chemical modifications ( Chem Bio Chem 2001, 2, 686-694), and this method may lead to denaturation of immobilized proteins and crosslinking reactions when immobilizing proteins on two-dimensional surfaces. This has the disadvantage of being tricky ( J. Immunol. Methods 2001, 255, 1-13). To supplement this, PEG hydrogel was used.

PEG hydrogels have been applied biologically due to their high water content and biocompatibility ( Adv. Mater . 2006, 18, 1345-1360). This three-dimensional method can increase the immobilization yield of biomarkers and can protect biomarkers from the surrounding environment due to chemical bonding and are stable ( Langmuir 2004, 20. 270-272). PEG hydrogels are widely used to fix biomaterials such as biomarkers and cells in terms of high water content and excellent biocompatibility. When the cells are fixed inside the hydrogel, three-dimensional culture can be obtained similarly to the actual body to obtain more accurate information from the cells. When the biomarkers are fixed, biomarkers are prevented from being denatured from environmental factors and It has the advantage of being able to measure strong signals by increasing the response of.

Likewise, in the case of silica nanoparticles, it is easy to manufacture and surface modification, and is used as a material for various fields such as immobilization of biomaterials to measure activity or use in drug delivery systems. The desired size can be synthesized, and methods of immobilizing biomaterials by increasing the surface area by making small pores on the surface of silica nanoparticles are also widely studied. By using tertiary structure of silica nanoparticles, more biomaterials can be attached. Has the advantage.

However, if the biomarker is encapsulated in the hydrogel using only the hydrogel as a support, the biomarker easily leaks. If the biomarker is fixed using only silica nanoparticles, the biomarker is easily changed by the surrounding environment. There are problems that can be denatured.

An object of the present invention is to prevent the leakage of the biomarker in the biosensor, and to maintain the activity of the biomarker continuously to fix the biomarker to the silica nanoparticles and biomarker-fixed nanoparticles biocompatible hydrogel It is to provide a hydrogel containing silica nanoparticles fixed with a biomarker, and encapsulating the biomarker.

Another object of the present invention is to provide a biosensor comprising a hydrogel having excellent physical properties.

In order to achieve the above object, the present invention

[Formula 1]

Where

X is silica gel or composite silica gel,

Y is substituted or unsubstituted alkylene, alkenylene or alkynylene having 1 to 12 carbon atoms,

Z is a biomarker,

o, p and q each independently represent an integer of 0 to 3.

The invention also

Modifying the surface of the silica nanoparticles;

Fixing the biomarker to the surface-modified silica nanoparticles; And

It provides a method of producing a biomarker-fixed silica nanoparticle-containing hydrogel comprising the step of adding a biomarker-fixed silica nanoparticles to the polymer precursor solution to produce a hydrogel.

The present invention also provides a protein biosensor comprising the hydrogel.

The present invention can prevent the biomarker leakage from the hydrogel by fixing the biomarker on the silica nanoparticles, and by using the biocompatible hydrogel to solve the biomarker activity by solving the biomarker degeneration due to environmental factors It can maintain the structure of the biomarker continuously, and the reaction with the substrate can be more stable, and the silica nanoparticles with the biomarker immobilized can be encapsulated in the hydrogel in the desired amount, and various shapes and It has the advantage of producing a hydrogel having a size. Therefore, the present invention can be usefully used for the study of biomaterials such as biomarkers, cells, antibodies and the like.

EMBODIMENT OF THE INVENTION Hereinafter, the structure of this invention is demonstrated concretely.

The present invention

Represented by Chemical Formula 1, the present invention relates to a hydrogel comprising silica nanoparticles having a biomarker immobilized thereon:

[Formula 1]

Where

X is silica gel or composite silica gel,

Y is substituted or unsubstituted alkylene, alkenylene or alkynylene having 1 to 12 carbon atoms,

Z is a biomarker,

o, p and q each independently represent an integer of 0 to 3.

The present invention is to fix the biomarker on the silica nanoparticles and to encapsulate the biomarker into a hydrogel having a high moisture content and hydrophilic biocompatibility as a soft elastomer to prevent biomarker leakage and degeneration of the biomarker due to environmental factors Have

In the present invention, the biomarker may be a protein or peptide such as an enzyme, an antigen, an antibody, or the like; Nucleic acid molecules such as DNA, RNA, siRNA, aptamers, and the like; Means a substance that can be a specific label in vivo, including cells and the like. In the following examples of the present invention, one of such biomarkers uses an enzyme, but the biomarkers of the present invention are not limited thereto. In order to facilitate confirmation of the reaction between the biomarker and the substrate, it may be desirable for the biomarker to exhibit a luminescence or color reaction upon reaction with the substrate, for which the biomarker may be labeled to enable a luminescence or color reaction. Can be.

The silica nanoparticles are a process of activating by introducing an amino group on the surface of the silica nanoparticles, by reacting an aminoalkylsilane derivative of the formula (3) to a silica gel or a composite silica gel carrier of the formula (2) to the silane present on the surface of the carrier It is preferable that the compound introduce | transduces the amino group of following formula (4) through the silane coupling reaction between an oligo group and an aminoalkylsilane derivative.

[Formula 2]

(3)

[Formula 4]

Where

X is silica gel or composite silica gel,

R is a methyl group or an ethyl group,

o, p and q each independently represent an integer of 0 to 3.

In the silica nanoparticles of Formula 2, a hydroxyl group is introduced into the silica gel nanoparticles. Methods of introducing hydroxyl groups into silica nanoparticles are well known to those skilled in the art, and for this purpose in the following examples, were treated with pyrana solutions.

The aminoalkylsilane derivatives include β-aminoethyl-γ-aminopropylmethyldimethoxysilane and aminopropylmethyldi

Ethoxysilane, (gamma) -aminopropyl trimethoxysilane, (gamma) -aminopropyl triethoxysilane, etc. can be used individually or in combination of 2 or more types. The aminoalkylsilane derivatives are not limited to the compounds exemplified above, and may be any one that can provide amino groups while binding to the silica nanoparticles.

In addition, the biomarker may be fixed by reacting the activated silica nanoparticles with a crosslinking agent. Preferably, the crosslinking agent having an aldehyde group is reacted with the silica nanoparticles having the amino group of Formula 4 introduced therein, and the silica having an aldehyde group at the end thereof. By reacting the nanoparticles with a biomarker having an amino group to fix the biomarker to the silica nanoparticles, the silica nanoparticles to which the biomarker of Chemical Formula 1 is fixed can be obtained.

As the crosslinking agent, any compound can be used as long as it has a compound having an aldehyde group at one terminal. For example, polyaldehydes, such as glutaraldehyde, dialdehyde starch, succinate aldehyde, can be used, and it is preferable to use glutaraldehyde.

The biomarker is preferably contained in 0.1 to 10 parts by weight based on 100 parts by weight of the silica nanoparticles in order to be sufficiently fixed to the silica nanoparticles and to maintain the biomarker activity continuously.

In addition, the biomarker-fixed silica nanoparticles are preferably included in 5 to 20 parts by weight based on 100 parts by weight of the hydrogel. This is because improved mechanical strength and proper biomarker activity appear within the content range.

The hydrogel of the present invention may be prepared by polymerization of a polymer having high moisture content and excellent biocompatibility.

The component constituting the hydrogel is not particularly limited, but for example, polyacrylic acid, polyacrylamide, polyhydroxyethyl methacrylate, polyethylene glycol, poly (N, N-ethylaminoethyl methacrylate), hyaluronic acid Polysaccharides such as hyaluronic acid, chitosan, and dextran may be included. In the preparation of the hydrogel of the present invention, the polymer is used in the form of a polymer precursor having photocuring sites at both ends of the polymer so as to be polymerized by a photoinitiator. For example, polyethylene glycol is used for the preparation of hydrogel in the form of polyethylene glycol-diacrylate so that polymerization by a photoinitiator can occur.

The shape and size (diameter) of the hydrogel of the present invention may have a variety of shapes and sizes depending on the type of mold at the time of manufacture, the lattice size is depending on the molecular weight of the biocompatible polymer for producing the hydrogel, for example polyethylene glycol It can be variously adjusted is not particularly limited. One skilled in the art can easily control the rate of reaction of the biomarker with the substrate by adjusting the lattice size of the hydrogel.

In addition, the hydrogel of the present invention has a feature that the strength is improved compared to the conventional hydrogel by containing silica nanoparticles, preferably has a strength improvement of more than twice.

The invention also

Modifying the surface of the silica nanoparticles;

Fixing the biomarker to the surface-modified silica nanoparticles; And

It relates to a method for producing a biomarker-fixed silica nanoparticle-containing hydrogel comprising the step of adding a biomarker-fixed silica nanoparticles to the polymer precursor solution to produce a hydrogel.

The step of modifying the surface of the silica nanoparticles is to modify the surface to create a condition for fixing the biomarker on the surface of the silica nanoparticles, the aminoalkylsilane of the formula (3) on the silica gel or a composite silica gel carrier By reacting a derivative, silica nanoparticles to which an amino group of the following Chemical Formula 4 is introduced may be obtained through a silane coupling reaction between a silanol group present on a surface of a carrier and an aminoalkylsilane derivative.

[Formula 2]

(3)

[Formula 4]

Where

X is silica gel or composite silica gel,

R is a methyl group or an ethyl group,

m, n and o each independently represent an integer of 0 to 3.

Wherein the aminoalkylsilane derivative is β-aminoethyl-γ-aminopropylmethyldimethoxysilane, aminopropylmethyldi

Ethoxysilane, (gamma) -aminopropyl trimethoxysilane, (gamma) -aminopropyl triethoxysilane, etc. can be used individually or in combination of 2 or more types. The aminoalkylsilane derivatives are not limited to the compounds exemplified above, and may be any one that can provide amino groups while binding to the silica nanoparticles.

In detail describing the step of modifying the surface of the silica nanoparticles, the silica gel or the composite silica gel of the formula (2) is put in an organic solvent, the aminoalkylsilane derivative of the formula (3), and then refluxed at preferably 100 to 110 ℃ To obtain water-insoluble silica nanoparticles into which the amino group of Formula 4 is introduced by a silane coupling reaction. The amount of the aminoalkylsilane derivative is preferably 1 to 20 parts by weight, preferably 2 to 15 parts by weight based on 100 parts by weight of the silica nanoparticles.

In the biomarker fixing step, a crosslinking agent is reacted with silica nanoparticles of Formula 4 to obtain a compound of Formula 5, and then reacted with a biomarker to obtain silica nanoparticles to which the biomarker of Formula 1 is fixed.

[Formula 4]

[Chemical Formula 5]

[Formula 1]

상기 식에서,

Where

X is silica gel or composite silica gel,

R is a methyl group or an ethyl group,

o, p and q each independently represent an integer of 0 to 3.

The crosslinking agent may be any compound as long as it can provide an aldehyde group to the silica nanoparticles modified to have the amino group. For example, polyaldehydes such as glutaraldehyde, dialdehyde starch, maleic acid aldehyde and the like can be used. The crosslinking agent is present in a solution state, and is preferably a solution prepared by adding 10 to 30 parts by weight of the crosslinking agent with respect to 100 parts by weight of the buffer solution.

The immobilization process of the biomarker will be described in more detail as follows. First, a crosslinking agent is reacted with silica nanoparticles to which the amino group of Formula 4 is introduced to obtain a compound of Formula 5. The amount of the crosslinking agent is preferably 10 to 30 parts by weight relative to 100 parts by weight of the silica nanoparticles. The reaction is carried out by stirring at a temperature of 0 to 30 ° C. for about 0.5 to 4 hours. Thereafter, the biomarker is reacted with the compound of Formula 5 to obtain silica nanoparticles of Formula 1 having the biomarker fixed thereto. The reaction is carried out by stirring at a temperature of 10 to 30 ° C. for 0.5 to 15 hours, preferably 1 to 7 hours. PH and temperature of the reaction solution should be made in a range that does not reduce the activity of the biomarker.

The amount of immobilized biomarker is preferably contained in an amount of 0.1 to 10 parts by weight relative to 100 parts by weight of the silica nanoparticles in order to be sufficiently fixed to the silica nanoparticles and to maintain the biomarker activity continuously. After the crosslinking reaction, it is preferable to sufficiently wash the silica nanoparticles in which the biomarker is immobilized with a suitable buffer to remove the easily detachable biomarker and excess crosslinking agent having insufficient binding.

The hydrogel manufacturing step is a step of encapsulating the silica nanoparticles in which the biomarker is fixed in a hydrogel in order to prevent biomarker denaturation according to environmental factors,

Preparing a polymer precursor solution; And

It is preferable to include a step of inducing radical polymerization by exposing the mixed solution of silica nanoparticles to which the polymer precursor solution and the biomarker are fixed to a UV light source.

The molecular weight of the polymer is preferably in the range of 500 to 10000, since it has a stable mechanical properties as a carrier when it is in the above range, and contains moisture which can maintain the biomarker activity continuously. In addition, the higher the molecular weight may show a tendency to increase the biomarker activity.

The polymer precursor solution is preferably prepared by adding the polymer precursor to any one solvent of distilled water or buffer solution.

The polymer precursor is preferably included in an amount of 10 to 50 parts by weight based on 100 parts by weight of the solvent to prepare a hydrogel having a suitable moisture content and lattice size.

In addition, the polymer precursor solution may include 0.001 to 0.02 parts by weight of photoinitiator based on 1 part by weight of the polymer precursor to induce photopolymerization of the polymer.

Photoinitiators include, but are not limited to, 2,2-dimethoxy-2-phenylacetophenone (DMPA), 2-hydroxy-2-methylpropiophenone (2-hydroxy- 2-methylpropipphenone, HOMPP), or Acuracure 2959 can be used alone or in combination of two or more.

The precursor solution prepared in the above step is mixed with silica nanoparticles fixed with biomarkers, poured into molds of various types of silicon, and exposed to a UV light source to induce radical polymerization to prepare a hydrogel having a network structure. Can be.

That is, when polyethylene glycol having vinyl groups at both ends is exposed to UV light, radicals generated from photoinitiators break carbon double bonds of vinyl groups and cross-links are formed to form a network structure. The particles can be trapped.

In addition, the hydrogel may have various shapes and sizes according to the type of the mold, and the lattice size may be variously controlled by changing the molecular weight of the polymer.

In addition, the biomarker-fixed silica nanoparticles are preferably included in 5 to 20 parts by weight based on 100 parts by weight of the hydrogel. This is because improved mechanical strength and proper biomarker activity appear within the content range.

The radical polymerization may be performed for 1 to 10 seconds under UV of 100 W, and is not particularly limited because it is determined by the molecular weight of the polymer constituting the hydrogel.

The invention also relates to a biosensor comprising the hydrogel of the invention.

In the case of the conventional biosensor, there are problems such as denaturation of the fixed biomarker due to physicochemical factors, water leakage, and a difficult immobilization reaction. However, the biosensor of the present invention fixes the biomarker to silica nanoparticles to prevent biomarker from the biosensor. Hydrogel encapsulates silica nanoparticles to which biomarkers are immobilized and keeps biomarker activity long, protects against physical shocks and chemical reactions, and maintains three-dimensional conformation of biomarkers. It can have a characteristic that the reaction with the substrate can proceed more stably.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the scope of the present invention is not limited to the following examples.

Example 1 Fixation of Enzyme on Silica Nanoparticles

1.5 g of silica nanoparticles having a size of 400 to 500 nm were reacted with a PIRANHA solution (9 mL of sulfuric acid and 3 mL of hydrogen peroxide) at 80 ° C. and 600 rpm for 30 minutes, centrifuged, and washed alternately with water and ethanol. The APTES solution was then treated. APTES solution was prepared by mixing the ratio of 99.9% ethanol and APTES in 19: 1, after sealing to block the air and reacted for 2 hours at room temperature on a shaker (shaker). After washing three times with ethanol and poured into Petri dishes and heat-treated at 115 ℃ for 2 hours. 1 g of heat-treated silica nanoparticles, 1.25 mL of glutaraldehyde (25%) and 48.75 mL of PBS buffer (pH 7.4) were added to a beaker, reacted for overnight on a shaker, and washed twice with water.

Aqueous glucose oxidase solution at a concentration of 1 mg / mL was added to 30 mL per 0.5 g of silica nanoparticles, and then reacted for overnight on a shaker, and the enzymes not immobilized on the silica nanoparticles were washed with water.

Fixing the enzyme to the silica nanoparticles is shown in FIG.

Experimental Example 1 Determination of Activity of Enzyme Immobilized on Silica Nanoparticles

The activity of the enzyme immobilized on the silica nanoparticles was measured by using an o-dianisidine solution. In other words, when glucose reacts with oxygen, water, and glucose oxidase, gluconic acid and hydrogen peroxide are produced. When hydrogen peroxide encounters O-Dianisidine and peroxidase, it changes to water and oxidized O-Dianisidine, which turns the colorless solution into a red color, and the eye can confirm the activity of the enzyme.

O-Dianisidine solution was prepared by adding 3 mL of 50 mM glucose, 600 μl of 1 mg / mL peroxidase, and finally 14.4 mL of 0.21 mM O-Dianisidine aqueous solution. After fixing the concentration of the silica nanoparticles prepared in Example 1 to 200mg / mL and reacted for 30 minutes while changing the concentration of glucose oxidase (0, 0.1, 0.5, 1.0, 2.0mg / mL). In addition, an aqueous solution of glucose oxidase in the same concentration range was prepared and used as a control. After the reaction, the mixture was placed in a 2 mL transparent cuvette, and 100 µl of 2 mM hydrochloric acid was added to terminate the reaction. Enzyme activity was measured by absorbance at 400 nm using UV-vis spectroscopy

Figure 4 shows the activity of the free enzyme in the aqueous solution, Figure 5 shows the enzyme activity of the enzyme immobilized silica, it was confirmed that the enzyme is fixed on the silica nanoparticles, the activity of the immobilized enzyme and the activity of the free enzyme The concentration increased similarly.

Example 2 Preparation of PEG Hydrogel Containing Enzyme-immobilized Silica Nanoparticles

In order to encapsulate the enzyme-immobilized silica nanoparticles with a hydrogel, 1 mL of the enzyme-immobilized silica solution prepared in Example 1, 1 mL of PEG-DA, and 2,2-dimethyloxy-2-phenyl-acetphenone PEG precursor solution was prepared by adding 20 µl of (2-2-dimethoxy-2-phenyl-acetophenone). The precursor solution was poured into a circular mold made of silicon 1 cm in diameter and exposed to 100 W ultraviolet (see FIG. 3).

Example 3 Changes of Enzyme Activity According to Silica Nanoparticle Concentration and PEG Hydrogel Molecular Weight

When the concentration of silica nanoparticles was 0, 10, 50, 100, 200 mg / mL, and PEG molecular weight 575 was used, the hydrogel was prepared by exposing 100W ultraviolet rays for 2 seconds by the method of Example 2. When PEG molecular weight 8000 was used, hydrogel was prepared by exposing at 100W ultraviolet light for 5 seconds by the method of Example 2.

After preparing the hydrogel, the activity of glucose oxidase immobilized on silica was measured according to the method of Experimental Example 1.

As shown in FIG. 6, as the concentration of silica nanoparticles increased, the enzyme activity increased, and the enzyme activity of 8000 was higher than the PEG molecular weight of 575. This suggests that the larger the mesh size of 8000 than 575 is, the diffusion resistance of glucose, the substrate, decreases, which leads to a rapid reaction with glucose oxidase, thereby increasing the reaction rate.

Example 4 Change of Enzyme Activity According to Enzyme-Substrate Reaction Time and PEG Molecular Weight

Hydrogel was prepared by fixing the concentration of silica nanoparticles to which glucose oxidase of 1 mg / mL concentration prepared in Example 1 was fixed at 200 mg / mL, and having PEG molecular weights of 575 and 8000. According to the method, enzyme activity was measured by time period (10, 20, 30, 60 minutes).

As shown in FIG. 7, the enzyme activity increased with increasing reaction time, and the molecular weight of PEG-DA 8000 was higher than 575.

Example 5 Mechanical Strength of Hydrogel

200 mg / mL of the enzyme-immobilized silica nanoparticles prepared according to the method of Example 1 and silica nanoparticles without enzyme-fixed as a control, respectively, using PEG-DA 8000, were lengthwise according to the method of Example 2 A hydrogel of 1 cm, width 3 cm, and average thickness 0.35 mm was prepared.

The strength of each hydrogel was measured by measuring the degree of breaking while pulling at a constant speed of 50mm / min using a UTM machine.

8 shows the strength of the silica nanoparticle-containing hydrogel according to the concentration of the nanoparticles, and FIG. 9 shows the strength of the enzyme-fixed silica nanoparticle-containing hydrogel by the concentration of the nanoparticles.

As shown in Figures 8 and 9, it was confirmed that the strength increases as the concentration of the silica nanoparticles increases. This is thought to increase the strength of the hydrogel by agglomeration of silica nanoparticles to form a microstructure. It was also found that even with the same concentration of silica nanoparticles, the strength of the enzyme-immobilized silica nanoparticles increased, which was thought to be higher due to the van der waals force between the enzymes surrounding the silica nanoparticles. do.

Example 6 Stability Test of Enzyme Immobilized on Hydrogel

In order to test the stability of the enzyme immobilized on the hydrogel, three types of samples were prepared and their enzyme activities were measured at different time periods (1, 2, 3, 5, 6, 7 days).

Sample 1: Hydrogel prepared according to the method of Example 2 using a PEG molecular weight of 8000 fixed with a concentration of silica nanoparticles fixed in glucose oxidase prepared in Example 1 to 200mg / mL.

Sample 2: Hydrogel prepared according to the method of Example 2 using 1 mg / mL glucose oxidase in aqueous solution using PEG molecular weight 8000.

Sample 3: Silica nanoparticles aqueous solution fixed with glucose oxidase.

As shown in FIG. 10, Sample 3 is easily denatured because the enzyme is exposed to the external environment without protection, thereby showing the sharpest degradation of enzyme activity, and Sample 2 is easily leaked, resulting in a decrease in enzyme activity. Silver enzyme activity lasted the longest.

The results show that the encapsulation method using the hydrogel of the enzyme-fixed silica nanoparticles has the advantage of sustaining the activity of the enzyme for a long time from the external environment.

Example 7 Measurement of Enzyme Activity Fixed on Silica Nanoparticles According to Substrate Concentration

The concentration of the enzyme-immobilized silica nanoparticles prepared in Example 1 was fixed at 200 mg / mL, and hydrogel was prepared according to the method of Example 2 using PEG-DA molecular weight 575.

The enzyme activity was measured using different concentrations of substrate (0, 2, 4, 6, 8, 10 mM glucose) and amplex red. That is, 1 mL of 0, 2, 4, 6, 8, 10 mM glucose, 500 μl of 1 mg / mL peroxidase, and 300 μl of 0.1 mM Amplex Red were mixed and reacted for 30 minutes. This is because glucose is oxidized by oxygen, water, and glucose oxidase to produce gluconic acid and hydrogen peroxide, and the hydrogen peroxide is oxidized by encountering amplex red and peroxidase, and in the oxidation process, amplex red is resolufin. ), Which uses the principle of measuring enzyme activity by turning colorless Amplex Red into fluorescent resolupin.

As shown in FIG. 11, it could be seen that the enzyme activity increased as the concentration of glucose increased.

<Example 8> SEM image according to the concentration of silica nanoparticles

The concentration of silica nanoparticles was 0, 10, 50, 100, 200 mg / mL, and hydrogels were prepared using PEG-DA 8000. After cutting the central portion of the prepared hydrogel, a SEM (Scanning electron microscopy) image of the cross-sectional area was obtained.

As shown in FIG. 12, it can be seen that as the concentration of the silica nanoparticles increases, coagulation occurs with each other, thereby forming a microstructure. This is a result supporting the experimental results of Example 5.

1 exemplarily illustrates a process of immobilizing an enzyme on silica nanoparticles.

2 exemplarily shows a process of chemical modification of the surface of silica nanoparticles.

3 exemplarily illustrates a process of encapsulating enzyme-immobilized silica nanoparticles with PEG hydrogel.

Figure 4 shows the results of measuring the activity of the free enzyme.

Figure 5 shows the results of measuring the enzyme activity of the enzyme-fixed silica nanoparticles.

Figure 6 shows the results of the enzyme activity according to the concentration of the enzyme-fixed silica nanoparticles in the PEG hydrogel (molecular weight 575, 8000) of the present invention.

Figure 7 shows the results of the measurement of biomarker activity according to the reaction time of the enzyme-substrate of the enzyme-fixed silica nanoparticles in PEG hydrogel (molecular weight 575, 8000) of the present invention.

8 shows the strength change of the hydrogel according to the concentration of silica nanoparticles.

Figure 9 shows the change in strength of the enzyme-fixed silica nanoparticles containing hydrogels.

Figure 10 shows the result of measuring the enzyme activity in the enzyme-fixed silica nanoparticle-containing hydrogel, free enzyme and enzyme-fixed silica nanoparticles.

Figure 11 shows the results of measuring the activity of the enzyme according to the substrate concentration in the enzyme-fixed silica nanoparticle-containing hydrogel of the present invention.

12 is a SEM image of the concentration of silica nanoparticles in the hydrogel containing silica nanoparticles to which the enzyme of the present invention is immobilized [(a): 200 mg / ml, (b): 100 mg / ml, (c ): mg / ml, (d): mg / ml, (e): mg / ml].

Claims (18)

Represented by Chemical Formula 1, a hydrogel comprising silica nanoparticles having a biomarker immobilized thereon: [Formula 1]

Where X is silica gel or composite silica gel, Y is substituted or unsubstituted alkylene, alkenylene or alkynylene having 1 to 12 carbon atoms, Z is a biomarker, o, p and q each independently represent an integer of 0 to 3. The hydrogel of claim 1, wherein the biomarker is a biomaterial selected from the group consisting of proteins, peptides, nucleic acids, and cells. The method of claim 1, The silica nanoparticles are hydrogels, characterized in that the compound of formula (4) obtained by reacting an aminoalkylsilane derivative of formula (3) to a silica gel or a composite silica gel carrier of formula (2): [Formula 2]

(3)

[Formula 4]

Where X is silica gel or composite silica gel, R is a methyl group or an ethyl group, o, p and q each independently represent an integer of 0 to 3. The method of claim 3, Wherein the aminoalkylsilane derivative is at least one selected from β-aminoethyl-γ-aminopropylmethyldimethoxysilane, aminopropylmethyldiethoxysilane, γ-aminopropyltrimethoxysilane and γ-aminopropyltriethoxysilane Hydrogel. Modifying the surface of the silica nanoparticles; Fixing the biomarker to the surface-modified silica nanoparticles; And A method for producing a biomarker-fixed silica nanoparticle-containing hydrogel, comprising adding a biomarker-fixed silica nanoparticle to a polymer precursor solution to prepare a hydrogel. The method of claim 5, The step of modifying the surface of the silica nanoparticles comprises silica nanoparticles having a biomarker-immobilized silica nanoparticles, characterized in that by reacting an aminoalkylsilane derivative of formula (3) to a silica gel or a composite silica gel carrier of formula (2) Hydrogel manufacturing method: [Formula 2]

(3)

[Formula 4]

Where X is silica gel or composite silica gel, R is a methyl group or an ethyl group, o, p and q each independently represent an integer of 0 to 3. The method of claim 6, The aminoalkylsilane derivative is at least one selected from β-aminoethyl-γ-aminopropylmethyldimethoxysilane, aminopropylmethyldiethoxysilane, γ-aminopropyltrimethoxysilane and γ-aminopropyltriethoxysilane Method for producing a hydrogel containing silica nanoparticles to which the biomarker is fixed. The method of claim 5, In the biomarker fixing step, a crosslinking agent is reacted with a compound represented by the following Chemical Formula 4 to obtain a compound represented by the following Chemical Formula 5, and then reacted with a biomarker to obtain a compound represented by the following Chemical Formula 1 Gel preparation method: [Formula 4]

[Chemical Formula 5]

[Formula 1]

Where X is silica gel or composite silica gel, Y is substituted or unsubstituted alkylene, alkenylene or alkynylene having 1 to 12 carbon atoms, Z is a biomarker, R is a methyl group or an ethyl group, o, p and q each independently represent an integer of 0 to 3. The method of claim 8, A method for producing a hydrogel-fixed silica nanoparticle-containing hydrogel, wherein the crosslinking agent is polyaldehyde. The method of claim 5, wherein the hydrogel manufacturing step Preparing a polymer precursor solution; And A method of producing a biomarker-fixed silica nanoparticle-containing hydrogel, comprising the step of inducing radical polymerization by exposing the mixed solution of the silica nanoparticles to which the precursor solution and the biomarker are immobilized to a UV light source. The method of claim 10, The polymer precursor solution is prepared by adding a polymer precursor to any one of a solvent of distilled water or a buffer solution. The method of claim 11, The polymer precursor is a method for producing a hydrogel containing silica nanoparticles fixed with a biomarker is a photocured site is introduced into the biocompatible polymer. The method of claim 11, The polymer precursor is a method for producing a hydrogel containing silica nanoparticles fixed with a biomarker of polyethylene glycol diacrylate. The method of claim 5, A method for producing a hydrogel containing silica nanoparticles having a fixed biomarker, characterized in that the molecular weight of the polymer is 500 to 10,000. The method of claim 5, The polymer precursor solution is a method for producing a biomarker-fixed silica nanoparticle-containing hydrogel, characterized in that it comprises a photoinitiator. The method of claim 15, The polymer precursor solution comprises a 0.001 to 0.02 parts by weight of the photoinitiator with respect to 1 part by weight of the polymer precursor, the biomarker-fixed silica nanoparticle-containing hydrogel manufacturing method. The method of claim 15, Photoinitiators include 2,2-dimethoxy-2-phenylacetophenone (DMPA), 2-hydroxy-2-methylpropipphenone (HOMPP) And agacure 2959 (Irgacure 2959), characterized in that at least one selected from the group consisting of biomarker-fixed silica nanoparticle-containing hydrogel manufacturing method. Biosensor comprising a hydrogel according to any one of claims 1 to 4.

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