KR100962007B1 - Interpenetrating polymer network hydrogel bead comprising polyethylene glycol and polyacrylamide, and method for immobilizing enzymes by using the same - Google Patents

Abstract

The present invention relates to a hydrogel bead of polyethylene glycol and polyacrylamide-containing interpenetrating structure, and to an enzyme immobilization method using the same, and more specifically, by adding polyacrylamide (PAAm) to polyethylene glycol (PEG) hydrogel. By forming one type of interpenetrating structure (IPN), while retaining the advantages of PEG hydrogels, the mechanical strength is improved and the enzymes can be chemically covalently bonded to provide functional groups that allow the enzymes to be permanently immobilized on the hydrogels. The present invention relates to a hydrogel bead of polyethylene glycol and polyacrylamide-containing interpenetrating structure and an enzyme immobilization method using the same.

Polyethylene glycol, polyacrylamide, hydrogel, beads, interpenetrating structure (IPN), enzyme, functional group

Description

Interpenetrating polymer network hydrogel bead comprising poly (ethylene glycol) and poly (acrylamide), and method for immobilizing enzymes by using the same}

The present invention relates to a hydrogel bead of polyethylene glycol and polyacrylamide-containing interpenetrating structure, and an enzyme immobilization method using the same, and more specifically, by adding polyacrylamide (PAAm) to polyethylene glycol (PEG) hydrogel. By forming one type of interpenetrating structure (IPN), while retaining the advantages of PEG hydrogels, the mechanical strength is improved and the enzymes can be chemically covalently bonded to provide functional groups that allow the enzymes to be permanently immobilized on the hydrogels. The present invention relates to a hydrogel bead of polyethylene glycol and polyacrylamide-containing interpenetrating structure and an enzyme immobilization method using the same.

As a support for immobilizing enzymes, inorganic materials such as metals or semiconductors, or polymers have been used. However, these existing supports are mostly hard and cannot absorb water, and proteins such as enzymes lose activity due to changes or drying in three-dimensional structures when they are fixed on hard surfaces. In recent years in order to solve these problems was started using a hydrogel with an enzyme fixed support (Journal of Molecular Catalysis B: Enzymatic 2000, 9, 269-274).

Hydrogel is a cross-linked hydrophilic polymer that absorbs large amounts of water and expands. When using hydrogels, enzymes can be immobilized on or inside the hydrogels, and the hydrogels are soft and contain a large amount of water, providing an environment in which enzymes can perform biological functions without denaturation. ( J. Appl . Polym . Sci . 2001, 82, 1404-1409).

Among various hydrogels, hydrogels using poly (ethylene glycol) (PEG) are widely used in biosensors and tissue engineering because of their excellent biocompatibility ( Polymer 2001, 42, 4893-4901).

However, in using PEG hydrogel as an enzyme fixation support, firstly, the mechanical strength of PEG hydrogel is low, and second, there is no functional group capable of covalently binding a protein.

Accordingly, the present invention has been made to solve the above disadvantages of the prior art, the object of the present invention while retaining the advantages of the PEG hydrogel, while improving the mechanical strength and provided with a functional group capable of chemically binding enzymes interpenetration It is to provide a method for producing a novel hydrogel of the structure.

Another object of the present invention is to provide a method for immobilizing an enzyme using a hydrogel bead having an interpenetrating structure prepared from the above.

In order to achieve the above object, the present invention

Preparing PEG hydrogel beads; And

It provides a method for producing an interpenetrating hydrogel comprising the step of preparing a hydrogel of the interpenetrating polymer network by adding polyacrylamide to the PEG hydrogel bead.

The present invention also provides a hydrogel of an interpenetrating polymer network prepared according to the method for producing the hydrogel.

The invention also

Preparing a hydrogel of an interpenetrating polymer network of the present invention;

Activating the functional group of the hydrogel; And

It provides a method for immobilizing an enzyme on a hydrogel comprising the step of immobilizing the enzyme by inducing a covalent bond of the activated hydrogel and the enzyme.

The present invention is characterized by the first can be prepared hydrogel beads with a simple equipment, secondly by adjusting the amount of the precursor solution for the hydrogel production bead size as desired.

In addition, when a method of preparing a hydrogel beads as a single material, such as PEG hydrogel overcomes the disadvantages of low mechanical strength and enzyme fixed capacity interpenetrating structure (IPN, I nterpenetrating P olymer N etwork) technology.

In applying the IPN technology, the polyacrylamide is used to improve mechanical strength and provide an amine group as a chemical functional group to create an environment in which enzymes can be chemically covalently bonded. In addition, although the mechanical strength is improved and the enzyme fixation capacity is increased, there is an effect that can maintain the advantages of PEG intact by maintaining a high moisture content.

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

The present invention is a hydrogel of the internal PEG hydrogel to supplement the disadvantages of the PEG hydrogel poly (acrylamide) (poly-acrylamide, PAAm) a was subsequently cross-linked interpenetrating structure (IPN, I nterpentrating P olymer N etwork) beads It is a technique to fix the enzyme by preparing in the form.

The IPN technology is a technology for making a hydrogel using two or more materials. Existing single materials, in particular, a hydrogel using PEG, which is regarded as a representative polymer material, may contain a large amount of water as a soft elastomer, and may be hydrophilic. Although it has the advantage of excellent biocompatibility, it has the disadvantage of low mechanical strength. In addition, there is no functional group capable of chemically covalent bonding of enzymes to fix enzymes, so that the enzymes that have been physically immobilized are separated and have a low enzyme fixation capacity in the long term.

The present invention improves the mechanical strength while accommodating the advantages of PEG hydrogels to compensate for these disadvantages, and provides a functional group capable of chemically covalent bonding of enzymes to ultimately increase enzyme fixation capacity. It is a technique for preparing a gel, and to prepare a hydrogel in the form of beads (bead) in order to secure a space in which the enzyme can be fixed.

Therefore, the present invention

Preparing PEG hydrogel beads; And

The present invention relates to a method for preparing a hydrogel having an interpenetrating structure, by adding polyacrylamide to the PEG hydrogel beads to prepare a hydrogel of an interpenetrating polymer network.

Preparing the PEG hydrogel beads,

Preparing a polyethylene glycol-containing precursor solution; And

Preferably, the precursor solution is exposed to a UV light source to induce radical polymerization.

The polyethylene glycol-containing precursor solution is prepared by dissolving solid PEG-DA using distilled water or a buffer solution as a solvent.

The polyethylene glycol is preferably contained in 10 to 50 parts by weight with respect to 100 parts by weight of the solvent in order to form a hydrogel bead having a suitable moisture content and lattice size.

In addition, it may further include a photoinitiator to induce photopolymerization of the polyethylene glycol.

As the photoinitiator 2,2-dimethoxy-2-phenylacetophenone (DMPA), 2-hydroxy-2-methylpropiophenone (2-hydroxy-2-methylpropipphenone, HOMPP ), Or Acuracure 2959 (Irgacure 2959) and the like can be used alone or two or more.

The photoinitiator is preferably included in 0.001 to 0.02 parts by weight based on 1 part by weight of polyethylene glycol in consideration of the content of polyethylene glycol contained in the precursor solution.

In addition, the precursor solution prepared from the above step, as shown in Figure 2, the lower end of the area is a black opaque area to prepare a transparent measuring cylinder that does not transmit UV light, and the mineral to the measuring cylinder A certain amount of oil is filled, the UV light source is placed in an appropriate position, and then dropped into a measuring cylinder using a micro pipette to induce radical polymerization according to UV irradiation to prepare a hydrogel.

The mineral oil gradually lowers the precursor solution to secure the exposure time to UV for 2 to 4 seconds, more preferably for 3 seconds, and serves to make the shape of the precursor solution bead form. The hydrogel surface is hydrophilic and the mineral oil is hydrophobic. This is based on the principle that the hydrophilic material and hydrophobic material are separated so that the interface energy increases when the hydrophilic material and hydrophobic material meet.

In addition, the lower end of the measuring cylinder is a black opaque region, which prevents the UV light source from penetrating, and to prevent further exposure of the gel beads that have already been exposed to the light source.

In addition, the size of the hydrogel beads may vary depending on the amount of the precursor solution added to the mineral oil is not particularly limited, according to an embodiment of the present invention may have a diameter of 1 to 2mm. In order to obtain beads of this size, the content of the precursor solution may be added from 1 to 20 μl.

The hydrogel beads prepared from the above step may be washed with ethanol and distilled water to wash off residual substances and oils.

In addition, the method for preparing IPN hydrogel from the PEG hydrogel beads prepared above

Mixing PEG hydrogel and polyacrylamide to induce a crosslinking reaction; And

It is preferable to include a step of irradiating UV after the reaction is completed.

Polyethylene glycol is added to the polyacrylamide solution and left for at least 24 hours to induce a crosslinking reaction.

The polyacrylamide provides a denser net structure in proportion to the amount added to the PEG hydrogel beads. Therefore, it is preferable to include 10 to 40 parts by weight based on 100 parts by weight of PEG hydrogel to maintain a moisture content of 70% or more.

In addition, a crosslinking agent may be further added to assist in crosslinking the two materials.

As the crosslinking agent, triethylene glycol dimethacrylate or N, N'-methylenebisacrylamide (N, N'-methylenebisacrylamide) may be used alone or in combination of two or more.

The crosslinking agent may be included in an amount of 0.005 to 0.002 parts by weight based on 1 part by weight of PEG hydrogel in order to minimize the unreacted residue of the PEG hydrogel and polyacrylamide.

In addition, by adding an additional 0.001 to 0.002 parts by weight of the photoinitiator with respect to 1 part by weight of polyacrylamide to the polyacrylamide solution, the photopolymerization by the UV light source can be induced after the cross reaction.

The photopolymerization is carried out by removing the cross-reacted hydrogel beads from the polyacrylamide-containing solution in the above step, and then irradiating the UV light source for 1 to 10 minutes, thereby retaining the advantages of PEG hydrogel and improving mechanical strength. IPN hydrogel beads of the present invention are formed.

The present invention also relates to a hydrogel of an interpenetrating polymer network prepared according to the method for producing a hydrogel of the present invention.

The hydrogel has a lattice size of 10 to 50 mm 3, a diameter of 1 to 2 mm, and a water content of 70% or more.

The hydrogel of the interpenetrating polymer network of the present invention has a dense structure because the molecular chain of PAAm crosses and binds to the PEG hydrogel's net while maintaining the PEG structure of the PEG hydrogel. Strength is improved. That is, it is more difficult to break the network structure of the denser IPN hydrogel than to break the network structure of the less dense PEG hydrogel.

In addition, the amine groups of PAAm can covalently bind proteins, allowing the enzyme to be permanently immobilized on hydrogel beads for a long time.

In addition, despite the fact that the net structure is densified, the water content of the IPN hydrogel can be maintained at 70% or more, compared to the PEG hydrogel, thereby maintaining the advantages of the hydrogel. .

The invention also

Preparing a hydrogel of an interpenetrating polymer network of the present invention;

Activating the functional group of the hydrogel; And

The present invention relates to a method for immobilizing an enzyme on a hydrogel, the method comprising immobilizing an enzyme by inducing covalent bonding of an activated hydrogel with an enzyme.

In order to fix the enzyme on the hydrogel, the amine group (NH ₂ ) of the hydrogel is activated, and then the hydrogel is added to the enzyme solution to chemically covalently bond the amine group and the enzyme to fix the enzyme.

It is preferable to use glutaraldehyde as a substance for activating the amine group of the hydrogel.

The activating material is present in the solution to activate the amine group of the hydrogel, it is preferable that the solution prepared by adding 1 to 3 parts by weight of the activating material to the buffer solution.

The enzyme is not particularly limited, and according to one embodiment of the present invention, glucose oxidase may be used.

The enzyme immobilized on the IPN hydrogel of the present invention has a high relative activity depending on the content of polyacrylamide, and the decrease in the relative activity of the enzyme decreases with time after fixation, so that the IPN hydrogel of the present invention is PEG hydrogel. It can be seen that the enzyme fixation ability is significantly improved compared to the gel.

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 Preparation of Hydrogel Beads of Interpenetrating Polymer Network

Dissolve 0.5 parts by weight of solid PEG-DA per 1 part by weight of distilled water, and 5 µl of photoinitiator per 1 mL of PEG-DA (2,2-dimethoxy-2-phenylacetophenone (DMPA) in 1 mL of 1-vinyl-2-pyrrolidinone). Prepared by adding 300mg) and mixed well to prepare a precursor solution.

A measuring cylinder having a lower end as an opaque region was prepared, 20 mL of a dense oil and 40 mL of a small oil were filled with mineral oil, and a UV light source was placed at an appropriate position. After doing this, 10 μl of the precursor solution was dropped into the measuring cylinder by using a micro pipette.

Afterwards, ethanol and distilled water were used to wash off the remaining substances and oils.

The hydrogel beads were immersed in 10% acrylamide solution by weight with 1% crosslinking agent (triethylene glycol dimethacrylate, TEGDMA) to crosslink between 0.5% photoinitiator and PEG and AAm by weight, and left for 24 hours.

After removing the hydrogel beads from the solution and irradiated with a UV light source for about 5 minutes to become IPN hydrogel beads, washed with distilled water to remove the unreacted residue.

<Examples 2 to 4 and Comparative Example 1> Preparation of a hydrogel of interpenetrating structure according to the polyacrylamide concentration

Hydrogel beads of interpenetrating structure were prepared using the same method as in Example 1, except that polyacrylamide content was added at 20, 30, and 40%, respectively.

As a comparative example, PEG hydrogel without polyacrylamide was used.

(Moisture content measurement)

The moisture content is determined according to the following formula.

×100Moisture content =

× 100

PEG hydrogel prepared by UV polymerization in Example 1 is the same as FIG. In addition, the PEG hydrogel prepared in Examples 1 to 4 has a diameter of about 1 to 2 mm, and when the beads are immersed in a PAAm solution to form an IPN hydrogel, the distance between the water content (WC) and the crosslinking point of the polymer ( Grid size is shown in Table 1.

As shown in Table 1, although the lattice size of the PEG IPN hydrogel is denser than the PEG hydrogel, it can be seen that the water content is maintained at 70% or more, although it may be restricted to entering and exiting water molecules.

(Verification of IPN Hydrogel Formation)

Formation of PEG hydrogels was verified using Raman spectroscopy.

As shown in FIG. 4, when the wavenumber 1630cm ^-1 part is seen, it can be seen that the peak before the UV exposure is almost disappeared after the UV exposure. This is because the photopolymerization occurs while being exposed to UV, thus absorbing C = C double bonds. It is reduced. This is because most of the double bonds are reacted and consumed through the first crosslinking. Thus, hydrogels were prepared. Therefore, when the PAAm hydrogels are crosslinked inside the PEG hydrogels, the bonds between the two hydrogels are not chemical bonds but two hydrogels. This means that the networks are physically intertwined.

In addition, Figure 5 shows the changes in the mass of the polymer according to the PEG hydrogel becomes IPN hydrogel, ● and ○ are the percent by weight of the IPN hydrogel according to the PAAm concentration (PAAm in the mass of water + PEG + PAAm Occupancy rate) increase and the PAAm mass fraction (the ratio of PAAm in PEG + PAAm) in the IPN hydrogel (see left y-axis, right y-axis).

It can be seen that as the PAAm concentration increases, the mass percentage of PAAm in the IPN hydrogel increases linearly, and the change in the mass fraction of PAAm is rapidly changed in the early stage, and the increase is gradually decreased.

In addition, it is possible to verify the formation of the IPN hydrogel using an infrared spectrophotometer, as shown in Figure 6, it can be seen that the new wavelength of 1653cm ^-1 and 1540cm ^-1 , and C = O stretching peak and NH bending peak. This proves that PAAm hydrogel is formed inside and outside the PEG hydrogel, and demonstrates that the enzyme can be chemically covalently bonded through the formed amine group.

(Mechanical strength measurement)

The mechanical strength of hydrogels is of great biological importance. This is because when hydrogels are applied to a liquid environment, the mechanical strength may drop or lose while absorbing large amounts of water. Therefore, the mechanical strength of the IPN hydrogel was verified through a tensile tester.

To this end, IPN hydrogel specimens were dumbbell-shaped and fixed in a tensile tester, and then a tensile load was gradually applied to the tensile test specimens to measure mechanical properties of the material. The stress generated by the tensile load is called tensile stress. A stress-strain diagram representing the relationship between tensile stress and elongation is an important indicator of the properties of the material. The stress at the maximum point in this diagram is called the maximum tensile strength. The tensile test can always be carried out under the same conditions, so the reliability of the measurement results is high, and the mechanical strength was measured by averaging several values.

As shown in FIG. 7, the graph shows that the PEG and IPN hydrogels are stressed and elongated and then broken, and the breaking point means the maximum tensile strength. Compared to PEG hydrogels, IPN hydrogels and IPN hydrogels also have excellent mechanical strength of hydrogels having high PAAm concentrations, and particularly, when the PAAm concentration is 40%, the mechanical strength is significantly improved.

Example 5 Enzyme Fixation Using Hydrogel Beads of Interpenetrating Polymer Network

In order to activate the amine group (NH ₂ ) of PAAm of the hydrogel beads prepared in Example 1, glutaaldehyde (glutaraldehyde) corresponding to 2.5% of the volume of the buffer solution was added to prepare a solution. IPN hydrogel was immersed in the solution for 2 hours at room temperature and washed with a buffer solution after 2 hours. IPN hydrogel beads were immersed at 4 ° C. for 24 hours in an enzyme solution (hereinafter referred to as GOx solution) —a solution made by adding 1 mg of glucose oxidase (GOx) per 1 mL of buffer. After 24 hours, the beads were washed with distilled water.

The procedure was carried out using PEG hydrogel beads as a control.

 (Relative activity investigation)

The activity of the immobilized enzyme was confirmed by measuring spectrophotometer of the discoloration of the dye called o -dianisidine. The overall reaction is divided into two stages: in the first stage, glucose is broken down into gluconic acid and hydrogen peroxide by the catalysis of glucose oxidase, and in the second stage, hydrogen peroxide is converted to water by peroxidase. The dyeing material is oxidized to become discolored. At this time, since the oxidized dye material absorbs light in the 405 nm region, the absorbance can be measured using a spectrophotometer to measure the activity of the enzyme.

As described above, since the IPN hydrogel has a chemically amine group, the enzyme can chemically covalently bond to the hydrogel so that the IPN hydrogel beads can play a sufficient role as the enzyme fixation support.

As shown in Figure 8, the relative activity immediately after immobilization of GOx to the hydrogel shows a similar pattern with PEG, IPN hydrogel.

However, it can be seen that the aspect after 3 days is completely different. As shown in FIG. 9, GOx is attached to or permeated into the surface of the hydrogel, while PEG hydrogel cannot covalently bind enzymes, while most of the enzymes are physically immobilized inside the hydrogel. Is fixed to the hydrogel surface and interior through covalent bonds. Therefore, IPN hydrogel was able to maintain relatively high enzyme activity even after 3 days. In addition, higher PAAm concentration indicates higher activity, indicating that PAAm effectively covalently binds the enzyme.

Meanwhile, the result of measuring the relative activity of the hydrogel in which the enzyme is fixed for 6 days for each time zone is shown in FIG. 10.

As for PEG hydrogels, the enzymes immobilized rapidly after 1 day of enzymatic immobilization, resulting in a significant decrease in activity. However, IPN hydrogels showed less activity decrease over time than PEG hydrogels, especially 40% PAAm concentration. Phosphorus IPN hydrogel shows high activity for a long time.

Figure 1 shows the sequential process of IPN hydrogel production of the present invention.

Figure 2 shows a schematic diagram of a manufacturing method of the IPN hydrogel of the present invention.

Figure 3 is a photograph of PEG hydrogel made through UV polymerization according to Example 1 of the present invention.

Figure 4 shows the PEG-DA (molecular weight 3400) Raman spectroscopic graph before and after UV exposure.

Figure 5 shows the change in mass fraction of the IPN hydrogel of the present invention according to the polyacrylamide concentration.

Figure 6 shows the analysis graph of the infrared spectrophotometer of the PEG hydrogel and IPN hydrogel of the present invention.

Figure 7 shows a graph of the stress-stretch relationship of PEG, IPN hydrogel according to the polyacrylamide concentration.

Figure 8 shows the relative activity immediately after fixation of glucose oxidase (GOx) on the hydrogel of the present invention.

Figure 9 shows the relative activity of the hydrogel fixed GOx measured after 3 days.

Figure 10 shows the relative activity of the enzyme-fixed hydrogel over time.

Claims (17)

Preparing PEG hydrogel beads; And Method of producing a hydrogel of interpenetrating structure comprising the step of preparing a hydrogel of interpenetrating polymer network by adding polyacrylamide to the PEG hydrogel beads. The method of claim 1, Preparation of PEG hydrogel beads Preparing a polyethylene glycol-containing precursor solution; And Method of producing a hydrogel of interpenetrating structure comprising the step of inducing radical polymerization by exposing the precursor solution to a UV light source. The method of claim 2, A polyethylene glycol-containing precursor solution is prepared by adding polyethylene glycol to a solvent of any one of distilled water or a buffer solution. The method of claim 3, Polyethylene glycol is a method for producing an interpenetrating hydrogel, characterized in that contained in 10 to 50 parts by weight with respect to 100 parts by weight of the solvent. The method of claim 2, A method for producing an interpenetrating hydrogel, comprising adding a precursor solution to a measuring cylinder containing mineral oil and exposing it to a UV light source. The method of claim 2, Method for producing a hydrogel of interpenetrating structure, characterized in that it further comprises 0.001 to 0.02 parts by weight of a photoinitiator with respect to 1 part by weight of polyethylene glycol to induce photopolymerization of polyethylene glycol. The method of claim 6, Photoinitiators include 2,2-dimethoxy-2-phenylacetophenone (DMPA), 2-hydroxy-2-methylpropipphenone (HOMPP) And agacure 2959 (Irgacure 2959). A method for producing an interpenetrating hydrogel, characterized in that at least one member selected from the group consisting of. The method of claim 1, The step of preparing the interpenetrating hydrogel Mixing PEG hydrogel and polyacrylamide to induce a crosslinking reaction; And Method of producing a hydrogel of the interpenetrating structure, characterized in that it comprises the step of irradiating UV after the reaction. The method of claim 1, Polyacrylamide is a method for producing an interpenetrating hydrogel, characterized in that added to 10 to 40 parts by weight based on 100 parts by weight of PEG hydrogel. The method of claim 8, A method for producing an interpenetrating hydrogel, further comprising adding a crosslinking agent for crosslinking the PEG hydrogel and the polyacrylamide. The method of claim 10, The crosslinking agent is at least one member selected from the group consisting of triethylene glycol dimethacrylate and N, N'-methylenebisacrylamide (N, N'-methylenebisacrylamide). The method of claim 10, A crosslinking agent is added in an amount of 0.005 to 0.002 parts by weight based on 1 part by weight of PEG hydrogel. A hydrogel of an interpenetrating polymer network prepared according to any one of claims 1 to 12. The method of claim 13, The hydrogel has a lattice size of 10 to 50 mm 3. Preparing a hydrogel of an interpenetrating polymer network according to any one of claims 1 to 12; Activating the functional group of the hydrogel; And A method of immobilizing an enzyme on a hydrogel, comprising the step of immobilizing an enzyme by inducing covalent bonding of an activated hydrogel with an enzyme. The method of claim 15, A method for immobilizing an enzyme on a hydrogel, wherein the substance activating the functional group is glutaaldehyde. The method of claim 15, The enzyme is a method of immobilizing the enzyme on a hydrogel, characterized in that the glucose oxidase.

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