KR100742421B1 - Method of enzyme immobilization on nanofiber web using electrospinning - Google Patents

Abstract

By immobilizing the enzyme using nanofibers, an enzyme is uniformly distributed and an immobilization method of an enzyme having high fixation efficiency is provided. In an embodiment of the present invention, a polymer solution such as polyvinyl alcohol is electrospun to obtain a sheet composed of nanofibers having a three-dimensional network structure, and the enzyme solution is dropped on a desired site of enzyme fixation, and then on the dropped enzyme solution. The enzyme is fixed by covering the sheet to entrap the enzyme and then drying. In another embodiment of the present invention, the enzyme may be immobilized by mixing the enzyme with the polymer solution, followed by stirring, and electrospinning the polymer solution containing the enzyme onto the desired site for the enzyme fixation. Alternatively, the enzyme solution may be applied to a desired site for enzyme fixation, followed by drying, followed by electrospinning the polymer solution to selectively fix the enzyme at the desired site. According to the present invention, since the enzyme is immobilized in the form of the enzyme penetrated to the nanofibers, the enzyme may be uniformly fixed.

Enzyme, immobilized, nanofiber, electrospinning, web, electrode

Description

Method of enzyme immobilization on nanofiber web using electrospinning}

1 is a view showing the application of the enzyme solution on the electrode of the hydrogel patch substrate

FIG. 2 is a view illustrating attaching a nanofiberized polymer sheet on an electrode to which an enzyme solution is applied. FIG.

Figure 3 is a photograph showing the appearance of a nanofiber web prepared using electrospinning.

Figure 4 is a photograph showing the appearance of the enzyme immobilized on the nanofiber web by applying the method of the present invention.

Figure 5 is a graph showing the response characteristics of the electrochemical biosensor for each concentration of the recognition material measured by fixing the enzyme using the nanofiber web according to the present invention.

<Description of Symbols for Main Parts of Drawings>

10 Hydrogel patch substrate 11 electrode

20 enzyme solution 30 polymer sheet

40 pipettes.

The present invention relates to a method for fixing an enzyme on a nanofiber web using electrospinning, and more particularly, to a method for fixing an enzyme with a uniform distribution and good fixation efficiency by fixing an enzyme using a nanofiber.

In general, in the case of preparing a target substance by using an enzyme, the use of a water-soluble enzyme has a disadvantage of low stability to temperature and pH, and difficult to separate from the reactant or product, and thus cannot be used repeatedly. The research to be continued.

For example, Korean Patent Publication No. 1999-028884 discloses a hydrogel patch for extracting glucose from human skin by electrochemical reaction by immobilizing an enzyme on a hydrophilic gel. As a method of immobilizing an enzyme on a carrier in a gel form, Korean Patent Publication Nos. 199-0005581, 1990-0002839, and the like are known.

However, when the enzyme is immobilized on a hydrophilic gel such as the hydrogel patch disclosed in Korean Patent Publication No. 1999-028884, the more uniformly distributed the enzyme is in the gel, the more uniform the reaction between the enzyme and the reactant can be obtained. The variation of the reaction result can be reduced. However, the enzyme fixation method according to the conventional method has a limitation in uniformly distributing the enzyme in the gel, and since the volume of the hydrophilic gel decreases as the reaction proceeds, it adversely affects the uniform distribution of the enzyme.

This invention is made | formed in view of this point, Comprising: It aims at providing the fixing method of the enzyme which can distribute an enzyme uniformly, and has a high fixed efficiency.

In an embodiment of the present invention, a polymer solution such as polyvinyl alcohol is electrospun to obtain a sheet of a three-dimensional network structure made of nanofibers, and the enzyme solution is dropped on a site where enzyme is to be fixed, and then on the dropped enzyme solution. The enzyme is fixed by covering the sheet to entrap the enzyme and then drying. In another embodiment of the present invention, the enzyme may be immobilized by mixing the enzyme with the polymer solution, followed by stirring, and electrospinning the polymer solution containing the enzyme onto the desired site for the enzyme fixation. In another embodiment of the present invention, the enzyme solution is applied to a desired portion of the enzyme fixation and dried to fix the enzyme by electrospinning the polymer solution. When electrospinning directly to the site where the enzyme is to be fixed, it is preferable to electrospin while the site where the enzyme is to be fixed is grounded to the ground of the electrospinning machine.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings.

In the present invention, the polymer solution is electrospun to obtain a sheet of a three-dimensional network structure made of nanofibers, or the polymer solution is selectively electrospun at a desired site for enzyme fixation.

As the polymer solution, generally used polymers such as polyvinyl alcohol (PVA), polyacrylonitrile, nylon, cellulose and the like can be used.

One example of a method of preparing the polymer solution used in the present invention is as follows. First, a PBS solution having a concentration of 1 M , pH5.1 to 7.4 is prepared. pH7.4 in PBS solution is then made so that the mixed solution of K ₂ HPO ₄ pH7.4 to a KH ₂ PO ₄ solution and 1 1 M concentration of M, can be prepared by mixing a 0.14 M NaCl. Then, a polymer capable of forming a chain such as PVA or polyacrylonitrile, nylon, cellulose, or the like is added to the PBS solution so as to have a weight concentration of 5 to 35 wt%, preferably 10 wt%, and then room temperature, preferably 24 degrees Celsius. A polymer solution can be obtained by stirring and dissolving at a temperature of about 25 degrees for a certain time, for example, 3 to 4 hours.

Techniques for forming mesh nanofibers using electrospinning are widely known (for example, Korean Patent Publication No. 10-2005-0032656). As the electrospinning apparatus used to electrospin the polymer solution in the present invention, a general electrospinning apparatus capable of applying a high voltage (5 to 50 kV) may be used.

The applied voltage range during radiation is preferably performed at 5 to 35 kV, more preferably 15 to 25 kV. The distance between the spinneret and the integrated plate is 5 to 30 cm, more preferably 10 to 15 cm. Spinning time is preferably 2 to 6 hours. 3 is a photograph of the state of the nanofiber web obtained by electrospinning.

In the present invention, the immobilization of the enzyme may be performed by dropping the enzyme solution at the site where the enzyme is to be immobilized, and then covering the sheet of the three-dimensional network structure of nanofibers formed using the polymer solution thereon, or the enzyme solution is The mixed polymer solution can also be carried out by electrospinning directly to the site where enzyme immobilization is desired. Alternatively, it is possible to drop the enzyme solution at the site where the enzyme is to be immobilized and to dry it, and then to selectively electrospin the polymer solution thereon. Hereinafter, each method will be described in detail.

<Example 1>

In Example 1, the enzyme is immobilized by dropping the enzyme solution at a desired site for enzymatic immobilization, then encapsulating the enzyme by covering the nanofiber web sheet of nanofibers formed using a polymer solution thereon and drying the enzyme.

First, the above-described polymer solution is electrospun according to the above-described conditions to obtain a nanofiber web sheet composed of nanofibers. The nanofiber web sheet thus obtained is cut to an appropriate size according to the intended use. When the enzyme is to be immobilized on the hydrogel patch disclosed in Korean Patent Publication No. 1999-028884, the size of the polymer sheet may be equal to or slightly larger than the area of the patch.

Then, the enzyme solution is dropped to the site where the enzyme is to be fixed. For example, in the case of the hydrogel patch disclosed in Korean Patent Laid-Open Publication No. 1999-028884, as shown in FIG. 2, the enzyme solution 20 is formed on the electrode 11 formed on the substrate 10 on which the hydrogel patch is placed. Is applied using a pipette 40 or a dispenser such as an airbrush. Alternatively, it is possible to drop the enzyme solution onto the hydrogel patch instead of the electrode.

The concentration of the enzyme solution may be appropriately adjusted depending on the purpose of use. On the other hand, the Republic of Korea concentration of the enzyme if it is desired to hold the enzyme in the hydrogel patch disclosed in Patent Publication No. 1999-028884 is preferably 5 ~ 50m M. In addition, the amount of the enzyme solution to be used may be appropriately adjusted according to the area of the desired site for the enzyme fixation or the size of the nanofiber web sheet, and in the case of the hydrogel patch described above, about 5 to 20 μl is preferable.

Thus, the enzyme solution 20 is dropped to the desired site 11 to fix the enzyme. Then, the enzyme solution is covered with the nanofiber web sheet 30 cut to an appropriate size as shown in FIG. Entrap and dry.

Since the nanofiber web sheet is a nanofiber, the enzymatic solution penetrates uniformly into the crosslinker sheet, and as the enzymatic solution is dried, the nanofiber web sheet is adhered to the site where the enzyme is to be immobilized by the polymer component of the nanofiber web sheet. The enzyme is fixed uniformly at the desired site.

<Example 2>

In Example 2, the enzyme is fixed by electrospinning the polymer solution in which the enzyme solution is agitated directly to the site where the enzyme is to be fixed.

First, the enzyme is dissolved in the above-mentioned polymer solution, while the concentration is stirred for 5 to 30 minutes such a period of time, for example at room temperature, preferably at a temperature of about 20 ° C such that after loading the 50m M.

The polymer solution including the enzyme thus produced is electrospun to the site where the enzyme is to be immobilized in accordance with the above-described spinning conditions so that the polymer nanofiber web sheet is formed at the site where the enzyme is to be immobilized.

At this time, since the nanofiber web sheet may be formed in addition to the desired portion, the sheet formed on the unnecessary portion is removed. Alternatively, when the electrospinning is performed while the part to which enzyme is desired to be fixed is grounded to the ground of the electrospinner, the nanofiber web sheet is formed only at the grounded part, and thus it is not necessary to remove the unnecessary nanofiber web sheet.

In this way, the polymer solution containing the enzyme is formed in the form of nanofibers at the site where the enzyme is to be immobilized, and the enzyme can be fixed evenly according to the characteristics of the nanofibers.

<Example 3>

In Example 3, the enzyme solution is applied to the desired site for enzyme fixation and dried, and then the enzyme is fixed by electrospinning the polymer solution directly to the desired site for enzyme fixation.

First, as shown in FIG. 1, the enzyme solution 20 as described above is dropped to the site 11 to which enzyme is to be immobilized. And the object (electrode in the example of FIG. 1) to which the enzyme solution was apply | coated is dried at room temperature.

Then, the polymer nanofiber web sheet is formed at the site where the enzyme is fixed by electrospinning the polymer solution to the site where the enzyme solution is dried and the enzyme is fixed according to the above-described spinning conditions.

In this case, since the nanofiber web sheet may be formed in addition to the desired portion, the nanofiber web sheet formed on the unnecessary portion is removed. Alternatively, when the electrospinning is performed in a state where the part to which enzyme is desired to be fixed is grounded to the ground of the electrospinner, the nanofiber web sheet is formed only at the grounded portion, and thus it is not necessary to remove the unnecessary nanofiber web sheet.

In this way, the polymer solution containing the enzyme is formed in the form of nanofibers at the site where the enzyme is to be immobilized, and the enzyme may be uniformly adsorbed and fixed on the nanofiber web according to the characteristics of the nanofiber.

Figure 4 is a photograph of a state in which the enzyme is fixed according to the method of the present invention. As can be seen in Figure 4, according to the present invention, the enzyme is immobilized in a form in which the enzyme is adsorbed to the nanofiber of the three-dimensional network structure made of polymer, so that the enzyme is uniformly and firmly fixed.

Figure 5 is a graph showing the change over time of the reaction current when the glucose extraction reaction using the enzyme-fixed hydrogel patch according to the method of the present invention, x-axis is the passage of time, y-axis of the enzyme over time It represents the intensity of the current generated by the electrochemical reaction. That is, the graph shows the change over time of the current value of the oxidation-reduction electrochemical reaction between the enzyme and the recognizer according to the concentration of the enzyme. In FIG. 5, the maximum current value means that the enzyme reacts with the recognition material. The larger the slope of the current decreases, the better the response characteristic of the enzyme to the recognition material. In other words, the maximum current value during the reaction and the sudden decrease in the intensity of the current at the end of the reaction means that the enzymes are uniformly distributed and all the enzymes react at the same time. On the other hand, when the enzymes are not evenly distributed, some of the enzymes react late and do not show a sharp slope. In some cases the concentration of the enzyme in 10m M 5, but a sharp slope of the slope is the concentration of the enzyme Beyond the 50m M dull can be seen that.

As mentioned above, although this invention was demonstrated to the several example, this invention is not limited to a specific Example. Those skilled in the art will appreciate that various changes and modifications can be made without departing from the spirit of the invention.

According to the present invention, the enzyme is uniformly and firmly fixed, and according to the inherent characteristics of the nanofibers formed by microfibers of nano units, it is expected to improve the fixing efficiency of the enzyme and increase the maintenance period of the enzyme activity.

In addition, by ground-grounding and electrospinning a desired site for enzyme fixation, it is possible to form a nanofiber web sheet in which the enzyme is selectively fixed only at the desired site. In addition, when used in a hydrogel patch can reduce the problems of the prior art that results in non-uniform reaction by deformation of the hydrogel due to the evaporation of water.

Claims (11)

At least one selected from polymers including polyvinyl alcohol, polyacrylonitrile, nylon or cellulose in a PBS solution having a concentration of 1 M and pH 5.1 to 7.4 in a PBS solution so as to have a weight concentration of 5 to 35wt%. Electrospinning the polymer solution produced by stirring and dissolving for a predetermined time at to obtain a web sheet of a three-dimensional network structure composed of nanofibers, And dropping an enzyme solution having a concentration of 5 to 50 m M in the desired enzyme stage fixing portion, Covering the nanofiber web sheet on the dropped enzyme solution to entrap the enzyme and then drying Fixing method of the enzyme having a. delete delete delete delete And the step of applying the enzyme solution at a concentration of 5 to 50 m M in the desired area the enzyme fixed, Drying the applied enzyme solution at room temperature, At least one selected from polymers including polyvinyl alcohol, polyacrylonitrile, nylon or cellulose in a PBS solution having a concentration of 1 M and pH 5.1 to 7.4 in a PBS solution so as to have a weight concentration of 5 to 35wt%. Electrospinning the polymer solution produced by dissolving by stirring for a predetermined time at a desired site for the enzyme fixation Fixing method of the enzyme having a. delete delete delete The method according to claim 1 or 6, The site where the enzyme is to be fixed is an enzyme fixing method, characterized in that the electrode. The method according to claim 1 or 6, The site where the enzyme is to be fixed is a method for fixing an enzyme, characterized in that the hydrogel patch.

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