KR20130084377A - Method of enzyme immobilization on electrode surface, enzyme sensor thereof and biosensor comprising the same - Google Patents

Abstract

PURPOSE: An enzyme fixation method is provided to fix biocatalysts such as an enzyme on the surface of an electrode by simple and effective electrodeposition (ED) and to have long-term enzyme activity and stability on the surface of the electrode. CONSTITUTION: An enzyme fixation method comprises the steps of: applying predetermined applied voltage to a mixture solution containing chitosan, an electron carrier, an enzyme, and a buffer solution; and electrodeposition by forming a complex layer of the chitosan and the enzyme on an electrode. The method also comprises the step of fixing the complex layer through adsorption by providing a predetermined quiet time before electrodeposition. An enzyme electrode is prepared by fixing the enzyme on the electrode and comprises the complex layer fixed in a thickness of 100-1000 nm. A biosensor contains the enzyme electrode on the electrode.

Description

Enzyme immobilization method, enzymatic sensor and biosensor comprising the same {Method of enzyme immobilization on electrode surface, Enzyme sensor about and Biosensor comprising the same}

The present invention relates to an enzyme fixation method, an enzyme sensor and a biosensor comprising the same. More specifically, the enzyme fixation method for fixing a large amount of enzyme to the electrode surface by a simple method, an enzyme sensor and a bio-containing device Relates to a sensor.

The basic feature of the bioelectrode device is the use of an electron transfer mechanism of a biofunctional group bonded to a biomatrix or a biomaterial fixed on a conductor or a semiconductor electrode. Immobilization of biomaterials such as redox enzymes on the electrode surface includes simple chemical physisorption, polymer solution casting, sol gel technique, covalent bonding, and crosslinking.

Recently, biosensors based on the Electrodeposition (ED) principle have been introduced, which have high selectivity and sensitivity and can be used without any pretreatment even if the sample is turbid and can accurately measure within a short time. Such biosensors generally use ferrocene, ferrocene derivatives, quinones, quinone derivatives, organic conducting salts, or viologens as electron transporters.

The electrochemical principle of the biosensor is as follows.

Glucose + GOx -FAD Glucose + GOx -FADH2

GOx -FADH2 + electron transporter (oxidized state) GOx -FAD + electron transporter (reduced state)

In the above scheme, GOx represents glucose oxidase, and GOX-FAD and GOX-FADH2 represent the oxidation state and reduction state of FAD (flavin adeninedi nucleotide), which are active sites of glucose oxidase, respectively.

By the catalysis of glucose oxidase, glucose is oxidized to glucose acid. At this time, FAD which is an active site of glucose oxidase is reduced to FADH2. The reduced FADH2 undergoes a redox reaction with the electron transporter, whereby the FADH2 is oxidized to FAD and the electron transporter is reduced. The reduced electron carrier diffuses to the surface of the electrode. At this time, the current generated by applying the oxidation potential of the reduced electron carrier is measured.

In the operation of such a biosensor, a method of uniformly distributing a large amount of oxidase on the electrode surface and immobilizing has been studied. However, there is still a need for a biosensor manufacturing method in which an enzyme is immobilized on an electrode surface efficiently and stably by a simpler process.

The present invention provides a method for immobilizing an enzyme on an electrode surface efficiently and stably by a simple process.

The present invention is to provide an enzyme electrode and a biosensor, the enzyme is maintained at a large amount on the electrode surface for a long time to improve the sensitivity.

One aspect of the present invention includes an electrodeposition step of forming a complex layer of chitosan and enzyme on an electrode by applying a predetermined applied voltage to a mixed solution containing chitosan, an electron carrier, an enzyme, a buffer solution,

The method relates to an enzyme fixation method for forming on an electrode comprising providing a predetermined wait time prior to the electrodeposition step to immobilize a complex layer of chitosan and enzyme on the electrode by adsorption.

Another aspect of the invention relates to an enzyme electrode and a biosensor comprising a chitosan-redox enzyme complex layer fixed at a thickness of 100-1000 nm on a working electrode.

The production method according to the present invention can fix biocatalysts such as enzymes to the electrode surface by a simple and effective electrodeposition method. In addition, according to the present invention, since a large amount of enzyme is fixed uniformly, and enzyme activity and stability on the electrode surface are maintained for a long time, it can be used repeatedly. Therefore, the biosensor manufactured by the present invention can improve the stability and sensitivity of the sensor.

1 is a schematic diagram of an electrode system formed by screen printing on a substrate in a reaction unit.
Figure 2 is a SEM image of the enzyme fixed on the electrode by the present invention.
3 is a graph showing the distribution value of the (catalyst) current flow according to the concentration of chitosan.
Figure 4 is a graph showing the distribution value of the (catalyst) current flow with increasing GOx concentration.
5 is a graph showing the distribution value of the current flow according to the phosphate buffer concentration.
6 is a graph showing the current flow after electrodeposition according to the pH change of the mixed solution while changing the pH of McIlvaine buffer to 3-6, respectively.
7 is a curve showing the current flow according to the waiting time.
8 is a graph illustrating a current flow according to an applied voltage.
9 is a graph showing the catalytic current flow of the electrodes (GOx) is fixed with each electrodeposition time.
Figure 10 (a) is not injected with glucose (b) is injected with 30mM glucose, (c) is a case of injecting 30mM glucose into the GOx aqueous solution to the SPCE electrode does not fix the enzyme Cyclic voltammograms are measured.
(A) of FIG. 11 is a graph in which cyclic voltammograms are measured when 0.12 mM H2O2 is injected when (b) is not injected.
12 shows (a) when nitrite (Nitrite) is not injected into a NiC-fixed SPCE electrode (b) when 0.1mM nitrite is injected, and (c) circulating voltage current after injection of 0.5mM nitrite (Nitrite) (Cyclic voltammograms) is a graph measured.
FIG. 13 shows cyclic voltage currents (Cyclic) for the case where (a) nitrite was not injected into the SPCE electrode fixed with NiR and CHIT-V obtained in Example 11, and (b) 0.5 mM nitrite was injected. voltammograms)

The present invention is an enzyme fixation method comprising an electrodeposition step of forming a complex layer of chitosan and enzyme on an electrode by applying a predetermined applied voltage to a mixed solution containing chitosan, an electron carrier, an enzyme, a buffer.

The present invention can use a conventionally known electrodeposition system. There is no particular limitation on the electrode system, but a three-electrode system including a working electrode, a counter electrode, and a reference electrode may be used. In the electrode system, the electrodes may be formed on one substrate and may be formed on two to three substrates, respectively.

1 is a schematic diagram of an electrode system formed by screen printing on a substrate in a reaction unit. Referring to FIG. 1, the electrode system 1 is a screening-printing screen-printed carbon paste electrode (SPCE) electrode on an insulating substrate on a strip. counter electrode (CE), reference electrode (RE) can be formed.

The working electrode serves to sense an electric current by a reduction reaction by fixing an enzyme, and there is no particular limitation on the material, but as a carbon electrode, a carbon paste electrode or a modified carbon paste electrode is used. desirable. Carbon paste is inexpensive and can be used for single use, mass production is possible, and the enzyme immobilized there is a relatively stable advantage.

The working electrode may be a hydrophilic surface treatment. The hydrophilic surface treatment can be performed by a known method. For example, the surface of the working electrode can be subjected to oxygen plasma treatment to perform hydrophilic surface modification.

The counter electrode pairs with the working electrode to flow a current, and a material similar to that of the working electrode may be used.

In addition, the reference electrode functions to maintain a constant operating voltage between the working electrode and the counter electrode without a current flowing, and the material is mainly used Ag / AgCl.

In order to make the reference electrode, the silver paste layer may be oxidized with a saturated solution of iron chloride (FeCl 3), washed with distilled water, and dried in air.

The reaction unit 2 provides a space in which an electrochemical reaction for fixing the enzyme occurs.

In the present invention, a compound of chitosan or chitosan-electron transporter can be used. However, in the case of using a compound of chitosan-electron transporter, there is a convenient side because it is not necessary to put the electron transporter separately in the electrochemical test for detecting a target substance after the enzyme is immobilized on the electrode.

Since the method of immobilizing the enzyme on the electrode of the present invention is the same when using chitosan alone or a compound of chitosan-electron transporter, the chitosan-electron transporter is also referred to the same except in the case where chitosan is mentioned below. Seen as one.

The chitosan has biocompatibility, high affinity with proteins, and antimicrobial properties and is suitable as a substance for fixing enzymes. In addition, the chitosan film produced by the electrodeposition method can hold well the immobilized bioactive agent.

Chitin is a polysaccharide high molecular material formed by a myriad of N-acetyl-D-glucosamine units, and chitosan is obtained by deacetylation of chitin. However, deacetylation is not complete and chitosan is generally a polymer in which chitin and deacetylated chitin are mixed. The primary amine at the C-2 site of the glucosamine residue plays an important role in the reactivity and dissolution metastasis of chitosan. Under strong acid conditions, the amine is protonated to have solubility in aqueous chitosan solution, and under basic conditions, the amine is deprotonated to make chitosan insoluble. Dissolution-insoluble transition occurs at ~ pH6.3. Therefore, it is possible to electrodeposit chitosan on the electrode by locally increasing the pH near the electrode (cathode).

The weight average molecular weight of chitosan usable in the present invention may be 150,000 to 360,000. The degree of deacetylation of the chitosan which can be used in the present invention is 70 to 90%, preferably 75 to 85%.

In the present invention, an electron transporter may be used separately, or a compound (hereinafter, chitosan-electron transporter) in which an electron transporter is coupled to chitosan may be used. Preferably, the compound may synthesize an electron carrier in a backbone of chitosan by covalent bonds, ionic bonds, hydrogen bonds, or coordinative bonds, and may be more easily synthesized by covalent bonds.

The electron transporter is an inorganic or organic fine powdery compound which promotes the reduction and oxidation of enzymes, and includes ferrocene, ferrocene derivatives, quinones, quinone derivatives, organic conducting salts, and biorogen ( viologen) can be selected from the group consisting of. More specific examples of electron transporters include ferricyanated alkali metal salts (preferably potassium metal salts), ferrocene or alkyl substituents thereof, p-benzoquinone, methylene blue (MB), β-naphthoquinone 4-sulfonic acid potassium, phenazineme Tosulfate, 2, 6- dichloro phenol indophenol, methyl viologen (MV), etc. are mentioned.

In the present invention, the chitosan and the electron transporter may be preferably covalently used. When the electron transporter has a functional group such as an amine group or a carboxyl group, the chitosan may be easily covalently bonded to the chitosan.

The enzyme may be an oxidoreductase, a hydrolase, a transfer enzyme, an isomerase, and the like, but there is no particular limitation thereto.

Examples of the redox enzymes include glucose oxidase, horseradish peroxidase (HRP), nitrite reductase (NiR), cholesterol oxidase, glutamic oxalacetic transaminase (GOT) and glutamic pyruvic And Glutamic Pyruvic Transaminase (GPT).

The mixed solution may be used a buffer such as phosphate, MOPS (3- (N-morpholino) propanesulfonic acid), MES (2- (Nmorpholino) ethanesulfonic acid), McIlvaine, and more preferably phosphate. .

In the enzyme fixation method of the present invention, since a strong pH change occurs in the reaction part, a buffer is important. That is, during the electrodeposition reaction, a pH gradient occurs from the electrode surface to the bulk solution, and this change in pH affects the solubility of chitosan.

In the present invention, the concentration of the buffer solution is about 5-20 mM, preferably about 10 mM. Too weak buffering induces instantaneous electrodeposition, resulting in a less organized layer, and too strong buffering prevents the increase of pH near the electrode. This is because it induces a firm fixation of the chitosan layer.

In the enzyme fixation method, it is preferable to adjust the pH of the mixed solution to 4 to 6, preferably 5 to 5.5. The pH of the mixed solution can be adjusted to the pH of the buffer solution.

The enzyme electrode electrodeposited at a low pH concentration has a small amount of catalytic current, which results in a low pH value of the mixed solution that prevents the increase of the local pH near the electrode, resulting in a small fixed amount of chitosan and enzyme. On the other hand, the enzyme electrode electrodeposited at pH 5-6 has almost no change in the amount of catalyst current, because these pH values are close to pH 6.3, the dissolution transition point of chitosan, and the effect on pH change is relatively small.

In the present invention, a mixture of chitosan, an electron transporter or a compound of chitosan-electron transporter and an enzyme is added to a buffer at a predetermined ratio to form a mixed solution.

The mixed solution includes 0.5 to 10 mg / mL of the chitosan or chitosan-electron transporter compound, 0.1 to 5 mM of electron transporter, 2 to 10 mg / mL of enzyme, and 5 to 20 mM buffer.

The chitosan or chitosan-electron transporter compound in the solution may be in the range of 0.5 to 10 mg / mL, preferably 1 to 5 mg / mL, and most preferably 1 to 3 mg / mL.

For example, the mixed solution may be formed by including 1 to 3 mg / mL of the compound of chitosan or chitosan-electron transporter and 2 to 10 mg / mL of the chitosan or chitosan-electron transporter in 5 to 20 mM of phosphate buffer. This is because chitosan concentrations with too high a concentration of chitosan are disadvantageous in that layer aggregation occurs to trap or transfer enzymes, and too low a concentration can form an unstable enzyme layer.

Enzyme concentration, especially GOx concentration, ranges from 2 to 10 mg / mL, preferably from 6 to 10 mg / mL, where the amount of enzyme is too small, the amount of fixation is small, and too much can interfere with the fixation function as a matrix of chitosan. Because there is.

The enzyme is preferably one selected from the group consisting of glucose oxidase, horseradish peroxidase (HRP) and nitrite reductase (NiR).

On the other hand, it also has two current flow slopes in the GOx concentration range. In other words, a high concentration (tilt; 0.192) of 6-10 mg / mL shows a current flow about 6.6 times higher than the low concentration (tilt; 0.0298) range of 2-6 mg / mL.

The present invention includes a step (hereinafter, an adsorption step) of forming a complex layer of chitosan or a compound of the chitosan-electron transporter and an enzyme by adsorption for a waiting time prior to electrodeposition.

The term "quiet time" used in the present invention refers to a time when electrodeposition is started after injecting a mixed solution into a reaction part provided with an electrode.

However, the compound layer used in the present invention is a compound of chitosan or chitosan-electron transporter and an enzyme to form a composite state as described above, which is formed by adsorption or electrodeposition on the electrode. Refers to all layers.

The waiting time is less than 3 minutes, that is, more than 0 minutes and less than 3 minutes, preferably 30 seconds or more and less than 2 minutes, most preferably about 1 minute.

After one minute of waiting time, the catalytic current of the electrode with the chito-enzyme electrodeposited is four times higher than that of electrodeposition immediately without waiting time. This is because the chitosan-enzyme complex layer previously adsorbed on the electrode during the waiting time has a strong affinity for the chitosan-enzyme that is subsequently electrodeposited.

However, too much waiting time decreases the catalytic current of the electrode electrode slowly, because the longer waiting time prevents the thicker preadsorbed chitosan-enzyme complex layer from following electrodeposition.

The present invention includes an electrodeposition step of continuously forming a composite layer by electrodeposition after the fixing step by adsorption.

Applying a voltage to the electrode system generates hydrogen near the working electrode, resulting in a locally increased pH near the working electrode. Since the dissolution-insoluble transition of chitosan occurs around pH6.3, it is possible to electrodeposit chitosan on the working electrode by locally increasing the pH to above 6.3 near the working electrode (cathode).

The second fixing step is to form a composite layer on the electrode by electrodeposition, the voltage can be applied in the range of -1.8 to -2.2V, preferably -1.9 to 2.1V, most preferably about -2.0V have.

Too high an applied voltage may cause excessive hydrogen flow and interfere with the deposition of chitosan on the electrode surface, while too low an applied voltage is difficult to increase the local pH near the electrode surface.

In the electrodeposition step, the voltage application time may be within 3 minutes, preferably between 30 seconds and 3 minutes, more preferably between 30 seconds and 2 minutes, and most preferably between 1 minute and 2 minutes.

This range is preferred because the amount of GOx electrodeposited increases with time, while the thickness of the chitosan layer increases simultaneously, which acts as a barrier to mass transfer.

In another aspect, the present invention relates to an electrode in which an enzyme is immobilized on an electrode produced by the above method. The enzyme electrode may include a complex layer of a compound of chitosan or chitosan-electron transporter and an enzyme electrodeposited to a thickness of 100 ~ 1,000nm, preferably 150 ~ 200nm on the working electrode. The composite layer has a non-uniform surface of macroporous structure.

Figure 2 is a SEM image of the enzyme fixed on the electrode by the present invention.

Referring to FIG. 2, the composite layer has a macroporous structure due to the generation of intense hydrogen gas during electrodeposition, and the surface of the layer is also uneven. Moreover, the average thickness of the said composite layer is about 150-200 nm. The thickness is thicker than that of the electrodeposited layer (about 40 nm in similar conditions), since it undergoes a two-step fixing mechanism and the size of GOx is larger than that of chitosan.

In another aspect the invention relates to a biosensor comprising an electrode to which an enzyme is immobilized by the method. The biosensor uses an electrode having an enzyme fixed therein as a working electrode, and the working electrode contacts the target material. In addition, the biosensor includes a counter electrode and a reference electrode. The biosensor can detect a target substance by electrochemical measurement using the enzyme electrode.

As an electrochemical measurement by a biosensor, for example, the chronoamperometry method, the coulometry method, the cyclic voltammetry, etc. which measure oxidation current or a reduction current, etc. A well-known measuring method can be used. As a measuring method, any of a disposable method, a batch method, a flow injection method, etc. may be used.

Hereinafter, the present invention will be described in more detail with reference to examples, but these examples should not be construed as limiting the protection scope of the present invention.

Example 1

A screen printed carbon paste electrode (SPCE; TE100-EK, Zensor R & D Co., Twiwan) was prepared. (SPCE is a three-electrode system consisting of a carbon working electrode (r = 0.15 cm), a carbon counter electrode, and a reference electrode (Ag / AgCl). The surface of the SPCE electrode was then pretreated with oxygen plasma for 10 minutes to modify the surface of the working electrode to be hydrophilic.

(1) Chitosan (molecular weight 190,000-310,000, 75-85% deacetylated) (1 wt%, 0.2 ml) (2) GOx (EC 1.1.3.4, Aspergillusniger, 100-250 U / mg) (2 mg / ml), (3) 1,1'-ferrocenedimethanol (Fc-dmol, 98%) (1 mM) and (4) McIlvaine buffer (Citric acid + Na ₂ HPO ₄ ) (10 mM) in vigorous 0.25 mL Eppendorf tubes The mixture was vortexed and uniformly mixed, and (5) the pH of the mixed solution was maintained at 5.5.

(6) A 1 minute standby time was then provided to form a composite layer of chitosan and enzyme on the working electrode by adsorption.

Subsequently, electrodeposition was performed by applying (7) -2 V vs Ag / AgCl (which is a reference voltage) to the SPCE electrode (8) for 1 minute. After performing electrodeposition, the SPCE surface was washed with PB solution and dried to obtain an SPCE electrode having a composite layer.

Example 2

While maintaining the rest of the conditions in the above embodiment (1) by changing only the concentration of chitosan (mg / mL) to measure the change in the current is shown in Figure 3 this.

Example  3

While maintaining the rest of the conditions in the above Example (2) by changing only the GOx concentration (mg / mL) to measure the change in the catalyst current is shown in Figure 4 this.

Example  4

While maintaining the rest of the conditions in the above embodiment (4) only by changing the McIlvaine buffer content was measured to change the catalyst current value is shown in FIG.

Example  5

In the above embodiment while maintaining the rest of the conditions (5) by changing the pH of the mixed solution to measure the change value of the catalyst current is shown in Figure 6 this.

Example  6

In the above example, the change value of the catalyst current was measured by changing only the waiting time (5) while maintaining the remaining conditions, and the results are shown in FIG. 7.

Example  7

In the above example, the change value of the catalyst current was measured by changing only the applied voltage (6) while maintaining the rest of the conditions, and the results are shown in FIG. 8.

Example  8

In the above embodiment while maintaining the rest of the conditions (7) by changing only the application time during electrodeposition, the change in the catalyst current was measured and shown in FIG.

Example  9

The SPCE electrode was obtained using 4 mg / mL of HRP (EC 1.11.1.7, 200-300 U / mg) instead of (2) GOx while maintaining the rest of the conditions in the above example.

Example  10

In the above example while maintaining the rest of the conditions (2) instead of GOx Cu-NiR (Copper containing nitrite reductase, EC 1.7.2.1, denitrificans, 2U / mg) 3 mg / ml to obtain a SPCE electrode.

Example  11

0.7 mL of water was added to 0.2 mL of a 1 wt% chitosan solution and 50 mg of 1-aminopropyl-1'-methyl-4,4'-bipyridinium was added thereto. The pH was adjusted to about 7 using dilute NaOH solution, and then 0.1 mL of glutaraldehyde (~ 25%) was added. The mixture was reacted at 60 ° C. for 4 hours to reduce Schiff's base to sodium cyanoborohydride, and then purified by centrifuge (microcon, Utracel YM-3, 3000 MWCO) to obtain CHIT-V. Obtained CHIT-V (0.3 wt%), Cu-NiR (Copper containing nitrite reductase, EC 1.7.2.1, denitrificans, 2U / mg) 3mg / ml, McIvavaine buffer (Citric acid + Na ₂ HPO ₄ ) (10 mM) Vortex vigorously and mix uniformly in 0.25 mL Eppendorf tubes (indicated final concentration in parentheses). Subsequently, under the same conditions as in Example 1, given the waiting time and electrodeposition conditions, an SPCE electrode fixed with CHIT-V and NiR was obtained.

Experimental Example  One

When (a) glucose was not injected into the SPCE electrode obtained in Example 1, (b) 30 mM glucose was injected, and (c) 30 mM glucose was added to the SPCE electrode without the enzyme immobilization therein, followed by adding GOx aqueous solution. In the case of injection, cyclic voltammograms were measured. The experiment was performed in 0.1 mL phosphate buffer, pH 6.5, electron carrier in a 5 mL cell at 1 mM Fc-dmol, scanning rate 0.002V / s and shown in Figure 10.

Experimental Example  2

(A) When H2O2 was not injected into the SPCE electrode fixed in the HRP obtained in Example 9 (b) When 0.15 mM H2O2 was injected, cyclic voltammograms were measured for each. The experiment was performed at 0.1 M phosphate buffer, pH 6.5, MV 0.2 mM in an electron carrier, and a scanning rate of 0.002 V / s in a 5 mL cell, which is shown in FIG. 11.

Experimental Example  3

(A) When nitrite (Nitrite) was not injected into the NiC-fixed SPCE electrode obtained in Example 10, (b) When 0.1mM nitrite was injected, (c) After injecting 0.5mM nitrite (Nitrite) Cyclic voltammograms were measured. The experiment was performed at 0.1 M phosphate buffer, pH 6.5, MV 1 mM with an electron transporter, and a scanning rate of 0.002 V / s.

Experimental Example  4

(A) When nitrite was not injected into the SPCE electrode having NiR and CHIT-V fixed in Example 11, and (b) Cyclic voltammograms were respectively applied to the case where 0.5 mM nitrite was injected. Measured. The experiment was performed in 0.1 M phosphate buffer, pH 6.5, scanning rate 0.002V / s and shown in FIG.

3 is a distribution value of the (catalyst) current flow according to the concentration of chitosan. Referring to Figure 3, the concentration of chitosan is preferably in the range of 1 ~ 3mg / mL, it can be seen that the optimization is shown when the content is 2mg / mL.

4 is a distribution value of (catalyst) current flow with increasing GOx concentration. Referring to Figure 4, it can be seen that the high concentration (tilt; 0.192) of 6-10mg / mL shows a current flow of about 6.6 times the low concentration (tilt; 0.0298) range of 2-6mg / mL.

5 is a distribution value of the current flow according to the phosphate buffer concentration. Referring to FIG. 5, a bell type curve showing an optimization when the concentration of phosphate is 10 mM can be obtained.

6 is a graph showing the current flow after electrodeposition according to the pH change of the mixed solution while changing the pH of McIlvaine buffer to 3-6, respectively. Referring to FIG. 6, the fixed amount of chitosan and enzyme is small at low pH, while the pH 5 to 6 is close to pH 6.3, which is the dissolution transition point of chitosan, and thus the effect on pH change is relatively small. .

7 is a curve showing the current flow according to the waiting time. Referring to FIG. 7, a bell type curve may be obtained with a maximum point of 1 minute. After one minute of waiting time, the catalytic current of the electrode with the chito-enzyme electrodeposited was four times higher than that of electrodeposition immediately without waiting time.

8 is a graph illustrating a current flow according to an applied voltage. Referring to FIG. 8, a bell-shaped profile was obtained, showing a maximum value at -2.0.

9 is a graph showing the catalytic current flow of the electrodes (GOx) is fixed with each electrodeposition time. Referring to FIG. 9, it can be seen that as time increases, the amount of GOx electrodeposited increases, while the thickness of the chitosan layer simultaneously increases, which acts as a barrier to mass transfer.

Figure 10 (a) is not injected with glucose (b) is injected with 30mM glucose, (c) is a case of injecting 30mM glucose into the GOx aqueous solution to the SPCE electrode does not fix the enzyme Cyclic voltammograms are measured. Referring to Figure 10, comparing (a), (b), it can be seen that the GOx-fixed electrode according to the present invention generates a high current flow change when the glucose is injected. Alternatively, comparing (c) and (b) shows that the current flow change is almost similar to the case where the enzyme is provided in solution, and thus the method according to the present invention can confirm that a large amount of enzyme can be immobilized on the electrode by a simple method. Can be.

(A) of FIG. 11 is a graph in which cyclic voltammograms are measured when 0.12 mM H2O2 is injected when (b) is not injected. Comparing FIGS. 11A and 11B, it can be seen that the HRP-fixed electrode according to the present invention generates a high current flow change when H 2 O 2 is injected.

12 shows (a) when nitrite (Nitrite) is not injected into a NiC-fixed SPCE electrode (b) when 0.1mM nitrite is injected, and (c) circulating voltage current after injection of 0.5mM nitrite (Nitrite) (Cyclic voltammograms) is a graph measured. Referring to Figure 12 (b), it is sensitive even when a small amount of nitrite is injected, it can be confirmed the superiority of the fixed enzyme electrode of the present invention.

FIG. 13 shows cyclic voltage currents (Cyclic) for the case where (a) nitrite was not injected into the SPCE electrode fixed with NiR and CHIT-V obtained in Example 11, and (b) 0.5 mM nitrite was injected. voltammograms)

Comparing FIGS. 13A and 13B, it can be seen that the NiR and CHIT-V-fixed electrodes according to the present invention generate a high current flow change when nitrite is injected.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims (13)

It includes an electrodeposition step of forming a complex layer of chitosan and enzyme on the electrode by applying a predetermined applied voltage to a mixed solution containing chitosan, electron transporter, enzyme, buffer,
The method comprises the step of immobilizing a complex layer of chitosan and enzyme on the electrode by adsorption by providing a predetermined wait time before the electrodeposition step. 2. The method of claim 1, wherein said wait time is within 3 minutes. The method of claim 1, wherein the electrodeposition step is an enzyme fixation method, characterized in that the applied voltage within the range of -1.8 to -2.2V, the application time within 3 minutes. The method of claim 1, wherein the mixed solution comprises 0.5 to 10 mg / mL chitosan, 0.1 to 5 mM electron carrier, 2 to 10 mg / mL enzyme and 5 to 20 mM buffer. The method of claim 1, wherein the chitosan and the electron transporter are present as a compound of the chitosan-electron transporter. The method of claim 5, wherein the mixed solution comprises 0.5-10 mg / mL of the compound of the chitosan-electron transporter, 2-10 mg / mL of enzyme, and 5-20 mM of buffer. The method of claim 1, wherein the pH of the mixed solution is 4 to 6, characterized in that the enzyme immobilization method. The method of claim 1, wherein the electron transporter is selected from the group consisting of ferrocene (ferrocene), ferrocene derivatives, quinones (quinones), quinone derivatives, organic conducting salts and biologen (viologen) Enzyme Fixation Method. 2. The enzyme of claim 1 wherein the enzyme is glucose oxidase, horseradish peroxidase (HRP), nitrite reductase (NiR), cholesterol oxidase, glutamic oxalacetic transaminase (GOT) and glutamic pi Enzyme fixation method, characterized in that selected from the group consisting of Glutamic Pyruvic Transaminase (GPT). An enzyme electrode immobilized on an electrode prepared according to any one of claims 1 to 9, wherein the enzyme electrode is a composite layer of electrodeposited chitosan and enzyme immobilized at a thickness of 100 to 1000 nm on a working electrode. Enzyme electrode comprising a. The enzyme electrode according to claim 10, wherein the composite layer comprises a non-uniform surface having a macroporous structure. A biosensor comprising an enzyme electrode immobilized on an electrode manufactured according to any one of claims 1 to 9. The biosensor of claim 12, wherein the biosensor detects a target substance by electrochemical measurement using the enzyme electrode.

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