KR19980075989A - Enzyme-fixed membrane of biosensor using acicular fine hydrogen peroxide electrode as converter and its manufacturing method - Google Patents

Abstract

The present invention relates to a method for producing an enzyme-immobilized membrane of a biosensor using a needle-like hydrogen peroxide electrode as a converter. More specifically, the present invention comprises a three-layer structure of an inner layer, an enzyme immobilization layer, and an outer layer attachable to the electrode sensitive part to increase the reproducibility of the microenzyme electrode and the measurable range and to exclude the influence of the measurement inhibitor. The present invention relates to a method for preparing an enzyme-immobilized membrane by a simple process. In the method of preparing an enzyme-fixed membrane of the present invention, an electrode tip, which is a sensitive part of acicular fine hydrogen peroxide electrode, is immersed in a polymer solution dissolved at a concentration of 1 to 10 percent in an appropriate solvent for 0.5 to 10 seconds and dried at room temperature for 1 to 5 minutes. Inner layer manufacturing process of the enzyme-immobilized membrane to repeat the process 1 to 15 times according to the required degree; Oxidase; 0.5 to 10 microliters of enzyme-immobilized solution containing bovine serum albumin and standard buffer solution in the composition of 2 to 10 mg and 10 to 100 microliters, respectively, was added to the tip of the electrode and divided into electrode tips. Dry for 10 to 90 minutes, immediately immersed in 0.1 to 7 percent glutaraldehyde dilution solution for 0.5 to 10 seconds, and then crosslinking for 10 to 90 minutes in a 2 to 15 ° C. refrigerator, or 25 to 50 percent high concentration of glutar A process for preparing an enzyme-immobilized layer of an enzyme-immobilized membrane which performs a crosslinking reaction for 0.5 to 5 hours at a temperature of 20 to 60 ° C. in a sealed container using a vapor of an aldehyde solution and washes 5 to 20 times with a standard buffer solution and distilled water, respectively; The electrode tip on which the enzyme immobilization layer is formed is immersed in a polymer solution dissolved at a concentration of 1 to 10 percent in an appropriate solvent for 0.5 to 10 seconds, dried at room temperature for 1 to 5 minutes, and the process is performed 1 to 15 times according to the required degree. An outer layer manufacturing step of the enzyme-immobilized membrane repeatedly carried out; And a step of preparing the enzyme-fixed membrane of the biosensor having a three-layered structure in which the enzyme-fixed layer is sandwiched by adding the inner layer, the enzyme-fixed layer, and the outer layer of the enzyme-fixed membrane to the needle-shaped fine hydrogen peroxide electrode tip in the order of the foregoing processes.

Description

Enzyme-fixed membrane of biosensor using acicular fine hydrogen peroxide electrode as converter and its manufacturing method

The needle-like fine hydrogen peroxide electrode, which can perform a specific component measurement in the food industry, the medical field, and the like by the selective specific action of the biological element of the present invention, is a transducer (hereinafter referred to as a transducer), and the enzyme is a biological element. The present invention relates to an enzyme immobilization membrane suitable for the operation of a sensitizer (hereinafter referred to as a biosensor) and a manufacturing technology thereof.

The biosensor converts changes in physicochemical factors such as ions, electrons, heat, mass, and light, which are generated as a result of specific reactions between biological elements and the measurement target, into electrical signals, amplifies them, and displays them in the appropriate form to measure specific components As a device that can be performed quickly, it has the advantages of being on-site, easy to operate, simplifying or eliminating sample preparation, and easy to use due to its automation and compact structure. And defense sectors (Biosensors and Bioelectronics, Vol. 9, p. 243, 373, 1994). Especially in the food industry, it can be used for food analysis, fermentation process control, quality control of fruits and vegetables, leading management of fish meat and livestock meat in the market. It can be used for pharmacokinetic analysis and medical device development. The market size of biosensor is growing rapidly, and it is estimated that it will be about 5 billion dollars worldwide in the early 2000s. In the 21st century, the quality evaluation of raw materials in the fermentation and pore process, agricultural and fisheries, environmental measurement and clinical As analytical systems are usually expected to be automated, the need for highly available biosensor systems is expected to increase.

Biosensors can be broadly classified into electrochemical biosensors, optoelectronic biosensors, piezoelectric (pizoelectric) biosensors, and biothermistors (Analyst, Vol. 114, p. 653, 1989; Biosensors). and Bioelectronics, Vol. 9, p. 243, 1994), among which electrochemical biosensors generate ions, electrons, etc., which are proportional to the concentration of substances, as a result of selective reactions between biological elements and the material to be measured. It converts into electrochemical signals such as impedance and impedance. Electrochemical biosensors were first manufactured by simple measuring principles and devices, as shown in the case of glucose biosensors using oxygen electrodes (Annals NY Acad. Sci., Vol. 102, p. 29, 1962). The prospect of commercialization is also very bright, making it the biosensor with the highest need for development in the future.

Representative of the electrochemical biosensors is the electrochemical enzyme sensor, and its action principle can be classified as follows according to the nature of the enzyme used as the biological element and the electron transfer method (In Food Biosensor Analysis, p. 31, Marcel Dekker, New York, 1994).

Enzyme reaction: [S] + O ₂ → [P] + H ₂ O ₂

Electrochemical reaction:

Oxygen ^{_{electrode: O 2 + 4H + + 4e}} - → 2H 2 O

Hydrogen peroxide _{electrode: H 2 O 2 → O 2} + 2H + + 2e -

Enzyme reaction: [S] + NAD (P ⁺ ) → [P] + NAD (P) H

The electrochemical reaction: NAD (P) H → NAD (P +) + H + + e -

Oxidase: [S] + M (ox) → [P] + M (red)

The electrochemical reaction: M (red) → M ( ox) + e -

Where [S] is the substrate, [P] is the reaction product, and M (ox) and M (red) are the oxidized and reduced electron transporters, respectively. Electrode materials for electrochemical enzyme sensors include precious metals such as platinum and gold (mainly measuring electrochemical reactions of hydrogen peroxide and oxygen), and carbon materials such as carbon paste and glass carbon (NAD (P) H, electrochemicals of electron transporters). Reaction is mainly measured, and the measurement potential is hydrogen peroxide, 100-800 mV for NAD (P) H electrode, -800 to -600 mV for oxygen electrode, and electron carrier for electrode using oxidation of electron carrier. The measurement potential varies depending on the nature. The converter of the electrochemical enzyme sensor is composed of a working electrode, a reference electrode, and a counter electrode, which exist independently or in combination. Enzymatic immobilization methods in the preparation of electrochemical enzyme sensors have been reported by non-covalent and covalent bonding methods. The former uses adsorption or physical envelopment by van der Waals forces, ionic bond forces, etc., and the latter uses covalent bonds of functional groups such as ε-amino groups, carboxyl groups, sulfhydryl groups and phenolic hydroxy groups in proteins and membranes and electrode surfaces. (Biosensors and Bioelectronics, Vol. 6, p. 681, 1991; In Sensors- A Comprehensive Survey, Vol. 3, p. 717, 1992).

Electrochemical enzyme sensor using hydrogen peroxide electrode as a converter can be easily and diversified electrode manufacturing, so it is easy to develop, its application is infinite, and the commercialization prospect is bright. The hydrogen peroxide electrode that has been used in the past is mostly a gelatin, cellulose acetate, viao, which can be adhered closely to the sensitive part of the working electrode when the enzyme is immobilized. Non-covalently binding enzyme preparations to membranes such as phosphorus immunoaffinity membranes (Z. Lebensm. Unters. Forsch, Vol. 177, p. 356, 1983; J. Chem. Tech. Biotechnol., Vol. 49, p. 255, 1990), and have been used covalently binding enzymes to activated nylon networks. Sometimes immobilization of metal oxide groups or carboxyl groups formed by electrochemically oxidizing the surface of precious metals to form amino groups and then covalently bonding amino groups of enzyme proteins by crosslinking with glutaraldehyde It has also been used (Methods Enzymol., Vol. 135, p. 141, 1987; In Sensors- A Comprehensive Survey, Vol. 3, p. 717, 1993).

However, in recent years, one of the major trends of biosensor development worldwide is micronization of electrodes as transducers, thereby ultimately converging the entire biosensor system to promote convenience and efficiency. If the miniaturization of the electrode is made efficiently, it is possible to develop multiple microsensors, which facilitates multicomponent analysis and reduces the production cost by mass production. Microsensors include ion-selective relay effect transistors (esp, ISFET), gas-selective metal oxide semi-conductors (MOS, MOS), thin-film electrodes, etc.The recent advances in electronic and semiconductor engineering technology make them inexpensive and reproducible. It became possible to manufacture. In the case of a biosensor system using a hydrogen peroxide electrode, the necessity of micronization has been greatly raised, and a biosensor using the acicular fine hydrogen peroxide electrode of the present invention as a converter is to meet this need.

An important issue addressed with biosensor micronization is the manufacture of immobilized biofilms. That is, the immobilized biofilm should be attached to the sensing site accurately and the attached film should be able to be manufactured so as to conform to the integrated circuit of the biosensor without easily falling off during use. As mentioned above, the biological element immobilization method for the membrane material cannot be applied to the enzyme immobilization of the inductive part of the micronization sensor. Therefore, the development of a new immobilization method is essential and the necessity for this has been constantly removed. .

After all, the present invention relates to a method for immobilizing an enzyme into a microsensing sensor induction unit which has been difficult to solve by a conventional method. Enzyme prepared by immobilizing enzyme on the sensitive part to obtain current reaction of repetitive and reproducible enzyme electrode while increasing the measurable range of specific substrate of enzyme electrode by selecting the appropriate polymer and excluding effects of measurement inhibitor To improve the measurement reliability and practical applicability of electrode in food industry and clinical medicine.

The present inventors have studied an enzyme fixation method that can improve the response characteristics of an electrochemical enzyme sensor using a needle-like hydrogen peroxide electrode as a converter. In addition, measurement inhibition by ascorbic acid, which is a reducing substance present in blood, can be eliminated, and an appropriate polymer can be coated on the electrode sensitive part where the enzyme is immobilized, thereby greatly increasing the measurable position of a specific substrate of the enzyme electrode. As a result of extensive research and discovery, the electrochemical enzyme sensor using a hydrogen peroxide electrode as a converter can eliminate the measurement disturbance that may appear as a problem and can also be measured by using a small needle electrode. Enzyme high, which greatly improves the range falling Hwacheung, developed an enzyme-immobilized film production method comprising a three-layer structure of the outer layer thus completing the present invention.

Hereinafter, the present invention will be described in more detail for each enzyme-fixed membrane manufacturing step.

1 is a schematic configuration diagram of a needle acicular fine hydrogen peroxide electrode.

* Description of the symbols for the main parts of the drawings *

1: working electrode (platinum wire) 2: reference electrode (silver / silver chloride wire)

3: FEP Tubing 4: Heat Shrink Tubing

5: electrode base (to wire)

2 is a schematic diagram of an enzyme-fixed membrane used in an electrochemical enzyme sensor using a needle-like hydrogen peroxide electrode as a transducer.

* Description of the symbols for the main parts of the drawings *

1: working electrode (platinum wire) 2: reference electrode (silver / silver chloride wire)

3: FEP Tubing 4: Heat Shrink Tubing

5: inner layer of enzyme fixation membrane 6: enzyme fixation layer of enzyme fixation membrane

7: outer layer of enzyme fixation membrane

The acicular fine hydrogen peroxide electrode used as a transducer of the biosensor in the present invention is a composite electrode integrated into one unit of a working electrode and a reference electrode, and a schematic configuration thereof is shown in FIG. 1. The electrode of FIG. 1 is made of platinum wire of 4 to 8 centimeters in length, 0.5 to 1 millimeter in diameter, and silver / silver chloride wire as the working electrode and the reference electrode, respectively, and the tip portion is polished using distilled water, acetone, and 1 mol nitric acid. Ultrasonic cleaning and silver electrodes are manufactured through electrolysis using 0.1 mol hydrochloric acid, coating and fixing using insulating FEP tubing, and electrode compounding using heat shrink tubing. At the same time, mass production by automated facilities is possible. On the other hand, the electrochemical enzyme sensor of the present invention is usually produced using a glycoprotein and excellent oxidase as a biological element.

Inner layer manufacturing process of enzyme fixation membrane

The inner layer production of the enzyme-immobilized membrane is carried out to exclude the influence of the measurement inhibitor. That is, ascorbic acid and cysteine of reducing substances capable of inhibiting the signal of a biosensor system using hydrogen peroxide in food, food ingredients, and blood, which are subject to the electrochemical enzyme sensor using the needle-like hydrogen peroxide electrode of the present invention as a converter. , Capsaicinoids, tocopherols and the like. In order to eliminate the measurement disturbance caused by these methods, a method of reducing the measurement potential or increasing the oxidation potential of the measurement inhibitor by using an electron carrier, pre-oxidation of the measurement inhibitor via a separate flow electrode, and measurement at the reduction potential Although the same method is used (Anal. Chem., Vol. 65, p. 2690, 1993), the present invention eliminates the influence of metrology inhibitors by using bulkiness or electrical properties. In order to achieve this purpose, a selected polymer is coated on the surface of the sensitive part of the acicular fine hydrogen peroxide electrode of the present invention, and the electrode coated with the polymer shows that ascorbic acid is present in the most prominent concentration in most samples among the measurement inhibitors. Measure the current response. The polymers that can be used include polyurethane, methyl cellulose, cellulose acetate, ethyl cellulose, nafion, and lauric acid. , Palmitic acid, and the like, and the transducer used in the present invention is a complex in which the working electrode and the reference electrode are integrated into one and the size of the sensitive part is small, thereby forming an inner layer by dip-coating. It is considered to be the most efficient. The concrete method of inner layer manufacturing is as follows.

The inner layer coating is performed with a polymer solution dissolved in benzene, dichloromethane, ethanol, acetone, etc. at a concentration of 1 to 10 percent. A bare electrode is immersed in each polymer solution for 0.5 to 10 seconds and dried at room temperature for 1 to 5 minutes. Repeat this process 1 to 15 times as necessary to complete the inner layer production. Immediately thereafter, at the oxidation potential of the coated electrode at 200 to 700 millivolts, the current response to the measurement inhibitor is measured in the standard buffer solution used for each enzyme electrode. At this time, the standard buffer solution is 0.05 to 0.3 mol phosphate buffer solution (including pH 6 to 8, 0.01 to 0.5 mol potassium chloride) for lactic acid biosensor, and 30 to 70 millimolar phosphate buffer solution (pH 6 to 8) for cholesterol biosensor. 8, 0.1 to 5 percent Triton X-100 with 1 to 10.5 percent isopropyl alcohol and 0.01 to 0.5 mol potassium chloride; 0.05 to 0.3 mol phosphate buffer solution (pH 6 to 8, 0.01 to 0.5 mol for glucose biosensors) Potassium chloride).

In the standard buffer solution of each oxidase electrode, ascorbic acid, which is a representative measurement inhibitor, was evaluated, and the degree of exclusion of the measurement inhibitor by the inner layer formation was up to 98 to 99 percent even after 3 to 5 coatings. Was reaching.

Enzyme Immobilization Layer Manufacturing Process of Enzyme Immobilization Membrane

The enzyme immobilization layer is prepared by immobilizing various oxidases on the inner layer of the fine needle hydrogen peroxide electrode of the present invention. Testing of the immobilizing agent such as glutaraldehyde and the photosensitive polymer FVA-SbQ (PVA-SbQ), the composite electrode tip consisting of a fine platinum wire and a silver / silver chloride line of 0.5 to 1 millimeter in diameter as in the present invention As a method of immobilizing the enzyme, cross-linking by glutaraldehyde is most preferred. When performing glutaraldehyde crosslinking, a method of immobilizing 0.5 to 5 hours at a temperature of 20 to 60 ° C. in a closed vessel using a vapor of 25 to 50 percent of a high concentration of glutaraldehyde solution and 0.1 to 7 percent glutar After immersing in aldehyde dilution solution for 0.5 to 10 seconds and drying in a refrigerating chamber at 2 to 15 ° C. to compare the crosslinking method, both methods were applicable to the preparation of enzyme-fixed membranes using oxidase, but in diluted glutaraldehyde solution. Dipping and crosslinking resulted in a more stable form of the enzyme immobilization layer, and the biosensor reaction was also reproducible. The specific method of preparing the enzyme immobilization layer is as follows.

0.5-10 microliters of well-enzyme-immobilized solution containing oxidase, bovine serum albumin, and standard buffer solution in the composition of 2-10 mg, 2-10 mg, 10-100 microliters, respectively. Take a microsyringe and divide it into 1 ~ 3 times on the tip of the sensitive electrode, and fix it in a rectangular parallelepiped container with 16 ~ 18 centimeters, 8 ~ 10 centimeters, and 2 ~ 3 centimeters in width, length and height, respectively. Dry for 10 to 90 minutes in a 2-15 ° C. refrigerator. Immediately thereafter, the mixture is immersed in a dilution solution of 0.1 to 7 percent glutaraldehyde for 0.5 to 10 seconds, and then crosslinked for 10 to 90 minutes in a refrigerator at 2 to 15 ° C. The cross-linked enzyme electrode is washed 5 to 20 times with standard buffer solution and distilled water, respectively, and then refrigerated or stored at room temperature until used in the rectangular parallelepiped container. When the enzyme-immobilized layer was stored in a dry state, the enzyme electrode of the present invention showed a constant level of current reaction at an infinite temperature at 30 to 50 days at room temperature and almost indefinitely.

Manufacturing process of outer layer of enzyme immobilization membrane

The enzyme fixation membrane of the electrochemical enzyme sensor using the acicular fine hydrogen peroxide electrode of the present invention as a converter has a three-layer structure, which provides the function of protecting the enzyme fixation layer in addition to the inner layer and the enzyme fixation layer and increases the measurable range of a specific component. This fact is one of the main features of the present invention. In other words, if the selected polymer is coated on the enzyme immobilization layer, which is an intermediate layer of the enzyme electrode of the present invention, the oxidase is a biological element, the measurable range of lactic acid, which is a substrate of the enzyme electrode, increases according to the characteristics of the polymer. At this time, the polymers used in the present invention include polyurethane, methyl cellulose, ethyl cellulose, cellulose acetate, cellulose triacetate, and the immersion coating method mentioned in the preparation of the inner layer is used as an efficient method for coating these polymers. The specific method of manufacturing an outer layer is as follows.

The outer layer coating is performed with a polymer solution dissolved in benzene, dichloromethane, ethanol, acetone, etc. at a concentration of 1 to 10 percent. The tip end of the enzyme electrode on which the enzyme immobilization layer is formed is immersed in 0.5 to 10 seconds in each polymer solution and dried at room temperature for 1 to 5 minutes. Repeat this process 1 to 15 times as necessary to complete the outer layer manufacturing. Immediately thereafter, an enzyme electrode coated in a standard buffer solution was measured at an oxidation potential of 200 to 700 millivolts, and the current response shown by increasing concentrations of lactic acid and cholesterol, which are oxidase substrates, was evaluated. It can be seen that even a simple process as in the invention can increase the measurable range of the oxidase substrate by 5 to 15 times.

3-layered enzyme-fixed membrane manufacturing process

The three-layered enzyme-immobilized membrane suitable for the electrochemical enzyme sensor of the present invention using a needle-like fine hydrogen peroxide electrode and an oxidase as a converter and a biological element, respectively, has the inner layer, enzyme-immobilized layer, and outer layer as described above. In order, it can be manufactured by forming by the process outlined above (FIG. 2). As shown in FIG. 2, the enzyme immobilization layer is sandwiched between the inner and outer layers of the working electrode and the reference electrode.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples according to the gist of the present invention.

Example 1:

When the biosensor measurement of the present invention using a needle-like hydrogen peroxide electrode as a transducer is carried out at an oxidation potential, the acetone is removed in order to exclude the influence of the inhibitory current generated by reducing substances such as ascorbic acid, which may be present in food, food ingredients, and blood. Inner layer coating was performed with% cellulose acetate solution. The bare electrode was immersed in each polymer solution for 10 seconds and dried at room temperature for 5 minutes. This process was repeated 15 times to complete the inner layer production. Immediately thereafter, the coated electrode exhibited a current response to ascorbic acid, a representative inhibitor of measurement, at an oxidation potential of 200 to 700 millivolts in the standard buffer solution used for each enzyme electrode.

Table 1 shows the results of examining the inhibition effect of ascorbic acid on the polymer and species obtained by coating the acicular acid peroxide electrode according to the present embodiment in the standard buffer solution of the lactic acid biosensor system. .

Experimental Example

When performing biosensor measurement in Example 1, the inner layer was formed by immersion coating of the polymer on the surface of the electrode under the combination of the following conditions in order to exclude the measurement damage by the reducing material.

Table 1: Effect of elimination of measurement inhibition by ascorbic acid when inner layer coating of needle-shaped fine hydrogen peroxide electrode using polymer in standard buffer solution of lactic acid biosensor system

^a : No measurement.

Table 2 shows the results of examining the inhibition effect of the measurement inhibition by ascorbic acid obtained when the inner layer coating of the acicular fine hydrogen peroxide electrode according to the present embodiment in the standard buffer solution of the cholesterol biosensor system. Is indicated.

Table 2: Effect of ascorbic acid on the measurement of the inhibition of measurement by the ascorbic acid when the inner layer of the needle-like fine hydrogen peroxide electrode was coated with polymer in the standard buffer solution of cholesterol biosensor system

^a : No measurement.

Ascorbic acid, which may exhibit measurement inhibition by oxidation current when measuring the biosensor using the acicular fine hydrogen peroxide electrode of the present invention as a representative reducible material that may be present in food and food raw materials, etc. It can be seen that the effect of is almost completely removed by the immersion coating using the polymer of the present embodiment, the same effect was also seen in the case of cysteine.

Comparative Example

In order to manufacture the biosensor of the present invention using the acicular fine hydrogen peroxide electrode as a converter, enzyme fixation was performed on the sensitive portion of the electrode coated with an inner layer according to Example 1. As a result of the comparison with the enzyme immobilization method, both glutaraldehyde crosslinking method and photopolymerization method using FV-SBIQ were applicable, but as in the present invention, a fine platinum wire having a diameter of 0.5 to 1 millimeter and silver / silver chloride line were used. As a method of immobilizing the enzyme at the tip of the composite electrode, crosslinking by glutaraldehyde was most preferred. To describe the glutaraldehyde crosslinking method in detail, take 0.5 to 10 microliters of the enzyme-immobilization solution as a microsyringe, divide it into 1 to 3 times at the tip of the electrode, which is the sensitive part, and fix it in a rectangular parallelepiped container with the tip down. 10 to 90 minutes in a refrigerating chamber at 2 to 15 ° C. and fixing the electrode tip at a temperature of 20 to 60 ° C. at a temperature of 20 to 60 ° C. in a sealed container with 25 to 50 percent of a high concentration of glutaraldehyde solution and 0.1 to It was immersed in 7% glutaraldehyde dilution solution for 0.5 to 10 seconds and then dried in a refrigeration room at 2 to 15 ° C to crosslink the crosslinking. Applicable to the production of enzyme-fixed membranes, but immersed in dilute glutaraldehyde solution The crosslinking method formed a more stable form of the enzyme immobilization layer and thus made the biosensor more reproducible and stable current response. The method of crosslinking by dipping in dilute glutaraldehyde solution will be described in more detail with the example of lactic acid biosensor as follows.

Example 2:

10 microliters of well-enzyme-immobilized solution containing lactic acid oxidase, bovine serum albumin and 0.3 mol phosphate buffer solution (including pH 8 and 0.5 mol potassium chloride) in a composition of 10 mg, 10 mg and 100 microliters, respectively. Taken with a micro syringe, divided into three times at the tip of the electrode, fixed in a cuboid container of 18 cm, 10 cm and 3 cm in width, length and height, respectively, with the tip side down and dried for 90 minutes in a 2-15 ° C. refrigerating chamber. Immediately thereafter, the mixture was immersed in a 7% glutaraldehyde dilution solution for 10 seconds and then crosslinked for 90 minutes in a 15 ° C. refrigerator. The crosslinked enzyme electrode was washed 20 times with the buffer solution and distilled water, and then refrigerated or stored at room temperature until use in the rectangular parallelepiped container. When the enzyme-immobilized layer was stored in a dry state, the lactic acid biosensor of the present example showed a stable level of current response at a room temperature for about 50 days and at almost indefinite temperature at refrigeration temperature.

Experimental Example

In Examples 2 to 4, in order to perform enzyme fixation on the electrode sensitive part coated with the inner layer of the biosensor, enzyme fixation was performed under a combination of the following conditions.

Experimental Example

Immediately after enzyme immobilization, the glataraldehyde crosslinking reaction was carried out under a combination of the following conditions.

Experimental Example

The enzyme-specific composition of the enzyme immobilization solution was carried out under a combination of the following conditions.

Example 3:

The method of crosslinking by dipping in dilute glutaraldehyde solution of Example 2 will be described in more detail with the example of the cholesterol biosensor as follows. Microshes 10 microliters of a well-enzyme-immobilized solution containing 10 mg and 100 microliters of cholesterol oxidase, bovine serum albumin and 70 mmol phosphate buffer solution (including pH 8 and 0.5 mol potassium chloride), respectively. It was taken as a rinse and added to the electrode tip three times, fixed in the rectangular parallelepiped container of Example 2 with the tip part down, and dried in a 15 degreeC refrigerator compartment for 90 minutes. Immediately thereafter, the mixture was immersed in a 7% glutaraldehyde dilution solution for 10 seconds and then crosslinked for 90 minutes in a 15 ° C. refrigerator. The crosslinked enzyme electrode was washed 20 times with the buffer solution and distilled water, and then refrigerated or stored at room temperature until use in the rectangular parallelepiped container. When the enzyme-immobilized layer was stored in a dry state, the cholesterol biosensor of this example showed a stable current response at room temperature and refrigeration temperature as in the lactic acid biosensor of Example 2.

Example 4:

The method of crosslinking by dipping in dilute glutaraldehyde solution of Example 2 will be described in more detail with the example of the glucose biosensor as follows. 10 microliters of well-enzyme-immobilized solution containing glucose oxidase, bovine serum albumin and 0.3 mol phosphate buffer solution (including pH 8 and 0.5 mol potassium chloride) in compositions of 10 mg, 10 mg and 100 microliters, respectively. Was taken in a microsyringe, divided into three portions at the tip of the electrode, fixed in the rectangular parallelepiped container of Example 2 with the tip down, and dried in a 15 ° C. cold room for 90 minutes. Immediately thereafter, the mixture was immersed in a 7% glutaraldehyde dilution solution for 10 seconds and then crosslinked for 90 minutes in a 15 ° C. refrigerator. The crosslinked enzyme electrodes were washed 5 to 20 times with the buffer solution and distilled water, respectively, and then refrigerated or stored at room temperature in the rectangular parallelepiped container. When the enzyme-immobilized layer was stored in a dry state, the glucose biosensor of this example showed a stable current response at room temperature and refrigeration temperature as in the lactic acid biosensor of Example 2.

Example 5:

In this embodiment, a polymer having an outer layer above the enzyme immobilization layer is selected to have a function as a protective layer of the enzyme immobilization layer and an increase in the measurable range to achieve a feature of the biosensor of the present invention using a needle-like hydrogen peroxide electrode as a transducer. It was formed as follows by the same immersion coating method at the time of forming the inner layer. The outer layer coating was performed with ethyl cellulose solution dissolved at 10 percent concentration in acetone. In the same process as in Example 2 to 4 using an oxidase, the enzyme electrode on which the enzyme immobilization layer was formed was immersed in each polymer solution for 10 seconds and dried at room temperature for 5 minutes, and this process was repeated 15 times as necessary. It carried out and completed manufacture of an outer layer.

Immediately thereafter, the current response of the enzyme electrode coated in the standard buffer solution was measured at an oxidation potential of 200 to 700 millivolts as the concentration of lactic acid and cholesterol, which are oxidase substrates, increased. In the case of enzyme electrode without outer layer when lactic acid oxidase is a bioelement, the enzyme electrode is saturated at substrate concentration of 15 to 25 milligram percent, whereas when the outer layer is formed by coating with the above polymer, the enzyme reaches a substrate concentration of 150 to 250 milligram percent or more. Since the electrode was not saturated, the measurable range increased by more than 10 times. On the other hand, polymer coating was performed on the enzyme electrode where cholesterol oxidase was a bioelement, and the result of measuring the current response shown by the enzyme electrode as the cholesterol concentration increased in the standard buffer solution was similar.

Experimental Example

In Example 5, the outer layer was formed by an immersion coating method using a polymer under the combination of the following conditions so as to have a function as a protective layer of the enzyme immobilization layer and an increase in the measurable range.

Example 6:

In this embodiment, the inner layer described above in Examples 1 to 5 at the electrode tip as a sensitive part for the preparation of an enzyme-immobilized membrane which can be used for an electrochemical enzyme sensor using a needle-like hydrogen peroxide electrode and an oxidase as a transducer and a bioelement. The enzyme immobilization layer and the outer layer were sequentially formed to prepare an enzyme immobilization membrane having a three-layer structure in which an enzyme immobilization layer was sandwiched between an inner layer and an outer layer, which are the final targets of the present invention. As an example, the lactic acid biosensors prepared according to Examples 1, 2, and 5 did not suffer any measurement loss in the ascorbic acid concentration range (5 to 50 milligram percent) in the kimchi sample, and the measurable range was greatly increased. The fermentation status was effectively used to measure the lactic acid concentration of various kimchi samples.

The enzyme-immobilized membrane of the present invention, which can be mounted on the sensitive portion of the needle-like fine hydrogen peroxide electrode, solves problems such as the reproducibility degradation that may appear in the case of the immobilized biofilm used to attach to the microelectrode, the reduction of the measurable range, and the biosensor using the hydrogen peroxide electrode. In the reaction, it is possible to contribute to the development of multiple microsensors through the densification of biosensor system, multi-component analysis, and production cost reduction by mass production by suggesting a method to exclude the influence of measurement inhibitors. It works.

Claims (6)

(i) in one solution selected from polyurethane, methyl cellulose, cellulose acetate, ethyl cellulose, Nafion, lauric acid, palmitic acid solution dissolved in benzene, dichloromethane, ethanol, acetone, etc. at a concentration of 1 to 10 percent. Bio using the needle fine hydrogen peroxide electrode as a converter, characterized in that the electrode tip, which is the sensitive part of the needle fine hydrogen peroxide electrode, was immersed in 0.5 to 10 seconds and dried at room temperature for 1 to 5 minutes, and the process was repeated 1 to 15 times. Inner layer manufacturing process of sensor enzyme-immobilized membrane; (ii) a microsyringe of 0.5-10 microliters of enzyme fixation solution containing oxidase, bovine serum albumin and standard buffer solution in the composition of 2-10 mg, 2-10 mg, 10-100 microliters, respectively. Add to the electrode tip divided into 1 to 3 times, fixed in the cuboid container with the tip down, and dried for 10 to 90 minutes in a 2 to 15 ℃ refrigeration chamber, and immediately immersed in 0.1 to 7 percent glutaraldehyde dilution solution for 0.5 to 10 seconds Crosslinking reaction for 10 to 90 minutes in a refrigerating chamber at 2 to 15 DEG C or 0.5 to 5 hours at a temperature of 20 to 60 DEG C in a sealed container using steam of 25 to 50 percent high concentration of glutaraldehyde solution. The crosslinking reaction was carried out and finally washed 5 to 20 times with standard buffer solution and distilled water, respectively. A process for preparing an enzyme-immobilized layer of a biosensor enzyme-immobilized membrane using a needle-like fine hydrogen peroxide electrode as a converter, which is refrigerated or stored at room temperature until use; (iii) Polyurethane, methyl cellulose, ethyl cellulose, cellulose acetate, cellulose triacetate in which the electrode tip in which the enzyme immobilization layer obtained in the above step is formed is dissolved at a concentration of 1 to 10 percent in benzene, dichloromethane, ethanol, acetone, etc. Using a needle-shaped fine hydrogen peroxide electrode as a converter, characterized in that the immersion coating 0.5 to 10 seconds in one solution selected from the solution and dried for 1 to 5 minutes at room temperature and repeating this process 1 to 15 times as necessary. Manufacturing an outer layer of the biosensor enzyme-immobilized membrane; And, (iv) A method for producing an enzyme-fixed membrane of a biosensor comprising a three-layered structure in which an enzyme-immobilized layer is prepared by applying the inner layer, the enzyme-immobilized layer, and the outer layer of the enzyme-immobilized membrane to the tip of a needle-like fine hydrogen peroxide electrode in the order of electric processes. The method of claim 1, wherein the enzyme for the production of the enzyme immobilization layer lactic acid oxidase, cholesterol oxidase, glucose oxidase using a needle fine hydrogen peroxide electrode characterized in that the use of a biosensor enzyme-fixed membrane production Way. According to claim 1, wherein the composition of the standard buffer solution used in the enzyme-immobilized solution for the enzyme-immobilized layer is 0.05 to 0.3 mol phosphate buffer solution (including pH 6 to 8, 0.01 to 0.5 mol potassium chloride) in the case of lactic acid oxidase 30 to 70 mmol phosphate buffer solution (including pH 6 to 8, 0.01 to 0.5 mol potassium chloride) for cholesterol oxidase, 0.05 to 0.3 mol phosphate buffer solution (pH 6 to 8, 0.01 to 0.5 mol for glucose oxidase) A method for producing an enzyme-immobilized membrane of a biosensor using a needle-like fine hydrogen peroxide electrode as a converter, characterized in that it comprises potassium chloride. The inner layer is formed by using one selected from polyurethane, methyl cellulose, cellulose acetate, ethyl cellulose, nafion, lauric acid, and palmitic acid dissolved in an organic solvent at the electrode tip, the sensitive part of the acicular fine hydrogen peroxide electrode. Enzyme fixation layer is formed by glutaraldehyde crosslinking, and the outer layer is formed by using one selected from polyurethane, methyl cellulose, ethyl cellulose, cellulose acetate, and cellulose triacetate dissolved in an organic solvent on the enzyme fixation layer. Enzymatic purification membrane of the biosensor consisting of a three-layered structure, the layer sandwiched between the inner layer and the outer layer of the immobilization membrane. 5. The polyurethane tip methyl cellulose, cellulose acetate, ethyl cellulose, Nafion, and La according to claim 4, wherein the electrode tip sensitive portion, which is a sensitive portion of the acicular fine hydrogen peroxide electrode, is dissolved at a concentration of 1 to 10 percent in benzene, dichloromethane, ethanol, acetone, and the like. 0.5 to 10 seconds immersion coating in one solution selected from a solution of uric acid and palmitic acid, and dried at room temperature for 1 to 5 minutes to repeat this process 1 to 15 times as necessary to form the inner layer of the enzyme-fixed membrane Take a microsyringe of 0.5-10 microliters of enzyme immobilization solution containing 2-10 mg, 2-10 mg, 10-100 microliters of oxidase, bovine serum albumin and standard buffer solution, respectively, on the inner layer. After dividing into 1 ~ 3 times, fix it in a rectangular parallelepiped container with its tip part down, then 2 ~ 15 ℃ Dry for 10 to 90 minutes in the refrigerator, immediately immersed in 0.1 to 7 percent glutaraldehyde dilution solution for 0.5 to 10 seconds, and then crosslinking for 10 to 90 minutes in a refrigerator at 2 to 15 ° C or at a high concentration of 25 to 50 percent. Using a vapor of a glutaraldehyde solution, the crosslinking reaction was carried out at a temperature of 20 to 60 ° C. for 0.5 to 5 hours in a sealed container and washed 5 to 20 times with a standard buffer solution and distilled water, respectively, to prepare an enzyme-immobilized layer of the enzyme-immobilized membrane. In a solution selected from polyurethane, methyl cellulose, ethyl cellulose, cellulose acetate, cellulose triacetate solution dissolved in benzene, dichloromethane, ethanol, acetone, etc. at a concentration of 1 to 10 percent over the enzyme immobilization layer. Immersion coating for 0.5 to 10 seconds and dry at room temperature for 1 to 5 minutes Enzyme of biosensor using needle-like fine hydrogen peroxide electrode composed of three-layer structure in which enzyme-immobilization layer is sandwiched by repeating 1 to 15 times as necessary to form outer layer of enzyme-immobilization membrane. Immobilized Film. The enzyme-fixed membrane of the biosensor using a needle-like fine hydrogen peroxide electrode as a converter, characterized in that the lactic acid biosensor, cholesterol biosensor and glucose biosensor equipped with the enzyme fixation membranes of claim 4 and 5.