JP2002162375A - Biosensor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To solve the problem that conventional biosensors cannot measure high levels of glucose nor achieve continuous measurement. SOLUTION: This biosensor is provided with a proton transmitter comprising a charge transfer element 1, a test article supply port 7, an oxygen supply port 8 and an enzyme 2, allowing a positive electrode 3 to measure highest glucose density level in no danger of insufficient oxygen supply on its oxygen- reactive surface when the positive electrode 3 is directly contact with a test article. Additionally since any blister generated on the positive electrode 3 may easily be discharged in the air, this biosensor allows continuous measurement without deterioration of charge transfer capability.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a biosensor for measuring a glucose concentration.

[0002]

2. Description of the Related Art Measurement of glucose concentration is used not only in the medical field but also in various fields such as the agricultural field and the brewing field. In particular, the measurement of glucose (hereinafter referred to as glucose) concentration in human urine has the following characteristics.

[0003] Urine is an important source of information on the health of individuals, and various functional disorders can be examined by quantitative analysis of urine components. As a means for responding to this need, there is a urine test paper used in health examinations and medical institutions. Urine test paper detects the discharge of sugar, protein, urobilinogen, occult blood, and the like into urine by a chemical reaction, and indicates the change in the color tone of the test paper. Test strips have the advantage that they are simple and can be used by anyone, but they are qualitative or semi-quantitative and have problems from the viewpoint of quantitative analysis.

Therefore, as a means for quickly and easily quantifying a specific component of urine, measurement using a biosensor that links an enzyme reaction to an electrode reaction has been performed.

FIG. 4 is an explanatory diagram for explaining the configuration and operation of a conventional biosensor. Urine that has entered from the specimen supply port 7 reaches the glucose oxidase layer 2 through the interfering substance removing film 13. The glucose oxidase layer 2 has glucose oxidase, and decomposes glucose in urine into gluconic acid and hydrogen peroxide. This hydrogen peroxide is
Furthermore, it is decomposed into hydroxyl ions and hydrogen ions. The hydrogen ions flow through the composite membrane 11 when a DC voltage is applied, and charge transfer occurs. The magnitude of this current depends on the amount of glucose contained in urine. Therefore, the amount of glucose in the urine of the subject is determined from the magnitude of the current. (Formula 1) shows this reaction.

[0006]

Embedded image

That is, electrons generated by the decomposition reaction of hydrogen peroxide shown in (Chemical Formula 1) are transmitted between the electrodes, and a current flows between the electrodes. A resistor is disposed in a circuit in which this current flows, and the voltage flowing across the resistor is measured to measure the current flowing, and this current is converted into the concentration of a specific substance.

[0008]

The above-mentioned conventional biosensor has a problem that the measurement range is limited to the measurement of glucose concentration up to 500 mg / dl, and furthermore, the continuous measurement cannot be performed. That is, the interference substance removing film has the following effects. It is to supply oxygen in the air to the urine and to limit glucose entry into the reaction layer.
However, this reaction system has the following problems (see FIG. 5). Oxygen dissolves in the urine through the interfering substance removing film, so that the dissolution rate of oxygen becomes the reaction rate-limiting.

Therefore, when a glucose concentration exceeding 500 mg / dl enters the reaction system, the proportional relationship between the glucose concentration, its output value, and the current converted to the glucose concentration is not established. There are also the following issues. An electrode active material generated by an enzymatic reaction, generally hydrogen peroxide, often adheres to the positive electrode and negative electrode surfaces of the electrode and prevents conduction between the electrode active material and the electrode. There is also a method of refreshing by applying a reverse charge between the positive electrode and the negative electrode of the electrode.
A second reverse charge conduction time and a refresh time for the electrode surface are required.

[0010]

SUMMARY OF THE INVENTION The present invention provides a biosensor for measuring the concentration of an electrode active material generated by an enzymatic reaction, which supplies an enzyme, a specimen supply section for supplying a specimen, and oxygen. It has an oxygen supply section and a detection section, and has a structure in which oxygen is dissolved in a test object and is directly supplied from the atmosphere.

Further, the detecting section comprises a charge transfer layer capable of moving the electrode active material, and a lower electrode which is electrically connected to the charge transfer layer.
One of the electrodes is brought into direct contact with the test liquid, that is, opened to the atmosphere, so that the electrode active material can be easily released into the atmosphere without deteriorating the charge transfer ability.
The biosensor is capable of continuous measurement. The biosensor having this configuration can be provided with features that are easy to use and can be processed easily.

[0012]

DETAILED DESCRIPTION OF THE INVENTION The invention according to claim 1 of the present invention provides an enzyme, an analyte supply unit for supplying an analyte to the oxidative enzyme, and an oxygen supply for supplying oxygen to the oxidative enzyme. If the biosensor is provided with a sensor and a detection unit for detecting the amount of the electrode active material, the enzyme, the decomposed substance, and oxygen necessary for the decomposition thereof are supplied at a saturated concentration, and the high-concentration decomposed substance can be measured.

In the invention according to claim 2 of the present invention, when the configuration of the detection unit is a biosensor comprising an electrode and a charge transfer body that moves ions reacting with the electrode active material, the decomposition target is decomposed and generated. A current flows through the electrode, assuming the concentration of the electrode active material as the movement of ions.

According to a third aspect of the present invention, there is provided a biosensor having a configuration in which a charge transfer body is connected to an oxygen supply unit and an oxidative enzyme is fixed to the oxygen supply unit of the charge transfer unit on the oxygen supply side. The reaction occurs at the interface between the solid layer where the reaction site of the decomposition product and the enzyme occurs, the liquid layer of the test object, and the gas layer of the oxygen supply section, and the oxygen supply failure which is the rate-limiting reaction of the enzyme does not occur.

According to the invention described in claim 4 of the present invention, if the configuration of the charge transfer body is a biosensor having a composite film, it can play the role of conducting the electrode active material and restricting the transmission of the test object.

The invention described in claim 5 of the present invention has shown the structure of the composite membrane. In other words, the membrane having pores has the effect of limiting the total amount entering the three-layer interface because the decomposed substance in the specimen reacts with the enzyme, and the substance other than the decomposed substance in the specimen reacts. This has the effect of making the structure difficult to penetrate into the system, and the effect of dissolving oxygen into the test object through the film. However, since this film is inferior in the ability to transfer electric charges, the biosensor has a composite film structure including a material capable of transferring electric charges.

The invention according to claim 6 of the present invention has a membrane structure having pores. If the biosensor is a polycarbonate film having pores, the pores penetrate straight from the front to the back of the membrane. Can be ensured, and conduction is achieved over a short distance. It is not necessary to consider the film degradation effect by the enzyme that decomposes the film mixed in the test object. Further, it is possible to obtain a material having a small thickness, which is useful for shortening the conduction distance.

The invention described in claim 7 of the present invention is a biosensor having a composite film structure of polyacrylamide capable of transferring electric charges to a polycarbonate film having pores. Polyacrylamide is acrylamide,
It is a material obtained by polymerizing bisacrylamide and ammonium persulfate. After the polycarbonate film is impregnated with a liquid in which acrylamide and bisacrylamide are dissolved, heat-polymerization or the like fills the pores of the polycarbonate film with the ion-rich gel-like substance. This polyacrylamide participates in conduction of the electrode active material.

The invention described in claim 8 relates to the degree of crosslinking of polyacrylamide. Polyacrylamide is a gel material commonly used for electrophoresis of proteins in the field of biochemistry. Proteins migrate through the polyacrylamide network. The degree of crosslinking determines the size of the network of the network structure. When the degree of cross-linking is increased, the network structure becomes smaller, and when the degree of cross-linking is lowered, the network structure becomes larger. That is, a faint net or a fine net can be formed depending on the degree of crosslinking. Explaining the reason for making the network structure fine in the present invention, the molecular weight of glucose is about 100. There are various components other than glucose, such as proteins and vitamins, but almost all of them are higher molecules than glucose. There are even thousands of proteins with high molecular weight. In other words, it has a smaller molecular weight that must be rejected as compared with those used at the biochemical research level. For the above reasons, the degree of polymerization was set to four times. Incidentally, the polymerization degree of polyacrylamide is limited to a tetraploid.

According to a ninth aspect of the present invention, there is provided a biosensor in which the size of the test object supply unit and the size of the oxygen supply unit are the same or the amount of oxygen supply is increased due to the larger oxygen supply port. An increase in the oxygen supply area has the effect of reducing the risk of limiting the reaction of the enzyme reaction. The present invention only secures the amount of oxygen supplied from the oxygen supply port, and it is sufficient that the amount of oxygen necessary for the reaction can be secured.

[0021] The invention described in claim 10 of the present invention describes an enzyme addition position. By applying the enzyme without spreading it over the entire circle of the oxygen supply port, oxygen is directly supplied to the reaction system from the oxygen supply port where the enzyme is not applied. This makes it possible to supply a biosensor that has the effect that the oxygen supply port fully fulfills its role.

In the invention according to claim 11 of the present invention, the enzyme is glucose oxidase. An enzyme that requires oxygen in an enzymatic reaction is glucose oxidase. Furthermore, the biosensor can be eliminated even if oxides are generated, and can perform accurate measurement.

According to the twelfth aspect of the present invention, since the electrode connected to the sample supply unit is a positive electrode, the electrode active material can be easily released into the atmosphere without deteriorating the charge transfer ability. And a biosensor capable of continuous measurement.

According to a thirteenth aspect of the present invention, the negative electrode is configured to be in contact with the composite membrane, and the composite membrane acts as an electron carrier, thereby enabling accurate measurement and continuous measurement. Sensor.

[0025]

Embodiments of the present invention will be described below. FIG. 1 is a cross-sectional view illustrating the configuration of the biosensor of the present embodiment. The biosensor of this embodiment has a base (5a to 5d).
And a negative electrode (4) provided on a base (5c to 5d)
And a charge transfer body (1) provided to conduct to the negative electrode (4), a positive electrode (3) provided on the charge transfer body (1), and a base (5a-b) thereon. There is. The upper and lower portions of the charge transfer body (1) are constituted by an analyte supply port (7), an oxygen supply port (8), and an enzyme (2).

There is no particular limitation on the material of the base (5a to 5c) as long as it has a strength capable of maintaining the structure. In this embodiment, a molded article of polyethylene terephthalate is used. . The base (5a) and the base (5b) in the uppermost layer have an appropriate space, and this space is a test object supply port (7) for sampling the test object. The lowermost base (5c) and the base (5d) have an appropriate interval, and this interval is an oxygen supply port (8) for collecting oxygen.

The negative electrode (4) is provided on a base (5c-5d), and the positive electrode (3) is provided on a charge transfer body (1) and is held down by the bases (5a-5b). The positive electrode (3) and the negative electrode (4) are partially open to the atmosphere.

FIG. 2 is a schematic view of the charge transfer member (1). The charge transfer body (1) is composed of a membrane film. The membrane film has a configuration in which pores are directly opened from the front to the back having pores with a very small diameter, and the pores are impregnated with polyacrylamide.
This membrane selectively permeates glucose (9), which is a test substance, and has a huge solid component other than glucose, for example,
Urine proteins (mainly albumin) are removed.
This membrane is also permeable to all oxygen.
Further, the charge transporter (1) has a role of holding the enzyme (2), and its position is at the oxygen supply port (8). The enzyme (2) is not applied to the entire oxygen supply port (8), but oxygen is supplied directly to the reaction surface between the glucose on the charge transfer body (1) and the enzyme (2). I have.

In this embodiment, glucose oxidase is used as the enzyme (2), and in this embodiment, the enzyme is provided by directly adsorbing the charge transfer body (1). Therefore, the free carbon atom of the charge transfer material (1) and the carbon atom of the enzyme (2) are directly bonded or bonded via an enzyme molecule. For this reason, the enzyme (2) adsorbed on the surface of the charge transfer body (1) never has a layer structure and has a porous structure.

FIG. 3 shows a new case of the biosensor of the present invention. A pair of electrodes (3, 4) is used as a charge transfer body (1)
The electrode active material can be measured by the enzyme (2) also in a method in which the electrode active material is placed above and below.

The operation of the product of the present invention will be described. The test article contains thousands of chemicals. The biosensor of the present embodiment measures the sugar content, which is one of the indices indicating the health information of the human body. The sugar content is measured by representing glucose.

The test object is supplied to the test object supply port (7) while a DC voltage of about 1 V or less is applied from a DC power supply (not shown) between the negative electrode (4) and the positive electrode (3). The test object supplied to the test object supply port (7) enters the charge transfer body (1). As shown in FIG. 3, the charge transfer body (1) is a straight hole from the front to the back having an extremely small diameter hole, and is a large solid component other than glucose, for example, protein, Albumin as the main component,
Are removed, glucose can pass through the pores to the reaction system, and oxygen can pass through the charge transfer body (1) as a whole. Therefore, the components that can enter the charge transfer body (1) are relatively low molecular weight components and oxygen. At this time, in this embodiment, the enzyme (2) is arranged above the charge transfer body (1). The enzyme (2) selectively reacts only with glucose and does not react with other components.

Thus, glucose is decomposed by the enzyme (2). At this time, in this example, glucose oxidase is used as the enzyme (2) as described above. Glucose oxidase decomposes glucose in a test substance into gluconic acid and hydrogen peroxide as described in the conventional example (Chem. 1). This hydrogen peroxide is further decomposed into hydroxyl ions and hydrogen ions. This hydrogen ion is
When a DC voltage is applied between the negative electrode (4) and the positive electrode (3), it flows through the charge transfer body (1) and flows between the negative electrode (4) and the positive electrode (3).

That is, the generated hydrogen ions are transmitted into the charge transfer body (1), exchanged with the hydrogen ions in the composite membrane, and the hydrogen ions are guided toward the negative electrode (4). Thus, a current flows between the positive electrode (3) and the negative electrode (4).

A resistor is arranged in a circuit through which this current flows, and the current flowing is measured by measuring the voltage across the resistor, and this current value is converted into a glucose concentration. The magnitude of this current depends on the amount of glucose contained in the test object. Therefore, the glucose concentration of the test substance is determined from the magnitude of the current.

At this time, the DC voltage applied between the negative electrode (4) and the positive electrode (3) is 1 as described above.
Less than about V is preferred. The reason for this is that when the applied DC voltage increases, the current flowing due to the electrolysis of water in urine does not correspond to the amount of glucose.

At this time, in the present embodiment, the positive electrode (3) is provided at a position in direct contact with the test object. For this reason, bubbles attached to the vicinity of the positive electrode (3) generated as a result of the reaction are easily released into the atmosphere. With the bubbles attached to the electrodes, the charge transfer ability is not reduced, and therefore, the sugar content of the test substance can be continuously measured.

Further, according to the present embodiment, the enzyme (2) does not come into direct contact with the specimen supply port (7), and is placed on the portion facing the charge transfer body (1) and the oxygen supply port (8). The test substance component that has penetrated into the inside is released into the atmosphere because it is applied and sustained release is ensured. For this reason, the biosensor of this embodiment does not have to be disposable each time it is used, but can be used repeatedly.

It is to be noted that a one-time-use disposable type biosensor is required to avoid contamination as much as possible in clinical tests, such as measuring glucose concentration in a patient's blood and checking fermentation processes in the brewing industry. Fields that must not be
It is useful in areas where infection must be prevented and numbers must be managed.

The biosensor developed this time measures glucose concentration, which is one of the indices indicating human health information. Means for measuring glucose concentration include those based on chemical reactions and those that measure optical rotation, refractive index, and infrared absorption in addition to the bio-based method using enzymes, but have lower substrate specificity than the bio-based method. The glucose concentration may be calculated to be lower than the actual value due to a negatively acting factor such as being easily affected by other urine components. The enzyme method adopted in the present invention has solved this problem. When glucose oxidase is used, it selectively reacts with glucose only and does not react with other substances.

Glucose (glucose) is decomposed by an enzyme (glucose oxidase) and decomposed into gluconic acid and hydrogen peroxide as a by-product. This hydrogen peroxide is decomposed into hydroxyl ions and hydrogen ions. The hydrogen ions serve as an electron carrier, migrate through the polyacrylamide portion impregnated in the charge transfer body (1), and are guided toward the negative electrode of the electrode. At that time, the current flows. If the electron carrier is a gel of an electrolyte having free anions capable of migrating, electrons are transferred even if the electrolyte is not polyacrylamide, and a current flows.

The polymerization of polyacrylamide, the flow of electric current and its durability will be described. Basically, there is no correlation between the polymerization of polyacrylamide and the current flow. This is a one-fold cross-link, and the number of ions necessary for the transmission of hydrogen ions is in a large or small state. Therefore, there are so many ions that the correlation cannot be obtained.

When preparing a polyacrylamide gel for biochemical research, tetramethoxyethylenediamine is used as a polymerization initiator. Since this medicine reacts instantaneously, it is difficult to add the medicine at a proper timing. In addition, there is a risk of uneven contact with the drug. Therefore, in the present invention, a biosensor is formed by means of crosslinking by heating for several minutes to several tens minutes. A polymerization initiator may be used as long as crosslinking can be performed evenly on the polycarbonate film. Further, the polymerization may be performed by ultraviolet irradiation.

Next, the features of the present invention will be described with reference to FIG. The specimen is guided into the biosensor from the specimen supply port (7). At the top of the sensor, a charge transfer body (1) made of porous polycarbonate impregnated with polyacrylamide gel is provided, only glucose and oxygen pass through the pores, and other large solid components, for example,
Proteins (mainly albumin in the case of urine) are removed. Further, oxygen can enter the inside of the reaction system from the entire surface of the film. Thus, components that can enter the reaction system constituted by the charge transfer body (1), the enzyme (2), and the oxygen supply port (8) are limited to relatively low-molecular urine components and oxygen. on the other hand,
In the present invention, by contacting the electrode with the sample before filtration, not only the anionic substance of the sample, but also by-products such as oxygen generated by the enzyme reaction are guided toward the positive electrode, and the sample supply port It can be easily released to the atmosphere via (7). The enzyme (2) that degrades glucose in the sample and glucose oxidase were directly adsorbed to the charge transfer material (1). The connection between the charge carrier and the enzyme is made physically.
Glucose oxidase adsorbed on the surface of the charge transfer body (1) is considered to have the above-mentioned bond, and thus does not take a layer structure but has a porous structure. In addition, the enzyme (2) is present at a location composed of the charge transfer body (1) and the oxygen supply port (8), has a role of retaining the enzyme (2), and is greatly involved in its sustained release. . The biosensor having such a configuration is not one-time disposable but can be repeatedly measured.

On the other hand, a one-time use disposable type biosensor must avoid contamination as much as possible in clinical tests, such as measuring glucose concentration in a patient's blood and checking fermentation processes in the brewing industry. In fields and in fields where numbers must be managed, there is an effect of disposable measurement.

FIG. 2 shows glucose (9) in the charge transfer medium.
FIG. 4 is a schematic diagram showing permeation of oxygen and oxygen (10).

FIG. 3 shows a configuration that can achieve the same effect as that of the biosensor shown in FIG. 1 and that includes a pair of electrodes.

FIG. 4 shows a conventional biosensor. By-products decomposed by glucose oxidase are guided to the electrode (12) in the lowermost layer of the biosensor, and when a foam is formed on the surface of the electrode, transmission of hydrogen ions is hindered, and the current value is reduced. In order to solve this phenomenon, it is necessary to apply a reverse current to forcibly remove the foam attached to the vicinity of the positive electrode. There is a problem that a biosensor that requires this process cannot perform continuous measurement. The present invention has solved this problem.

Hereinafter, the contents of the present invention will be specifically described with examples.

(Embodiment 1) (Table 1) shows the features of the present invention and the features of the conventional invention. The conventional invention uses the biosensor having the configuration shown in FIG. The measurement was performed using an artificial urine in which glucose was dissolved, and a test liquid having a concentration increased every 500 mg / dl. The number of continuous measurements was measured using an automatic measuring instrument, and glucose concentration of 0, 5,
The cycles were continuously measured in the order of 00, 2000, 750, 4000, and 100 mg / dl, and the number of measurements was determined from the number of cycles.

[0051]

[Table 1]

From Table 1, the measurement range is larger for the present invention and the conventional invention. The reaction system in which the enzyme reacts with glucose is a liquid layer and a solid layer in the conventional example. However, in the present invention, a three-layer interface of the liquid layer, the gas layer, and the solid layer is provided, and oxygen sufficient for the enzyme reaction is sufficiently supplied. It depends. Glucose concentration 500mg / d with biosensor for healthy people
If you can measure up to l, it is a sufficient measurement range to manage the physical condition from the change. On the other hand, the measurement range is not sufficient for measuring the glucose concentration excreted from urine of a diabetic patient. To measure glucose concentration from urine of diabetic patients, the ability to measure up to 2000 mg / dl is required. The present invention satisfies this need.

Example 2 In Example 2, the concentration of dissolved oxygen applied to the reaction was measured. The product of the present invention is the amount of oxygen generated from an electrode when the electrode is in direct contact with urine in a biosensor, and the amount of oxygen generated when the electrode is not in direct contact with urine is measured using a dissolved oxygen meter in a conventional product. . The measurement temperature was 34 ° C., and the concentration of glucose dropped on the biosensor was 500 mg / dl. Table 2 shows the results. The dissolved oxygen concentration is shown in ppm order.

[0054]

[Table 2]

As can be seen from the results, the amount of oxygen dissolved in the solution due to the foaming phenomenon is larger in the product of the present invention. In other words, if the positive electrode in contact with the raw urine is configured to be in contact with the atmosphere, the negatively charged substance in the raw urine is attracted to the vicinity of the positive electrode, from which it is easily released into the atmosphere. The glucose oxidase used in the present invention is decomposed into gluconic acid and hydrogen peroxide when glucose is decomposed. In this case, the hydrogen peroxide quickly becomes negatively charged ions in the solution. This is drawn to the positive electrode (3). Therefore, the combination of the electrode in contact with the raw urine is the positive electrode, the enzyme used for the reaction is the oxidase, and the combination of glucose oxidase is a pair.

Next, measurement of electric charge in the enzyme reaction will be described. According to Japanese Patent Nos. 2940007 and 2943700, a charge transfer body (1) in which a polycarbonate film is impregnated with polyacrylamide is used for charge transfer. In the present invention, the role of the hydrogen ion has been played as one involved in the electron transfer in the charge transfer body (1).

Example 3 The sensor shown in FIG. 1 was used for measurement. The glucose used for the measurement is from 50mg / dl to 50
It was dissolved in artificial urine to a concentration of 00 mg / dl. The measurement was expressed as voltage by passing the current through a resistor. Table 3 shows the results. As can be seen from Table 3, the enzyme,
The sensor without glucose oxidase only measured phosphate-buffered saline, and even measured only 500 mg / dl glucose, no increase in voltage was observed. Even when the enzyme was sealed, no voltage value was observed in the phosphate buffered saline. However, in the case where the enzyme was sealed, an increase in the voltage value was observed, and there was a relationship between the concentration gradient and the linearity. From these results, the polycarbonate film impregnated with polyacrylamide controls charge transfer. The enzyme used for the measurement was glucose oxidase (320,000 units) manufactured by Amano Pharmaceutical.

In the same manner as in Example 3, a control urine (manufactured by Bio-Rad) whose glucose was adjusted to 5000 mg / dl was measured for the voltage value. The voltage was not measured in urine, but in the case where glucose was added, the value measured when the glucose buffer was adjusted to 5000 mg / dl in the phosphate buffered saline shown in Example 2 was almost equal to the value. Similar values were measured.

[0059]

[Table 3]

(Embodiment 4) The composition of the charge transfer member will be described with reference to FIG. The charge transfer body is formed by layering a polyacrylamide gel on a polycarbonate film.
The pores and entirety of the polycarbonate film are buried in polyacrylamide. The polyacrylamide in which the pores are sealed is involved in the transfer of hydrogen ions. In other words, the pores are filled with an electrolyte, and hydrogen ions are exchanged between the electrodes. This problem is a means for impregnating a polyacrylamide into a polycarbonate film.

The polycarbonate film has vertical pores on both sides. Hydrogen ions are transmitted through the pores, and as a result, current flows. Considering that one hydrogen ion passes through the pore, the moving distance is such that an ion of 10 ⁻⁸ μm moves over a width of 40 μm, and travels an enormous distance. Therefore, when the electrolyte is sealed in the pores, the hydrogen ions generated in the enzymatic reaction do not need to travel a huge distance by themselves, and the hydrogen ions generated by the enzymatic reaction react with the electrolyte in the vicinity, The hydrogen ions propagate through the electrolyte, and the labor of one hydrogen ion is reduced.

Further, this film is characterized in that oxygen passes even where there are no pores in the polycarbonate film. Polyacrylamide has a network structure.
It is the degree of polymerization that determines the size of the mesh. As the degree of polymerization increases, the rejection molecular weight decreases, and it becomes difficult for those having a low molecular weight to enter the inside of polyacrylamide. What is necessary for the reaction system in the present invention is glucose, and the others should be excluded as much as possible. For the above reasons, the degree of polymerization was set to be equivalent to 4-fold polymerization.

Next, a method for polymerizing polyacrylamide will be described. The polymerization means includes a method using tetramethoxyethylene amide as a polymerization initiator, a method using heating, and a method using ultraviolet irradiation. A mild reaction cannot be expected by using a chemical, and the polycarbonate film cannot be held in a film form. Ultraviolet irradiation requires a protective device as well as an ultraviolet lamp, so it is not desirable to use it. Therefore, polymerization by heating was washed. When a polycarbonate film is impregnated with a liquid in which acrylamide and bisacrylamide are dissolved before the start of polymerization, the liquid is taken into the pores by capillary action. It was polymerized by heating. When the polymerization temperature is in the range of 30 ° C. to 70 ° C. and the polymerization is carried out under mild conditions, the polymerization can be carried out evenly in the pores of the polycarbonate film.

Based on the above, it will be described whether or not a current flows through the electrode of this configuration. In the electrode structure shown in FIG. 1, a vinyl chloride sheet was used instead of the charge transfer body (1). At the electrode, the reaction current of glucose oxidase was measured. Table 4 shows the results. The measurement was performed using a measuring instrument set so that the applied voltage was 1 V and the current value when glucose was not added was all 0 A. The glucose used for the measurement was 100 mg / dl and used was dissolved in phosphate buffered saline. No numerical value was detected for vinyl chloride. In the case where the charge transfer body is a single polycarbonate film, current is detected. However, since the pores of the polycarbonate film are not sealed, the enzyme flows out along with the reaction, and measurement cannot be repeated.

In this embodiment, a current flows if the film is a film having pores. Furthermore, any material involved in the transfer of electric charge by enclosing the electrolyte in the pores can be selected depending on the use environment. For example, when measuring the glucose concentration in urine, it is necessary to consider the following. There are various enzymes in urine. For example, diastase, cellulase and the like. There is a cellulose nitrate filter having pores and a membrane structure. Since a cellulose filter is decomposed by cellulase, a cellulose nitrate filter cannot be used for a sensor that is used repeatedly a plurality of times.

[0066]

[Table 4]

[0067]

Industrial Applicability The present invention provides a mechanism of a sensor in which an enzyme, a substance to be decomposed and oxygen required for its decomposition are supplied at a saturated concentration, and the accuracy of enzyme quantification is increased, and a high-concentration glucose is detected instantaneously. be able to.

[Brief description of the drawings]

FIG. 1 is a configuration diagram of a biosensor according to an embodiment of the present invention (1).

FIG. 2 is a schematic view showing the limited permeation of glucose and oxygen in a charge transfer body.

FIG. 3 is a configuration diagram (2) of a biosensor according to an embodiment of the present invention.

FIG. 4 is a configuration diagram of a conventional biosensor.

FIG. 5 is a diagram showing the function of an interference substance removing film of a conventional biosensor.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Charge transfer body 2 Enzyme 3 Positive electrode 4 Negative electrode 5a-d Base 6 Seal 7 Specimen supply port 8 Oxygen supply port 9 Glucose 10 Oxygen 11 Interference substance removal film 12 Electrode

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/66 G01N 27/46 338 F term (Reference) 2G045 AA16 CB03 DA31 FA34 FB01 FB05 4B029 AA07 BB16 CC05 CC11 FA10 FA12 4B063 QA01 QA05 QA18 QQ67 QR03 QR82 QS26 QS28 QS39 QX05

Claims (13)

[Claims] 1. An oxidative enzyme, an analyte supply unit for supplying an analyte to the oxidative enzyme, an oxygen supply unit for supplying oxygen to the oxidative enzyme, and detection for detecting an amount of an electrode active material. Biosensor provided with a part. 2. The biosensor according to claim 1, wherein the detection unit comprises an electrode and a charge moving body that moves ions that react with the electrode active material. 3. The biosensor according to claim 2, wherein the charge transfer body is connected to the oxygen supply unit, and the oxidative enzyme is fixed to the oxygen supply unit of the charge transfer unit on the oxygen supply unit side. 4. The biosensor according to claim 1, wherein the charge transfer member is a composite film. 5. The biosensor according to claim 4, wherein the composite film has a film structure having pores and is made of a material capable of transferring electric charges. 6. The biosensor according to claim 1, wherein said membrane having a membrane structure is a polycarbonate film having a pore processing. 7. The biosensor according to claim 1, wherein the charge transferable material is polyacrylamide. 8. The biosensor according to claim 7, wherein the polyacrylamide is cross-linked four times. 9. The biosensor according to claim 1, wherein the size of the analyte supply unit and the oxygen supply unit is large enough to ensure an absolute supply amount of oxygen. 10. The biosensor according to claim 1, wherein the enzyme does not spread over the entire circle of the supply port provided in the oxygen supply section. 11. The biosensor according to claim 1, wherein the enzyme is glucose oxidase. 12. The biosensor according to claim 1, wherein the electrode connected to the sample supply unit is a positive electrode. 13. The biosensor according to claim 1, wherein the electrode connected to the oxygen supply unit is a negative electrode.