JPH085601A - Enzyme sensor and its production - Google Patents

Abstract

PURPOSE:To obtain a stabilized high sensitivity enzyme sensor having long service life by providing an enzyme film layer containing a non-crosslinked hydrophilic polymer and an enzyme dispersed into the non-crosslinked hydrophilic polymer and crosslinked. CONSTITUTION:The enzyme sensor comprises a columnar working electrode 1 housed in a tubular reference electrode 2 through an insulator 6, and an enzyme film layer 3 touching the electrodes 1, 2 on the outside. Preferably, the electrode 1 is made of a novel metal, e.g. platinum or gold, and the electrode 2 is made of silver or silver chloride. The film layer 3 contains a non-crosslinked hydrophilic polymer, and an enzyme dispersed into the non-crosslinked hydrophilic polymer and crosslinked therewith through a crosslinking agent. The enzyme includes glucose oxidase, lactose oxidase, etc., and glutaraldehyde is preferably employed as a crosslinking agent. This method produces a stabilized miniature enzyme sensor having long service life and excellent in sensitivity.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an enzyme sensor and a method for producing the enzyme sensor, and more specifically, with high sensitivity,
The present invention relates to an enzyme sensor having excellent stability and long life, and a method for producing an enzyme sensor capable of manufacturing an enzyme sensor having high sensitivity, excellent stability, and long life by a simple method.

[0002]

2. Description of the Related Art Conventionally, an amperometric hydrogen peroxide electrode type glucose sensor is known as an enzyme sensor for measuring the glucose concentration in a sample solution.

This glucose sensor comprises a working electrode and a reference electrode, and an enzyme membrane which is a polymer membrane on which an enzyme acting on glucose is immobilized. Glucose oxidase is often used as the enzyme acting on glucose.

The glucose concentration in the sample solution is measured by this glucose sensor as follows. That is, this glucose sensor is immersed in a sample solution containing glucose. Upon immersion, gluconolactone and hydrogen peroxide are produced from glucose in the sample solution by the catalytic action of glucose oxidase immobilized on the electrode surface. The hydrogen peroxide produced is decomposed into water and oxygen at the working electrode. As a result of this, a current proportional to the glucose concentration flows between the working electrode and the reference electrode. Therefore, by measuring this current value, the glucose concentration in the sample solution can be indirectly calculated.

As a method for immobilizing an enzyme on an electrode in a conventional enzyme sensor, a cross-linking method, a covalent bond method, an entrapping method, an adsorption method and the like are known. Among these, the crosslinking method is widely adopted because the immobilization operation is simple. As the cross-linking method, a method of forming a cross-link between the enzymes by using a cross-linking agent such as glutaraldehyde to bond the enzymes to each other and immobilizing the enzyme, and adding a matrix substance such as albumin to the enzyme and the matrix substance An example is a method of immobilizing an enzyme together with a matrix substance by forming a bridge between them.

[0006] However, in the enzyme sensor provided with the enzyme membrane by the conventional cross-linking method, the permeation and diffusion of the substance is likely to be poor due to the close bonding of the enzymes, and as a result,
The sensitivity of the sensor is low, the reproducibility is poor, and the service life is short. Further, there is a problem that the stability against sterilization with ethylene oxide gas is poor.

Furthermore, in recent years, a glucose sensor has been attracting attention for the purpose of continuously monitoring the glucose concentration in blood vessels or subcutaneous tissues of diabetic patients and the like, and it can be used for such purposes. It has been desired to develop a glucose sensor, that is, a small size, high sensitivity, higher stability, and longer life glucose sensor. Further, not only glucose but also other substances are similarly measured, and similarly, development of an enzyme sensor having excellent sensitivity and stability and long life has been desired.

The present invention has been completed based on the above circumstances. That is, an object of the present invention is to provide an enzyme sensor which eliminates the drawbacks of the conventional enzyme sensors. Another object of the present invention is to provide an enzyme sensor that is small in size, has excellent sensitivity and stability, and has a long life. Another object of the present invention is to provide a method for producing an enzyme sensor, which can produce an enzyme sensor having such excellent properties by a simple method.

[0009]

As a result of intensive studies conducted by the present inventors in order to solve the above-mentioned problems, as a result, an electrode having a working electrode and a reference electrode or the outside of the working electrode has a non-crosslinking hydrophilic property. It has been found that an enzyme sensor characterized by having an enzyme membrane layer containing a molecule and a crosslinked enzyme dispersed in a non-crosslinking hydrophilic polymer can achieve the above object. The invention was completed. The present invention will be described in detail below. The enzyme sensor of the present invention has an enzyme membrane layer on an electrode composed of a working electrode and a reference electrode or on the outside of the working electrode. As the electrode composed of the working electrode and the reference electrode, an electrode in which the working electrode and the reference electrode are in contact with each other via an insulator can be preferably adopted.
Among them, an electrode in which a rod-shaped or column-shaped working electrode 1 is housed through an insulator 6 inside a cylindrical reference electrode 2 as shown in FIG. 1 can be preferably adopted.

The material of the working electrode or the reference electrode is
The working electrode is not particularly limited as long as it can function as a working electrode or a control electrode, respectively. The working electrode is preferably a noble metal such as platinum or gold, and the control electrode is preferably silver or silver chloride.

The electrode consisting of the working electrode and the control electrode has an enzyme membrane layer on the outside thereof. As an embodiment, as shown in FIG. 1, the enzyme membrane is formed on the surfaces of the working electrode 1 and the control electrode 2. Layer 3 may be in direct contact,
One or more other layers may be provided on the surfaces of the working electrode and the control electrode, and the enzyme membrane layer may be provided via these layers. Examples of the other layer include a hydrophilic semipermeable membrane layer described later and the like.

Similarly, in an enzyme sensor having an enzyme membrane layer on the outside of the working electrode, as an embodiment having an enzyme membrane layer, as shown in FIG. 2, the enzyme membrane layer is formed on the surface of the working electrode 1 having an insulator 6. 3 may be in direct contact, or may have one or more other layers on the surface of the working electrode, and may have an enzyme membrane layer via these layers.

When an enzyme sensor having an enzyme membrane layer on the outside of the working electrode is used to measure the concentration of glucose or the like, another reference electrode is necessary in addition to this working electrode.

The working electrode and the reference electrode may have any shape such as a rod shape, a needle shape, a cylindrical shape, a flat plate shape.

The enzyme membrane layer in the present invention contains a non-crosslinking hydrophilic polymer and an enzyme dispersed and crosslinked in the non-crosslinking hydrophilic polymer.

The term "non-crosslinkable" means that the hydrophilic polymer itself does not crosslink at neutral and at room temperature due to the crosslinking agent used for immobilizing the enzyme. Whether or not the hydrophilic polymer is non-crosslinkable can be determined by whether or not the solubility of the hydrophilic polymer is substantially changed before and after the crosslinking reaction. That is, if the solubility does not substantially change, it is judged to be non-crosslinking. The hydrophilic property means that the water absorption is 5% or more, preferably 15% or more.

The non-crosslinking hydrophilic polymer that can be used in the present invention is not particularly limited as long as it is a non-crosslinking and hydrophilic polymer, but it is not biocompatible. Good, that it can be uniformly mixed with the enzyme without adversely affecting the activity of the enzyme, and from the viewpoint of required properties such as being water-insoluble, polyvinyl alcohol, metal alginate, methyl cellulose, ethyl cellulose, Examples thereof include hydroxyethyl cellulose, carboxymethyl cellulose, sodium polyacrylate, polyethylene oxide and the like.

These polymers become water-soluble depending on the molecular weight and the number of functional groups, but they are dissolved in water or high-temperature water as a raw material, and after they are formed into a film by coating and drying, they are kept at room temperature. It is preferable to select a polymer having a molecular weight (degree of polymerization) or a number of functional groups that does not dissolve in water. However, if a solvent system is used in which the cross-linking agent and the enzyme can be mixed together and the cross-linking reaction of the enzyme is possible, a polymer that is insoluble in water may be used. A polymer that dissolves in water at room temperature after film formation cannot be used when the object to be measured is a component in an aqueous solution, which is not preferable in view of the intended use of this film.

In the cross-linking reaction in the present invention, since an enzyme is present, it is preferable to select a substantially neutral condition at room temperature that does not inactivate the enzyme. Examples of the cross-linking agent capable of forming cross-links under such conditions include glutaraldehyde and formaldehyde. However, when the glutaraldehyde is used as the cross-linking agent, the various polymers described above are non-cross-linkable. Can be used as the hydrophilic polymer. Among these, it is preferable to employ polyvinyl alcohol as the non-crosslinking hydrophilic polymer.
On the other hand, when formaldehyde having high reactivity is used as a cross-linking agent, it is required to be non-cross-linking. Therefore, among the above-mentioned various polymers, low-reactivity polymers such as polyethylene oxide are preferable. .

The non-crosslinkable hydrophilic polymer in the present invention can be appropriately selected according to the enzyme, crosslinking agent, etc. used in the system of the crosslinking reaction, and is not limited to a specific polymer. Absent.

When polyvinyl alcohol is adopted, the degree of polymerization of polyvinyl alcohol is usually 1,000.
To 2,500, preferably 1,500 to 2,000
0. When polyvinyl alcohol in the above range is used, an enzyme sensor having particularly excellent properties can be obtained.

The enzyme membrane layer in the present invention contains the enzyme dispersed in the non-crosslinkable hydrophilic polymer and crosslinked by the crosslinking agent.

The enzyme is not particularly limited, and examples thereof include glucose oxidase, lactose oxidase,
Examples thereof include alcohol oxidase and uricase. As the cross-linking agent, it is possible to use a cross-linking agent known per se suitable for each enzyme,
Among them, glutaraldehyde can be preferably used.

The method of forming the enzyme membrane layer on the outer side of the electrode composed of the working electrode and the reference electrode or the working electrode is as follows:
There is no particular limitation as long as the enzyme can be crosslinked in the presence of the non-crosslinking hydrophilic polymer, for example, a solution containing the non-crosslinking hydrophilic polymer and the enzyme, a working electrode and a reference electrode. After dipping and drying the electrode or working electrode consisting of, or applying a solution containing a non-crosslinking hydrophilic polymer and an enzyme to the electrode consisting of the working electrode and the control electrode or working electrode, and drying. After that, a method of treating with a cross-linking agent, a mixed solution of a non-cross-linkable hydrophilic polymer and an enzyme is applied on a support and dried to form an enzyme film, which is then peeled off from the support. Examples thereof include a method of forming an enzyme membrane layer by coating the electrode directly or via another layer.

Among these methods, the electrode consisting of the working electrode and the control electrode or the working electrode is immersed in a solution containing a non-crosslinking hydrophilic polymer and an enzyme and dried, or after the non-crosslinking treatment. A method of applying a solution containing a hydrophilic hydrophilic polymer and an enzyme to the electrode consisting of the working electrode and the control electrode or the working electrode, and drying and then treating with a crosslinking agent is simple in operation, It is particularly preferable because an enzyme membrane layer having a uniform thickness and adhered to the electrode can be obtained without damage to the membrane due to peeling or the like.

When glucose oxidase is used as the enzyme to be immobilized and polyvinyl alcohol is used as the non-crosslinking hydrophilic polymer, it is preferable to form the enzyme membrane layer as follows. That is, glucose oxidase is 1 to 10%, particularly preferably 3
~ 7% and polyvinyl alcohol 1-5%,
Particularly preferably, an electrode composed of a working electrode and a control electrode which may have other layers on the surface or a working electrode is immersed in a solution containing 2 to 3% and dried. Next, a crosslink is formed by immersing in a solution containing 1 to 10%, particularly preferably 3 to 7% of glutaraldehyde and drying. In this case, a method of sequentially applying the two solutions instead of dipping the electrodes in the two solutions can also be suitably adopted.

In the enzyme sensor according to the present invention,
It is possible to have an enzyme membrane layer on the surface of the electrode composed of the working electrode and the reference electrode or the working electrode, but between the electrode composed of the working electrode and the reference electrode or the working electrode and the enzyme membrane layer. More preferably, it has a hydrophilic semipermeable membrane.
By having such a hydrophilic semipermeable membrane, it is possible to eliminate the influence of interfering substances existing in the body such as ascorbic acid and uric acid on the measurement accuracy. As such a hydrophilic semipermeable membrane, a membrane of a cellulose derivative such as cellulose acetate or cellulose propionate can be preferably adopted.

The method for forming the hydrophilic semipermeable membrane is not particularly limited, and it may be formed, for example, by immersing an electrode consisting of a working electrode and a control electrode in a solution of a cellulose derivative or a working electrode and drying. it can.

Even when the hydrophilic semipermeable membrane is formed on the surface of the electrode as described above, the enzyme membrane layer can be formed on the surface of the hydrophilic semipermeable membrane by the method described above.

In the enzyme sensor of the present invention, as shown in FIG. 3, for example, it is preferable to have a porous membrane permeable to the substance to be measured on the outside of the enzyme membrane layer. The presence of the porous film improves the adhesion between the enzyme film layer and the electrode. As the porous film, a film made of polyurethane, polycarbonate or cellulose acetate can be preferably used.

The method for forming the porous membrane is not particularly limited, and it can be formed, for example, by immersing the electrode having the enzyme membrane layer in a polyurethane, polycarbonate or cellulose acetate solution and drying.

In the present invention, the thickness of the electrode comprising the working electrode and the control electrode or the layer including the enzyme membrane layer formed outside the working electrode is usually 5 to 50 μm, preferably 10 to 20 μm. When the thickness is within the above range, molecules such as glucose and oxygen can be rapidly permeated and diffused into the film, and the electrode having good responsiveness and small output variation is preferable.

[0033]

The present invention will be described in detail below with reference to examples. The present invention is not limited to the embodiments.

(Example) Five needle type electrodes consisting of an anode made of platinum and a cathode which is a silver / silver chloride electrode were prepared, and each was immersed in a 3% cellulose acetate solution and dried to obtain the above. A film made of cellulose acetate was formed on the electrode surface. Then, the tip of the electrode having the cellulose acetate coating was treated with glucose oxidase at 5% and polyvinyl alcohol having a degree of polymerization of 1700 at 2.
The tip surface of the electrode was further coated with an enzyme membrane layer by immersing in a mixed solution containing 5% and drying.
Then, the tip of this electrode was immersed in a 5% glutaraldehyde solution and dried to crosslink the glucose oxidase. Finally, the electrode after cross-linking treatment was immersed in a 6% concentration polyurethane solution and dried to obtain a glucose sensor.

The thickness of the laminated film including the enzyme membrane layer thus formed at the tip of the electrode was 10 to 20 μm.

The performance of the obtained glucose sensor was evaluated as follows.

The glucose sensor was immersed in a phosphate buffer, and a voltage of 0.65 V was applied between the working electrode and the control electrode for aging. Then, the output response of the glucose sensor to the glucose-containing phosphate buffer solution having glucose concentrations of 0, 100, 200, and 300 mg / dl was examined.

After the measurement, the glucose sensor was dried, and one week later, the reproducibility of the output response to the glucose concentration was examined in the same manner as above. Then, the glucose sensor was immersed in a phosphate buffer solution having a glucose concentration of 200 mg / dl, and thereafter, the measurement was continuously performed for 48 hours to examine the stability of the output of the sensor.

Comparative Example 5 glucose oxidase was used.
% And polyvinyl alcohol 2.5% were replaced with a mixed solution containing glucose oxidase 5% and bovine serum albumin 2.5% in the same manner as in Example. A glucose sensor was obtained.

The glucose sensor was evaluated in the same manner as in the examples. (Results) As shown in FIG. 4, the enzyme sensor obtained by the method shown in the example showed high output and good linearity in the first measurement, and there was little variation among the sensors.

Even after being left for one week in a dry state, good reproducibility was exhibited except that the output slightly decreased as shown in FIG.

Even in the continuous measurement for 48 hours, as shown in FIG. 6, almost no output fluctuation was observed and good stability was exhibited.

On the other hand, the enzyme sensor obtained by the method shown in the comparative example showed good linearity in the first measurement, but its output value was low, as shown in FIG.
There was also a large variation between sensors.

Further, after being left for one week in a dry state, as shown in FIG. 8, the linearity was lowered and the output value was greatly varied among the sensors.

In the continuous measurement for 48 hours, the sensor output decreased with time as shown in FIG.

[0046]

According to the present invention, it is possible to provide an enzyme sensor which is small in size, has excellent sensitivity and stability, and has a long life.

The enzyme in the present invention is crosslinked and immobilized in a state of being dispersed in a non-crosslinking hydrophilic polymer. Therefore, it is considered that the enzyme is maintained in an appropriately dispersed state without becoming dense. As a result, the permeation and diffusion of glucose, which is the object of measurement, is improved, and it is speculated that the enzymatic reaction proceeds efficiently. In addition, since the hydrophilic polymer used is non-crosslinkable, it is considered that crosslinks are not formed with the enzyme and the enzyme is immobilized with a high degree of freedom. It is speculated that the high degree of freedom of the immobilized enzyme prevented the inactivation of the enzyme, thereby achieving the excellent stability of the enzyme sensor of the present invention.

Further, the hydrophilic polymer has a function of protecting the enzyme from the cause of enzyme deactivation such as drying and sterilization. Due to the action of the hydrophilic polymer, the enzyme sensor is resistant to drying and sterilization. Seems to have improved.

Furthermore, since hydrophilic polymers such as polyvinyl alcohol are less likely to deteriorate than proteins such as albumin, it is considered that an enzyme sensor having excellent stability and long life can be obtained.

According to the present invention, it is possible to provide a method for producing an enzyme sensor which can produce an enzyme sensor having high sensitivity, excellent stability and long life by a simple operation.

[Brief description of drawings]

FIG. 1 is a schematic explanatory view showing an example of an enzyme sensor of the present invention.

FIG. 2 is a schematic explanatory view showing an example of the enzyme sensor of the present invention.

FIG. 3 is a schematic explanatory view showing an example of the enzyme sensor of the present invention.

FIG. 4 is a graph showing the output response of the enzyme sensor of Example to glucose concentration.

FIG. 5 is a graph showing the output response with respect to glucose concentration of the enzyme sensor of the example after standing for 1 week in a dry state.

FIG. 6 is a graph showing the relationship between time and sensor output when the glucose concentration was continuously measured for 48 hours using the enzyme sensor of the example.

FIG. 7 is a graph showing the output response of the enzyme sensor of the comparative example with respect to glucose concentration.

FIG. 8 is a graph showing the output response with respect to glucose concentration of the enzyme sensor of Comparative Example after being left for one week in a dry state.

FIG. 9 is a graph showing the relationship between time and sensor output when glucose concentration was continuously measured for 48 hours using the enzyme sensor of Comparative Example.

[Explanation of symbols]

1 ... Working electrode, 2 ... Control electrode, 3 ... Enzyme membrane layer, 4 ... Hydrophilic semipermeable membrane, 5 ... Porous membrane, 6 ...
·Insulator

Claims (5)

[Claims] 1. A non-crosslinking hydrophilic polymer and an enzyme dispersed and crosslinked in the non-crosslinking hydrophilic polymer on the outside of an electrode having a working electrode and a control electrode or the working electrode. An enzyme sensor comprising an enzyme membrane layer containing: 2. A hydrophilic semipermeable membrane, a non-crosslinking hydrophilic polymer and a non-crosslinking hydrophilic polymer dispersed on the surface of an electrode having a working electrode and a control electrode or the surface of the working electrode,
An enzyme sensor comprising an enzyme membrane layer containing a crosslinked enzyme and a porous membrane in this order. 3. The enzyme sensor according to claim 1 or 2, wherein the non-crosslinkable hydrophilic polymer is polyvinyl alcohol. 4. The enzyme sensor according to claim 1, wherein the enzyme is glucose oxidase. 5. An enzyme membrane layer is formed by cross-linking an enzyme in the presence of a non-crosslinking hydrophilic polymer on the outside of an electrode having a working electrode and a control electrode or a working electrode. Enzyme sensor manufacturing method.