JP2536780B2 - Pick-up type enzyme electrode - Google Patents

Description

TECHNICAL FIELD The present invention relates to an enzyme electrode, and more particularly to a hydrogen peroxide electrode and a disposable enzyme electrode using an oxidoreductase that converts oxygen into hydrogen peroxide. And an enzyme electrode configured to improve measurement accuracy.

[Problems to be Solved by Prior Art and Invention]

Since disposable enzyme electrodes do not require calibration of calibration curves or cleaning of electrodes in clinical tests, they are so-called maintenance-free and can be downsized. Is. However, the detection accuracy,
In terms of performance such as reproducibility, it is not always satisfactory, and there are problems such as high manufacturing cost to be solved in terms of technology and economy.

If the sensor is miniaturized, the electrode area can be reduced, and the amount of expensive enzyme used can be reduced accordingly, so it is theoretically possible to reduce the manufacturing cost of the sensor, but if the miniaturization is too much, In order to obtain reproducibility in manufacturing, it is necessary to improve the accuracy of electrode formation, which eventually leads to an increase in substrate material and manufacturing cost. There is also a risk that the operability, which is an advantage of the disposable sensor, may be sacrificed. In addition, in order to reduce the invasiveness to the living body and to facilitate the measurement, it is desired to reduce the amount of the sample.

Here, in the oxidoreductase using oxygen as one of the substrates, the diffusion of oxygen in the air to the sample becomes the reaction rate control, and the change depending on the concentration of the substrate to be originally measured may not be detected. For example, normal batch or flow H ₂
In the glucose sensor using O ₂ , the measurement limit of high concentration is determined by the rate of O ₂ diffusion. The diffusion rate of O ₂ changes depending on the distance between the bottom surface of the sample, which is the electrode arrangement surface, and the surface of the sample, that is, the distance between the air interface of the sample and the electrode. For example, when the sample is dropped on the electrode, the sample becomes a drop due to the surface tension of the sample and tends to diffuse nonuniformly on the electrode surface. Further, the size of the drop, that is, the distance between the air interface and the electrode, changes according to the amount of dropping. Therefore, the dropping of the sample must be performed very carefully because it needs to be performed uniformly and quantitatively on the electrode, but the detection error is unavoidable even with such careful handling.

[Means for solving the problem]

The present invention is configured to solve the technical problems in the above-described conventional techniques, has sufficient detection accuracy, reproducibility, etc., without impairing the advantages of disposable enzyme electrodes, and operates. The purpose of this is to realize a low-cost electrode with good performance, to reduce the amount of sample during inspection, and to solve the reaction limitation due to the O ₂ reaction rate-controlling. Also, an electrode portion is formed at the other end portion, and the electrode portion is composed of a measurement electrode group consisting of two working electrodes and a reference electrode forming a differential electrode and a counter electrode, and contains a redox enzyme in the resin. An enzyme-type electrode in which an enzyme-immobilized resin part is formed on the surface of at least one of the two working electrodes of the measurement electrode group by a resin, and a coating part of the same resin having no enzyme activity is formed on the other working electrode. And at the edge of the board A pole is formed, and the measurement electrode group is formed adjacent to the counter electrode, the counter electrode surface is layered with a material that infiltrates the sample to form a sample introduction part, and a space is formed above the measurement electrode group formation part. By forming the oxygen permeable film through the sample introduction portion, the air interface of the sample infiltrated into the space through the sample introduction part is arranged so that the oxygen permeable film is located at the position where the oxygen permeable film and the measurement electrode group are arranged. The disposable enzyme electrode is characterized in that the distance between and is a predetermined value.

[Action]

The sample dropped in the sample introduction part flows into the space formed in the adjacent measurement electrode group forming part, and the air interface of the sample filled in the space becomes this oxygen permeable membrane arrangement surface. Since the oxygen permeable membrane is arranged with an extremely small height from the measurement electrode group arrangement surface, the distance from the measurement electrode group arrangement surface to the air interface is set short and the space is evenly filled. .

[Basic Structure of the Invention]

FIG. 1 shows the basic structure of a disposable electrode according to the present invention.

This disposable electrode has a width of 4-10 mm and a total length of 30-
With a size of about 60 mm, its structure can be divided into three parts. First, reference numeral 20 is a main body connecting portion connected to the main body of the measuring apparatus, 21 is an insulating portion, and 22 is an electrode portion (H ₂ O ₂ electrode) connected to the main body connecting portion 20 side via the insulating portion 21. More specifically, the structure of the electrode portion is shown. Reference numerals 1 and 2 are working electrodes whose surfaces are made Pt, 3 is a reference electrode, and a pair of working electrodes 1, 2 and a reference electrode 3 are used for the measurement electrode group. Make up.

Reference numeral 4 is a counter electrode formed on the end portion of the substrate 5 adjacent to the measurement electrode group. Reference numeral 5 is a substrate for forming each of these patterns.

Next, the following resin layer is formed on the surface of the electrode portion having the structure shown in FIG. That is, in FIG.
Is an enzyme-immobilized resin portion formed on the working electrode 1 by a resin containing an enzyme, and 6 is a resin similar to the enzyme-immobilized resin portion 7 and covered with at least the working electrode 2 by a resin having no enzyme activity. It is the formed coating part.

In the present application, the following configuration is added to the above basic configuration.

That is, in FIG. 3 and FIG. 4, first, the counter electrode 4 is formed into a layer by being covered with a resin, a porous body or a non-woven fabric capable of infiltrating the sample, and the layer forming part is configured as the sample introducing part 10. . Reference numeral 8 denotes a sample holding area limiting wall (hereinafter simply referred to as “limiting wall” or “wall portion”) formed so as to surround at least three sides around the electrode portion 22.

Reference numeral 9 is a film body in which the formation portion of the measurement electrode group consisting of the working electrodes 1 and 2 and the reference electrode 3 is arranged so as to cover it through a space as shown in FIG. A film body (hereinafter, this film is referred to as an "oxygen permeable film").
Further, the distance from the surface of the coating portion 6 to the oxygen permeable membrane 9, that is, the height of the space, is 0.5 m, for example, as in the embodiment described later.
It is set to a very small value of m or less.

As the name implies, the oxygen permeable film 9 is formed of a material that allows oxygen to pass therethrough, such as an oxygen permeable resin or a porous body. Among them, as the oxygen permeable resin, silicone resin, silicone rubber or the like can be considered. In addition, as the structure of the porous body
A cloth or non-woven cloth of 120 mesh or more, a metal cloth of 120 mesh or more, which is electrically connected to a part of the counter electrode, is considered.

In the above configuration, the sample dropped in the sample introducing unit 10 flows into and fills the space of the adjacent measurement electrode group via the sample introducing unit 10. The space has a height of 0.5 m, for example.
The space is a very small space of about m, and the sample is uniformly filled with a small amount of the sample, and the air interface of the filled sample is the arrangement surface of the oxygen permeable membrane 9.

Since the diffusion rate of oxygen in the sample can be regarded as constant, the smaller the height (distance) of the space, the greater the amount of oxygen supplied,
It is possible to accurately measure even a high-concentration sample. Also, the formation of the oxygen permeable membrane itself reduces the supply of oxygen compared to the case where the specimen is directly exposed to air, but the oxygen permeable membrane can significantly reduce the quantity of the specimen and the height of the air interface of the specimen. Therefore, it is possible to increase the supply amount of oxygen relatively by installing the oxygen permeable membrane because the temperature can be lowered.

Here, the measurement electrode group, which is a hydrogen peroxide electrode, is required to have the ability to detect H ₂ O ₂ converted by the enzyme with sufficient accuracy and reproducibility, and to be inexpensive. In order to realize this sufficient accuracy and reproducibility, it is necessary to ensure the uniformity of the electrode area. That is, it is necessary to perform highly precise patterning. As a substrate for realizing highly precise patterning, a ceramics substrate, a glass substrate, etc. can be mentioned.
Patterning can be performed with a degree of difference and the pattern can be miniaturized, but the manufacturing cost cannot be denied. On the other hand, when a plastic substrate is used, the difference in patterning is generally about several tens of μm, which is inferior to the above-mentioned substrate in terms of miniaturization of patterning, but it is inexpensive and is generally used for forming a printed circuit. Glass epoxy substrates and phenolic substrates are inexpensive.

Also, considering the operability of the sensor, the size of the entire sensor must be such that it can be easily handled with a finger, and the operability becomes worse with a hydrogen peroxide electrode of a few millimeters. That is, a hydrogen peroxide electrode using a ceramics substrate or a glass substrate can be manufactured at a low cost by miniaturizing the pattern, but such a fine pattern is not appropriate from the viewpoint of operability. After all, a slightly larger pattern should be formed, and therefore, it is preferable to use an inexpensive plastic substrate as the substrate itself. Hydrogen peroxide electrode using a plastic substrate, for example if patterned within hidden ± 15 [mu] m,. 2 to errors in electrode area of 1 mm ²
It can be kept within 3%.

On the other hand, the H ₂ O ₂ oxidation potential is relatively low for H ₂ O ₂ detection,
It is desirable to use an electrode material that does not overlap with the O ₂ generation potential due to the electrolysis of water and is inert to Cl ⁻ ions contained in a large amount in the sample. For example, carbon has a nearer O ₂ generation potential than the hydrogen peroxide oxidation potential. For this reason, the oxidation currents overlap and the O ₂ current is considerably large, so it is difficult to separate the H ₂ O ₂ oxidation current. In addition, Au is not appropriate because Cl ⁻ ions in the sample cause a considerably large reaction current, which also adversely affects the H ₂ O ₂ oxidation current.

Next, the Pt electrode has an H ₂ O ₂ oxidation potential of 300 mV to 900 mV (pH7.2Ag /
AgCl) and lower, Cl ^- said to be suitable as a hydrogen peroxide electrode because there is no response to. Although Pt itself is expensive, cost reduction can be achieved by forming a pattern with copper and forming a Pt layer on it by a method such as Pt plating. Further, when the Pt layer is formed by Pt plating, the adhesion can be improved by performing Au plating between copper and Pt.

Next, as a H ₂ O ₂ oxidation current detection method, for example, 700 mV (A
g / AgCl) A method of determining the oxidation current when a constant potential is applied is used. In this method, the current value is determined by the concentration gradient created by H ₂ O ₂ diffusion, but in the potentiostatic method or the slow potential sweep method, the concentration gradient changes with time according to the Cottrell equation, The current value also changes accordingly. The distance covered by the concentration gradient becomes longer with time, and changes in the enzyme distribution and the diffusion coefficient distribution affect the current. This imposes great restrictions on the design of the film thickness of the enzyme-immobilized portion in close contact with the electrode surface, the distance to the liquid surface, and the like, which adversely affects the reproducibility. Therefore, by sweeping the potential at a constant rate (tens of mV / sec to hundreds of mV / sec) by the potential sweep method or the cyclic voltammetry method, it is possible to reduce the influence of changes in the enzyme distribution and the diffusion coefficient distribution.

The potential sweep method and the cyclic voltammetry method use a three-pole method of a working electrode, a counter electrode, and a reference electrode. The reference electrode, which is the standard for applying the potential, has a mechanism to extract a stable potential under certain conditions such as the ion composition and the concentration of the electrode active substance, like a silver / silver chloride electrode in a saturated KCl solution with a liquid junction. Therefore, due to the buffering property of the sample itself, or the stabilization of the pH by the addition of a buffer, etc., and the change in the composition ratio of the redox substance in the sample, the concentration, etc. being slight during the measurement, If it is an inactive metal, it can be used as it is as a reference electrode.

In the present invention, since the differential method is applied to the cyclic voltammetry method, a differential electrode of the working electrode 1 and the working electrode 2 has a four-pole structure in which a reference electrode and a counter electrode common to these electrodes are added,
In addition, the working electrode 1 and the working electrode 2 are coated with the same resin so that one working electrode has an enzymatic activity. With this configuration, it is possible to remove the effect of the redox wave of the redox substance in the sample, and also the difference in the diffusion coefficient distribution and the enzyme distribution that hinders the reproducibility, such as the change in the liquid volume and the thickness of the immobilization part, etc. The influence of can be reduced.

Further, in the present invention, the method of holding the sample on the electrode surface is adopted. As a result, it was possible to reduce the cost of manufacturing the sensor and reduce the amount of sample by downsizing the electrode. In addition, the miniaturization of the sample provided two advantages: reduced invasiveness and increased substrate concentration detection range.

Of these, the increase in the substrate concentration detection range will be described. That is, since the enzyme used for the electrode of this sensor requires the enzyme as one of the substrates, it is necessary to consider the supply of O ₂ from the solution. It is glucose that is considered to require the most O ₂ among the analyte components taken out from the living body. Therefore, taking a glucose sensor as an example, the enzyme glucose oxidase is immobilized in a sufficient excess in the usual enzyme electrode method, and the detection current is glucose diffusion limited, but when the concentration of glucose becomes high, the amount of O ₂ diffusion increases. Is insufficient, the H ₂ O ₂ conversion amount saturates and the detected current does not change,
The glucose measurement range is limited. However, in the present invention, by forming a space above the measurement electrode group and miniaturizing this space, the amount of the sample can be made small, and thus the distance between the enzyme immobilization membrane and air can be reduced. The distance is the same as that of, and the distance is greatly reduced compared to the past. As a result, although oxygen is supplied to the sample through the oxygen permeable membrane, the amount of oxygen supplied from the air to the sample will be increased due to the drastic decrease in this distance. The appearance of the saturation current of was shifted to the high concentration side, and the measurement range was expanded.

Next, the sample holding method is shown below (1) to (3)
The configuration and the like are conceivable.

(1) A structure in which a sample is contained in a porous body placed on the surface of an electrode (JP-A-61-294351, etc.).

(2) A configuration in which a small amount of container is formed by surrounding four sides of the electrode.

(3) A configuration in which a microvolume container is formed that surrounds the electrodes on three sides and has an oxygen-permeable film formed so as to face the electrode surface (configuration of the present invention).

Although the configuration (1) is low cost among the above configurations,
The O ₂ supply amount is relatively small, and the configuration of (2) has a problem that the O ₂ supply amount is large but the sample collection is difficult and the handleability is not good. On the other hand, although the method (3) is slightly expensive, it is easy to collect a sample using the capillary phenomenon,
By setting the property of the oxygen permeable membrane and the installation height of the oxygen permeable membrane to be low, it is possible to expect a higher O ₂ supply amount than the configuration of (1) in which the sample is brought into direct contact with air, and skill is required. Without any difficulty, the distance between the electrode and the oxygen permeable membrane can be made constant, and there are advantages in reproducibility such as constant capacity and constant O ₂ supply amount.

As a measure for reducing the manufacturing cost, which is a problem in the above methods (2) and (3), an adhesive agent is applied to a plastic film such as polyvinyl chloride, polyester, polyethylene, or polypropylene as a material for forming a wall surrounding an electrode. A method in which an object is die-cut and this is mounted as a wall is suitable. Further, instead of this method, the cost can be reduced by forming the wall portion on the substrate by resin in advance by the screen printing method.

FIG. 7 shows changes in the glucose measurement range and the air interface distance between the electrode and the sample (hereinafter referred to as “electrode-interface distance”) in the method shown in (2). In the figure, when the electrode-interface distance is 0.5 mm, the glucose measurement range is 40 mM (720 mg / d
l) It extends to more than the above, but the electrode-liquid level distance is 2.0 mm
In the case of, it is saturated at about 20 mM (360 mg / dl). When the detection range is defined as 20 mM (360 mg / dl) or more, the wall height that determines the liquid surface distance, the distance to the O ₂ permeable membrane, and the thickness of the porous body are 2.0 mm or less, preferably 0.5 mm or less. It is desirable to do.

Subsequently, as the enzyme immobilization method, it is effective to use the entrapping immobilization method using a hydrophilic resin. The entrapping immobilization method can prevent the enzyme, which is a protein from falling out of the resin, prevent the blood cell components and proteins in the sample from entering the membrane, and prevent the decrease in response reproducibility due to the adsorption of the protein to the electrode. . Further, as the resin for entrapping fixation, a photocurable resin or a photosensitive resin is used, so that the manufacturing cost can be reduced. In the differential electrode method of the present invention, an enzyme-immobilized film is formed on the working electrode 1, but the other electrodes other than the working electrode 1, that is, the working electrode 2 and the reference electrode 3 are made of the same resin containing no enzyme. Perform coating. As a result, it is possible to prevent the blood cell components and proteins in the sample from adsorbing to each electrode, and to make the diffusion coefficients of the respective components of the electrode active substance uniform.

 Hereinafter, the present invention will be described in more detail with reference to examples.

[Example 1] (i) Substrate 5 is a glass epoxy resin substrate (ii) Working electrode 1, working electrode 2, reference electrode 3 and counter electrode 4 are formed by patterning with copper, and Au plating is 1 μm on the upper part of the pattern.
Then, Pt plating is further formed to 1 μm.

(Iii) The sample shall have the following configuration.

NaCl 106mEq Ascorbic acid 5.9mg / dl Uric acid 4.3mg / dl BSA 7.6 / dl and 0.1M phosphate buffer pH7.3 added to the above.

A cyclic voltamograph with a constant pH by Buffer was obtained with good reproducibility.

Figure 6 shows the above solution (0.1M phosphate buffer added)
Cyclic voltammogram of the sample in which 0 to 1 mM H ₂ O ₂ was added to -600 mV to 1000 mV, sweep speed 100 mV / sec, 2cy
The current value of the working electrode 1 of 700 mV and the current value of the working electrode 2 of the sample to which H ₂ O ₂ was not added were subtracted from this current value at the time of the cle up, and the H ₂ O ₂ concentration was plotted horizontally. It is a calibration curve taken on each axis. In this example, a correlation coefficient of 0.9967 and n25 were obtained, and it was confirmed that sufficiently good reproducibility was obtained. That is, it was confirmed that the reference electrode having the above-mentioned configuration was sufficiently functioning.

In the Pt electrode, the H ₂ O ₂ oxidation current is flat between 400 mV and 800 mV in the cyclic voltammetry method, so even if the reference potential changes slightly due to sensor manufacturing tolerances, differences in analyte composition, etc. Few.

Example 2 First, the structure of the electrode was the same as in Example 1. A water-soluble photosensitive resin obtained by adding a stilbazolium residue to polyvinyl alcohol on the working electrode 1 of this constitution (JP-A-56-5761).
No., polymerization degree 1700, saponification degree 88%, SbQ1.3mol%, 1.1wt
%) Glucose oxidase (EC, 1,1,3,4, S
igma, 136units / mg ・ Aspersillus ・ niger) 4.1mg added 2μl spread coating, dried and UV irradiation 500
It was cured at μW (340 nM) for 10 minutes.

Next, as shown in FIG. 3 to FIG. 5, the sample holding area limiting wall 8 was formed by sticking an adhesive tape (product name Nitto 5010) on a polyester film and a thickness of 188 μm, and stamping it to make an electrode. 10 μl of the above water-soluble photosensitive resin containing no enzyme was dropped on the entire surface of the electrode, dried and cured.

Further, Tetoron mesh # 200 was attached as an O ₂ permeable film on the polyester film wall so as to cover the measurement electrode portion using an adhesive. The volume of the sample holding area in this example was about 10 μl.

 A calibration curve of the sensor having the above configuration is shown in FIG.

An immobilized enzyme amount of 0.15 Units / mm ² was the calibration curve of this sensor, and a similar calibration curve was obtained with the enzyme amount of 0.23 Units. 0.1
When it was 5 Units / mm ² or more, the same calibration curve was obtained, showing that the reproducibility was good regardless of the enzyme activity. The reproducibility was Cv2.49% at a glucose concentration of 10 mM (180 mg / dl), and the correlation coefficient was 0.9954 in the concentration range of 1 mM to 20 mM (20 mg / dl to 360 mg / dl).
was gotten.

〔effect〕

The present invention provides a space for filling a specimen by forming and disposing an oxygen permeable film at a predetermined arrangement height in the measurement electrode group arrangement portion, and the specimen by filling the space with the specimen. Since the height between the air interface and the measurement electrode group is a preset height, it is possible to increase the amount of oxygen supplied to the sample by setting the height to a small value, and to measure a high-concentration sample. Will also be possible.

Further, since the sample dropped in the sample introducing part is configured to flow into and fill the adjacent space, accurate measurement can be performed without requiring skill in dropping the sample.

[Brief description of drawings]

FIG. 1 is an electrode plan view showing a lower layer structure of a disposable enzyme electrode showing an embodiment of the present invention, FIG. 2 is an enlarged partial plan view of an electrode showing an intermediate layer structure of the electrode, and FIG. 3 shows an upper layer structure of the electrode. FIG. 4 is a sectional view taken along line AA in FIG. 3, FIG. 5 is a sectional view taken along line BB in FIG. 6, FIG. 6 is a calibration curve in Example 1, and FIG. 7 is a glucose measurement range. FIG. 8 is a diagram showing the calibration curve shown, and FIG. 8 is a diagram showing the calibration curve showing the glucose measurement range in the configuration of Example 2. 1, 2 ... Working electrode, 3 ... Reference electrode, 4 ... Counter electrode, 5 ...
Substrate 6 ... Coating part, 7 ... Enzyme-immobilized resin part 8 ... Wall part for forming sample introduction part, 9 ... Oxygen permeable membrane 10 ... Sample introduction part, 20 ... main body connection part, 21 ... insulation Part 22 …… Electrode part

Claims (2)

(57) [Claims] 1. A measurement comprising a working electrode and a reference electrode which form a body connecting part on one end of the same plane of the substrate and an electrode part on the other end, and the electrode part constitutes a differential electrode. It is composed of an electrode group and a counter electrode, and an enzyme-immobilized resin part is formed on the surface of at least one working electrode of the two working electrodes of the measurement electrode group by a resin containing a redox enzyme in the resin, and on the other working electrode. Is an enzyme-type electrode in which a coating part of the same resin having no enzyme activity is formed, a counter electrode is formed at the end of the substrate, and the measurement electrode group is formed adjacent to the counter electrode, and the counter electrode surface infiltrates a specimen. The sample introduction part is formed by layering the material, and the oxygen permeable film is formed on the upper part of the formation part of the measurement electrode group through the space, so that the sample infiltrated into the space through the sample introduction part Position of oxygen permeable membrane at the air interface Disposable enzyme electrode, characterized in that the distance between the air interface and the measuring electrode group of the sample is constructed to be a predetermined value by configured to be. 2. The disposable enzyme electrode according to claim 1, wherein the oxygen permeable membrane is an oxygen permeable resin or a porous material.