JPH03277956A - Enzyme immobilized electrode device - Google Patents

Abstract

PURPOSE:To prevent the decrease in detecting sensitivity with the elapse of time and to obtain stable detecting performance for a long period by forming a protecting film on the surface of a counter electrode. CONSTITUTION:The patterns of a measuring electrode 2 and a counter electrode 3 are formed on the surface of a ceramic substrate 1. A ground film 4, an interference removing film 5 and an enzyme immobilized film 6 are sequentially formed on the measuring film electrode 2. The surface of the film 6 is exposed. A protecting film 7 is provided on the counter electrode 3. The protecting film 7 is formed by applying the aqueous solution wherein bridging agent is added into macromolecular material such as gelatin, albumin and polyallylamine on the surface of the counter electrode 3 and performing bridging reaction. In this way, attachment of impurities such as protein on the surface of the counter electrode 3 is prevented when the current and the potential difference between the measuring electrode 2 and the counter electrode 3 are measured and the material to be measured is detected. The decrease in sensitivity of the enzyme immobilized electrode 6 with the elapse of time is prevented, and the stable detecting performance can be displayed for a long period.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to an enzyme-immobilized electrode device that is used as a sensor for detecting a substrate such as glucose and is equipped with an electrode having an immobilized enzyme. It is.

[Conventional technology]

The conventional manufacturing method for enzyme-immobilized electrodes involves applying a dilute enzyme solution containing gelatin and glutaraldehyde as a crosslinking agent to a platinum electrode, forming a film on the surface of the electrode body, and sharing the enzyme. There is a method of binding and immobilizing enzymes to form an enzyme-immobilized membrane on the surface of the electrode body.

In addition, in order to perform various detections and measurements using an enzyme-immobilized electrode, it is necessary to combine a measurement electrode on which an enzyme is immobilized and a counter electrode on which no enzyme is immobilized, and measure the potential difference and current between the two electrodes. Therefore, an enzyme-immobilized electrode device that obtains detection information in the form of an electrical signal is used.

Here, gelatin is a matrix component of the enzyme-immobilized membrane, and if only a dilute enzyme solution and glutaraldehyde as a cross-linking agent are used, the strength of the enzyme-immobilized membrane obtained by cross-linking reaction is weak. Used to increase strength. For example, in the case of a membrane with immobilized glycol oxidase, gelatin is added in an amount of about 5 to 10 times the amount of the enzyme, and the membrane is crosslinked with glutaraldehyde to form a membrane.

When using such an enzyme-immobilized electrode device, care must be taken to ensure that the detection of the analyte by the enzyme-immobilized measurement electrode is not interfered with by various components other than the analyte contained in the sample solution. It is necessary to keep it. Therefore, between the surface of the electrode body of the measurement electrode and the enzyme-immobilized membrane, or on the surface of the enzyme-immobilized membrane, an interference removal membrane (hereinafter referred to as an ``interference removal membrane'') that has the effect of preventing or removing interference substances from passing through is installed. An interference-insensitive enzyme-immobilized electrode has been proposed.

For example, in Japanese Patent Application Laid-Open No. 62-88953, an electrode body provided with an enzyme-immobilized membrane of glucose oxidase on the surface of the electrode body is immersed in an electrolytic polymerization solution containing virol and NaCl, and subjected to electrolytic polymerization treatment. A technique has been disclosed in which an electrolytically polymerized membrane is formed on an immobilized membrane and the electrolytically polymerized membrane is used as an interference removal membrane.

In addition, the inventors of the present invention have invented an enzyme-immobilized electrode in which an interference removal film containing a humectant is interposed between the electrode body and the enzyme-immobilized film, and have filed a patent application for this invention as Japanese Patent Application No. 1-164797. There is.

Furthermore, as described above, in addition to forming an interference removal membrane between the electrode body and the enzyme-immobilized membrane, the inventors of the present invention have also developed a porous film made of polymer resin on the surface of the enzyme-immobilized membrane. He invented an enzyme-immobilized electrode in which a thin film was formed to protect the enzyme-immobilized membrane, and filed a patent application for the invention as Japanese Patent Application No. 1-127361. In this case, the porous thin film made of polymer resin has the effect of preventing proteins other than the measurement substance from physically adsorbing on the surface.

[Problem to be solved by the invention]

However, with conventional enzyme-immobilized electrode devices, when measuring the concentration of physiologically active substances contained in body fluids such as serum, the detection sensitivity gradually decreases when continuous measurements are repeated even for the same sample. There was a drawback that the detection performance became unstable and the detected concentration conversion value did not show the correct value.

This is because proteins other than the substance to be measured are physically adsorbed on the surface of the electrode. Like the prior art,
Physical adsorption of proteins, etc. can be prevented to some extent by forming a protective film made of polymer resin on the enzyme immobilization membrane, but if the enzyme immobilization membrane is covered with a polymer resin membrane, , the responsiveness when detecting the substance to be measured becomes worse.
A problem arises in that detection sensitivity also decreases.

Therefore, an object of this invention is to provide an enzyme-immobilized electrode device such as the one described above with good response and high detection sensitivity, without causing unstable detection performance due to a decrease in detection sensitivity over time. [Means for Solving the Problems] An enzyme-immobilized electrode according to the present invention that solves the above problems. The device is an enzyme-immobilized electrode device that includes a measurement electrode and a counter electrode on a substrate, and an enzyme-immobilized film is provided on the surface of the measurement electrode, and a protective film is formed on the surface of the counter electrode.

The materials and structure of the measurement electrode, counter electrode, substrate, etc., are the same as those used in ordinary enzyme-immobilized electrode devices.For example, the substrate may be ceramic, synthetic resin, glass, or other ordinary electronic element substrates. The same materials are used as the electrode materials, such as platinum, silver, and other electrode materials used in ordinary enzyme-immobilized electrode devices.

The materials and manufacturing method for the enzyme-immobilized membrane may be the same as those for ordinary enzyme-immobilized membranes. For example, by applying an enzyme such as glucose oxidase and an enzyme solution containing gelatin, a cross-linking agent, etc. and causing a cross-linking reaction, a film having gelatin as a matrix component is formed, and at the same time, the formed film is Enzymes can be immobilized. The type of enzyme and the combination of additives such as gelatin and crosslinking agents can be freely changed in addition to those mentioned above. For example, matrix components other than gelatin may be used. As a crosslinking agent, in addition to dialdehydes such as glutaraldehyde,
An appropriate crosslinking agent can be selected from among various crosslinking agents commonly used in film formation, such as formaldehyde, bismaleimides, aryl dihalides, and diisocyanates.

An interference removal film or a base film may be formed between the measurement electrode and the enzyme-immobilized film, if necessary. The interference removal membrane is interposed between the measurement electrode and the enzyme-immobilized membrane to prevent interference substances from interfering with detection at the measurement electrode. The materials and structure of the interference removal membrane are the same as those used for the above-mentioned purpose in ordinary enzyme-immobilized membranes. Specifically, a water-soluble polymer containing proteins such as albumin, such as a polyallylamine aqueous solution to which a crosslinking agent such as glutaraldehyde is added, is applied, and a crosslinking reaction is performed to form a film. The above-mentioned gelatin or the like can be used instead of polyallylamine, which serves as a matrix component for film formation. As the crosslinking agent, the same one as in the case of the enzyme-immobilized membrane described above can be used.

The base film is interposed between the interference removal film and the measurement electrode, and is used to improve the adhesion between the interference removal film and the electrode. For the base film, for example, a crosslinking agent such as glutaraldehyde may be added to an aqueous solution of a polymer such as gelatin, and the mixture may be applied onto the measurement electrode to cause a crosslinking reaction to form a film. The material and manufacturing method of the base membrane may be the same as those for a normal enzyme-immobilized electrode device. The crosslinking agent can be changed to the same one as in the case of the enzyme-immobilized membrane described above. However,
The above-described interference removal film and base film may be omitted.

In this invention, a protective film is provided on the surface of the counter electrode. Polymeric substances such as gelatin, albumin, and polyallylamine are used as materials for the protective film, and an aqueous solution containing this and the aforementioned crosslinking agent is applied to the surface of the counter electrode to cause a crosslinking reaction to form the film. Let it form.

The concentration of the polymer component in the film-forming solution for the protective film of the counter electrode is preferably set in a range of 20% by weight or less from the viewpoint of permeability, responsiveness, etc., and a range of 0.5 to 5% by weight is preferable. More desirable. The amount of film-forming liquid applied to the counter electrode surface is particularly limited.
However, when using a film forming solution with a concentration of 0.5 to 50% by weight, it is preferable to apply 0.1 to 5 μl per 1 m” of counter electrode area. Not only one type but also two or more types can be used in combination.In order to prevent impurities such as proteins from being directly physically adsorbed on the counter electrode surface, gelatin or other polymeric substances can be used as the polymeric substance constituting the protective film. Although it is preferable to use biopolymers such as proteins, it is also possible to use substances other than those mentioned above, as long as they can perform the same function. For example, collagen etc. can also be used. Any ordinary crosslinking agent can be used as long as it can cause a crosslinking reaction to the polymeric substance, but specifically, for example, aldehyde compounds such as glutaraldehyde, phthalic dialdehyde, glyoxazal, diisocyanate, etc. Polyfunctional compounds such as dicarboxylic acids, dialcohols, diglycidyl compounds, etc. can be used.

[Function] Providing a protective film on the surface of the measurement electrode and counter electrode that make up the enzyme-immobilized electrode device will ensure that impurities such as proteins do not adhere to the surface of the counter electrode. It can be prevented. As a result, performance changes such as a decrease in sensitivity over time of the enzyme-immobilized electrode device can be prevented, and stable performance can be exhibited over a long period of time.

In other words, in conventional enzyme-immobilized electrode devices, an interference removal film or a protective film is provided on the measurement electrode to prevent detection from being interfered with by interfering substances, but there is nothing to prevent impurities from adhering to the counter electrode. No countermeasures were taken. Detection of a measuring substance in an enzyme-immobilized electrode device is carried out by measuring the current, potential difference, etc. between the measuring electrode and the counter electrode, so impurities adhering to the counter electrode as well as the measuring electrode are also susceptible to enzyme immobilization. This will have a significant impact on the performance of the chemical electrode device. In particular, since the electrode surface of the counter electrode is directly exposed, if even a small amount of impurity adheres to the surface of such a counter electrode, the detection performance will be greatly affected.

Therefore, in the present invention, by providing a protective film on the counter electrode, which has not been given much importance in the past, and preventing the adhesion of impurities, it is possible to effectively prevent performance deterioration over time of the enzyme-immobilized electrode device. becomes possible.

Moreover, as mentioned above, if the surface of the enzyme-immobilized membrane of the measurement electrode is covered with a protective film, the reaching of the measuring substance to the enzyme-immobilized membrane or the action and reaction will be inhibited, resulting in a slow response speed. However, if it is the surface of the opposite electrode, such a problem does not occur at all, and the response speed is as good as when there is no protective film.

Note that even if the surface of the enzyme-immobilized membrane of the measurement electrode is not covered with a protective film, it does not significantly affect the decrease in detection sensitivity over time. This is because the measurement electrode does not directly expose the electrode metal, but instead has an enzyme-immobilized membrane made of a polymer material, etc., so it is less likely to physically adsorb impurities or affect the electrode function than the counter electrode. This is thought to be due to the fact that there are few.

〔Example〕

Specific examples and comparative examples of the present invention will be described below.

FIG. 1 and FIG. 2 show the schematic structure of the enzyme-immobilized electrode device according to the present invention.

A measurement electrode 2 and a counter electrode 3 made of platinum, silver, etc. are patterned on the surface of a substrate 1 made of ceramic or the like using a common electrode forming technique. The pattern of the measuring electrode 2 and the counter electrode 3, for example, as shown in FIG. 2 is used, but it can be freely changed according to the purpose.

On the measurement electrode 2, a base film 4, an interference removal film 5, and an enzyme immobilization film 6 are sequentially formed. The surface of the enzyme-immobilized membrane 6 is exposed, and no protective film or the like is formed thereon. A protective film 7 is provided on the counter electrode 3.

An enzyme-immobilized electrode device having the basic structure as described above was manufactured through the following steps.

- Preparation of an enzyme-immobilized electrode device - A base film preparation solution, an interference removal membrane preparation solution, and an enzyme-immobilized membrane preparation solution having the compositions shown in Table 1 below were used.

Table 1: After forming the measurement electrode 2 made of platinum and the counter electrode 3 made of silver on the substrate 1, 0.5 μl of the base film preparation solution is applied to the surface of the measurement electrode 2, and a crosslinking reaction is carried out. A geomembrane 4 was formed. The size of the area to be coated with this base film preparation solution is 1 x 1 f.
It was i. Next, 0.5 μl of the interference removal membrane preparation solution was applied to the surface of the base film 4, and a crosslinking reaction was performed to form the interference removal membrane 5. Furthermore, 0.5 μl of the enzyme-immobilized membrane preparation solution was applied to the surface of the interference removal membrane 5, and a crosslinking reaction was performed to form the enzyme-immobilized membrane 6. The steps up to this point are similar to those of the conventional enzyme-immobilized electrode device.

Next, protective Ni 17 is formed on the surface of the counter electrode 3.

As the protective film preparation solution, solutions having respective compositions shown in Table 2 below were used. In the table, Comparative Example 1 is a case where the protective film 7 was not formed and the surface of the counter electrode 3 was exposed as it was, as shown in FIG.

Apply 15μ of the above protective film preparation solution to the surface of the counter electrode 3.
The protective film 7 was formed by coating and performing a crosslinking reaction. The size of counter electrode 3 was 4.5 x 5.75 m. In this manner, the enzyme-immobilized electrode devices of Examples 1 to 3 were manufactured. In addition, as a conventional enzyme-immobilized electrode device, Comparative Example 1 does not have a protective film 7 on the counter electrode 3, and a polymer resin film serving as a protective film is placed on the enzyme-immobilized film 6 of the measurement electrode 2 instead of the counter electrode 3. An enzyme-immobilized electrode device of Comparative Example 2 in which .

Comparison of performance of enzyme-immobilized electrode devices For each of the enzyme-immobilized electrode devices of Examples 1 to 3 and Comparative Example 1, changes in detection sensitivity over time were compared. Evaluation of sensitivity fluctuation is performed by detecting glucose contained in serum as a measurement target, and when measuring the same serum 20 times, the change between the first sensitivity measurement value and the 20th sensitivity measurement value is evaluated. The ratio was determined by the following formula.

Sensitivity was measured by connecting the enzyme-immobilized electrode device to a measuring device such as a polaro analyzer, and measuring a voltage of 0.6 V (potential difference between measurement electrode 2 and counter electrode 3) + tumble amount of 20 μl.

The sample has a glucose concentration of 59 mg/d! Glucose in the serum was detected using the serum of The results are shown in Table 2 above.

As can be seen from Table 2, in the enzyme-immobilized electrode devices of Examples 1 to 3, there was almost no decrease in sensitivity even when serum was measured repeatedly, and the sensitivity maintenance rate after 20 measurements was 99 to 10.
0%, demonstrating extremely stable performance over time. In Comparative Example 1, the same sensitivity maintenance rate was quite large at 93%, so much so that it affected the reliability of measurement.

FIG. 4 graphically displays the sensitivity (density conversion value) for each number of measurements for Example 1 and Comparative Example 1. Looking at this graph, it can be seen that in Example 1, there is no change in sensitivity at all, whereas in Comparative Example 1, the sensitivity decreases each time the measurement is repeated.

Next, FIG. 5 shows Example 1 and Comparative Example 1.2.
In the first measurement, the change in sensitivity (detected current) from the start of measurement until the sensitivity stabilized is shown in a graph. Looking at this graph, it can be seen that in Example 1 and Comparative Example 1, the sensitivity rapidly increases after the start of measurement and settles at a constant value, whereas in Comparative Example 2, the sensitivity increases very slowly.

That is, when a protective film is provided on the enzyme-immobilized film 6 of the measurement electrode 2 as in Comparative Example 2, the response speed becomes extremely slow compared to Comparative Example 1 in which no protective film is provided. On the other hand, it has been demonstrated that when the protective film 7 is provided on the counter electrode 3 as in Example 1, an excellent response speed that is almost the same as that of Comparative Example 1 in which no protective film is provided is demonstrated. It was done.

〔Effect of the invention〕

According to the enzyme-immobilized electrode device of the present invention as described above, by forming a protective film on the surface of the counter electrode, it is possible to effectively prevent a decrease in detection sensitivity over time, and to maintain stable detection performance over a long period of time. At the same time, it also provides excellent responsiveness during measurement.

In other words, for example, even if body fluids such as serum are measured continuously or repeatedly, proteins in the body fluids will not adhere to the counter electrode, so there will be no decrease in sensitivity and stable reproducibility. It is possible to make good measurements. Moreover, unlike when a protective film is provided on the #element immobilization film of the measurement electrode, the detection response speed does not decrease, making it possible to quickly obtain accurate measurement results. This will greatly contribute to increasing the efficiency and precision of various measurements using electrode devices.

[Brief explanation of the drawing]

FIG. 1 is a schematic cross-sectional structural diagram showing an example of the enzyme-immobilized electrode device according to the present invention, FIG. 2 is a plan structural diagram, and FIG. 3 is a schematic cross-sectional structural diagram of a comparative example showing the prior art. FIG. 4 is a graph showing changes in sensitivity over time in Examples and Comparative Examples, and FIG. 5 is a graph showing detection responsiveness. l...Substrate 2...Measuring electrode 3...Counter electrode 6...
・Enzyme immobilization membrane 7...Protective film Figure 3 Figure 4 Figure 5 - Name of the invention No. 079802 Relationship to the case of the person who corrected the enzyme-immobilized electrode device Patent applicant address 1048 Oaza Kadoma, Kadoma City, Osaka Name (583) Representative of Matsushita Electric Works Co., Ltd. ((1m position) Toshio Miyoshi

Claims (1)

[Claims] 1 An enzyme immobilization electrode device comprising a measurement electrode and a counter electrode on a substrate and an enzyme immobilization film on the surface of the measurement electrode, characterized in that a protective film is formed on the surface of the counter electrode. chemical electrode device.