JPH05164725A - Biosensor - Google Patents

Abstract

PURPOSE:To reduce the number of manufacturing processes and the cost by simplifying the construction of a biosensor by dispensing with a mediator layer and to improve the precision in measurement of a substance to be measured, by excluding the effect of a disturbing substance in a solution to be measured, by the simplified construction. CONSTITUTION:A biosensor 1 is provided with a discrimination layer 9 which is formed by making glucose oxidase and Pt powder (having a particle size of 5mum or below and a content of 5 pts.wt. or above to 100 pts.wt. of an organic substance) be borne and fixed on the upper surface of a working electrode 5 formed on an insulating substrate 3. By setting the particle size and the content of this Pt powder at specified values, the Pt powder is made to operate as a mediator in the discrimination layer and makes it execute donation and acceptance of electrons on the working electrode 5 side. Therefore, an independent mediator layer is dispensed with.

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 substance to be measured using a biological substance having a function of identifying the substance to be measured.

[0002]

2. Description of the Related Art This type of biosensor measures a substance to be measured in a solution to be measured such as urine by utilizing a biochemical reaction that progresses between a substance to be measured and a biological substance such as an enzyme or a microorganism. There are various known types. For example, Japanese Patent Application Laid-Open No. 61-50262 proposes a flat plate type biosensor as an electrode type biosensor. That is, as shown in FIG. 4, a flat plate type biosensor 100.
Is an insulating substrate 101 such as ceramic or plastic,
A working electrode 103 and a counter electrode 105 formed on the insulating substrate 101, an identification layer 107 formed by stacking on the working electrode 103, an insulating layer 108 for insulating between the working electrode 103 and the counter electrode 105, and a function. The electrode 103 and the counter electrode 105 are respectively connected to the terminal portions 109 and 111, and an electric measuring portion (not shown) for measuring a current value therebetween is provided. The identification layer 107 carries and immobilizes a biological substance having an identification function for the substance to be measured, and a sol-like substance containing the biological substance is applied onto the working electrode 103 and then dried and solidified (gelation). Is formed. The side of the identification layer 107 serves as the sensitive section 113 of the biosensor 100.

In order to measure the substance to be measured with this biosensor 100, the sensitive portion 113 is brought into contact with the solution to be measured containing the substance to be measured. As a result, a biochemical reaction proceeds between the biological substance on the working electrode 103 and the substance to be measured contained in the solution to be measured, and oxygen is consumed to generate hydrogen peroxide, for example. The substance to be measured is detected by measuring the electric current obtained by the electrode reaction of the electrode active substance which is consumed or generated in this way, by the electric measuring unit.

The biosensor 100 includes an identification layer 107.
By using various enzymes or microorganisms as the biological substance, the substance to be measured that reacts with it can be detected. For example, when glucose oxidase is used as the biological substance, it becomes a biosensor for detecting glucose. Further, the use of glucuronic acid oxidase serves as a biosensor for detecting glucuronic acid.

[0005]

It is generally known that such a biosensor is affected by dissolved oxygen in a solution to be measured and its measurement accuracy is lowered. Therefore, for the purpose of preventing the influence of dissolved oxygen, a derivative liquid of ferrocene for electron transfer is applied and dried on the discrimination layer 107 to form a mediator layer. As described above, by providing the mediator layer, electrons are exchanged on the working electrode 103 side and the electrode reaction accompanying the biochemical reaction is reflected as the response current on the working electrode 103, thereby lowering the measurement accuracy due to dissolved oxygen. I'm avoiding it.

However, since it is necessary to form the identification layer and the mediator layer with high precision, it is not only complicated to adjust the material for forming each layer, but also the manufacturing process and equipment are complicated and the cost is high. ..

Particularly, in urine, in addition to glucose and glucuronic acid, there are substances having a reducing action such as ascorbic acid and uric acid. Therefore, when measuring glucose and the like in urine, the measurement accuracy of dissolved oxygen In addition to the decline, the following problems have been pointed out. Since ascorbic acid, uric acid, etc. in urine are charged to the negative electrode and have a reducing property, the response current at the electrode is increased more than the current obtained by the electrode reaction accompanying the biochemical reaction by the biological material of the discrimination layer. That is, ascorbic acid, uric acid, and the like act as substances that interfere with the measurement of urinary glucose. Therefore, even if glucose is directly measured from urine, the glucose concentration may not be accurately measured. In order to solve this, conventionally, it has been dealt with by removing interfering substances in the solution to be measured by pretreatment, and the work has been complicated.

The present invention has been made in order to solve the above problems, and its main object is to omit the mediator layer to simplify the structure of the biosensor and to reduce the number of steps required for its manufacture and the cost. Another object is to eliminate the influence of interfering substances in the solution to be measured with a simple structure and improve the measurement accuracy of the substance to be measured.

[0009]

[Means for Solving the Problems] The first means adopted by the present invention in order to achieve the above object is to measure the amount of electrical change accompanying a biochemical reaction between a substance to be measured in a solution to be measured and a biological substance. In the biosensor for detecting the substance to be measured by measuring, a pair of electrodes formed on an insulating substrate and a biological substance having an identification function for the substance to be measured are carried, and the pair of electrodes is provided. An identification layer formed on the surface of one of the electrodes, the identification layer carrying one or more precious metal powders selected from Au, Ag, Pd, Rh, Ir, and Pt in addition to the biological material. The gist of the supported noble metal powder is that the particle size is 5 μm or less and 5 parts by weight or more is contained with respect to 100 parts by weight of the biological substance.

A second means adopted by the present invention is a property of electrically neutralizing a charged substance in the solution to be measured, in addition to the biological substance and the noble metal powder, in the discrimination layer in the biosensor described above. The gist of the invention is to carry an antistatic substance having

[0011]

In the biosensor having the above structure, an identification layer carrying a biological substance is formed on the surface of one electrode of a set of electrodes formed on an insulating substrate. The substance to be measured is measured based on the amount of electrical change generated between the electrodes in association with the biochemical reaction that proceeds with the substance to be measured. In this case, in the biosensor of the present invention, the identification layer is loaded with noble metal powder having a particle size of 5 μm or less in an amount of 5 parts by weight or more with respect to 100 parts by weight of the biological material, and fixed. Made into Then, this noble metal powder is made to act as a mediator in the discrimination layer.
As a result, even if the mediator layer is not formed as an independent layer, electrons are exchanged at the electrode (working electrode) on the side where the discrimination layer is formed, and the electrode reaction accompanying the biochemical reaction is used as the response current at the working electrode. This is reflected to avoid a decrease in measurement accuracy due to dissolved oxygen.

[0012] Furthermore, by supporting the charge eliminating substance on the discrimination layer, the charge substance in the solution to be measured is electrically neutralized,
The charged substance is made harmless and does not interfere with the electrode reaction accompanying the biochemical reaction. Therefore, this biosensor
It is possible to accurately measure a substance to be measured in a solution to be measured in which a charged substance that acts as an interfering substance exists.

[0013]

Preferred embodiments of the present invention will be described below in order to further clarify the constitution and operation of the present invention described above. FIG. 1 is a perspective view of the biosensor 1 of the embodiment.

As shown in FIG. 1, the biosensor 1 has substantially the same structure as the biosensor 100 described above, but differs in the structure of the discrimination layer formed on the working electrode. That is, the biosensor is polyethylene terephthalate (P
An insulating substrate 3 having a plate thickness of 1.5 mm, a working electrode 5 and a counter electrode 7 formed on the insulating substrate 3, and an identification layer 9 formed by stacking on the upper surface of the working electrode 5. An insulating layer 11 that insulates the working electrode 5 and the counter electrode 7 from each other, and an electrical measurement unit (not shown) that is connected to the terminal portions 13 and 15 of the working electrode 5 and the counter electrode 7 and that measures the current value therebetween are provided. The identification layer 9 side is used as the sensitive section 17. Hereinafter, the biosensor 1 will be described in detail, but the manufacturing process and the like common to the biosensor 100 will be simplified.

Working electrode 5 in the biosensor 1 of the embodiment
The counter electrode 7 and the terminal portions 13 and 15 were formed through screen printing of a graphite paste on the upper surface of the insulating substrate 3 and drying treatment at 50 ° C. for 1 hour. At this time, as the graphite paste, a paste obtained by kneading 60 wt% of graphite fine powder having a particle size of 0.5 μm and 40 wt% of liquid paraffin with a roll mill was used.

After that, the insulating layer 11 and the identification layer 9 were sequentially formed. The formation of the insulating layer 11 is completed by a known process of printing and drying an appropriate insulating agent such as an epoxy resin. The discriminating layer 9 is formed by carrying and immobilizing glucose oxidase having a discriminating function for glucose and platinum (Pt) powder, and was formed as follows. First, 90 wt% collagen and 10 wt
%, Pt powder is added to an aqueous solution mixed with glucose oxidase (GOD) to prepare a GOD-Pt paste. Then, approximately 2 GOD-Pt paste was applied to the upper surface of the end of the working electrode 5 on the side opposite to the terminal portion 13 with a microsyringe.
The coating was applied in a thickness of 0 μm, and then naturally dried for 24 hours for solidification (gelation) to form the identification layer 9. Thus
The biosensor 1 of the example is completed.

In preparing the GOD-Pt paste for forming the identification layer 9, the particle size of Pt powder and the identification layer 9 are used.
The Pt powder content (weight ratio of Pt powder to the GOD aqueous solution, that is, parts by weight) in Table 1 was set to a particle size of 5 μm or less and a content of 5 parts by weight or more.

Next, an evaluation test of the biosensor 1 of the embodiment will be described. This evaluation test is performed in advance of a predetermined concentration (2
Glucose reagent (dissolved oxygen concentration: 8.0 ppm) prepared to 5 mg / dl) was prepared, the glucose concentration of this glucose reagent was measured with a sensor, and the measured concentration and the above-mentioned predetermined concentration of glucose (25 mg / dl) It was done by comparing with. The biosensor used in the evaluation test has a Pt powder particle size and content within a predetermined range (particle size: 5
Biosensor 1 with μm or less, content: 5 parts by weight or more)
(Example product) and a biosensor in which at least one of the particle size and the content of Pt powder is outside the above predetermined range (comparative example product).
And a conventional biosensor in which an identification layer is formed from a GOD paste containing no Pt powder (conventional product) and a biosensor in which a mediator layer is laminated from a ferrocene derivative liquid on the identification layer (conventional improved product). Using. The results are shown in Table 1.

[Table 1]

The GOD content in the discriminating layer of the conventional biosensor and the conventional improved biosensor is the same as that of the biosensor 1 of the example, and the thickness of the mediator layer in the conventional improved biosensor is about 10 μm. Yes,
The ferrocene content in the mediator layer is about 2w
t%. In addition, the measured value for each sensor in Table 1 is the current value measured by the electrical measurement unit of each sensor converted to glucose concentration, and is the average of three measured values.

As is clear from Table 1, according to the biosensors of the respective Examples, the glucose concentration of the glucose reagent (25 mg / 25 mg / 25 mg / kg) is equivalent to that of the conventional improved biosensor in which the discrimination layer and the mediator layer are formed in an overlapping manner. dl) can be measured accurately. On the other hand, even if the Pt powder is carried on the identification layer, the measurement accuracy is low in the biosensor of Comparative Example in which at least one of the particle size and the content is out of the predetermined range.

That is, the Pt powder is allowed to act as a mediator in the discrimination layer not only by supporting and fixing the Pt powder on the discrimination layer, but also by setting the particle size and the content thereof to specific values. Electrons can be transferred and received on the working electrode 5 side. Therefore, even if the mediator layer is not formed as an independent layer, electrons are transferred in the working electrode 5 to reflect the electrode reaction accompanying the biochemical reaction as the response current in the working electrode 5, and the measurement caused by the dissolved oxygen is performed. It is possible to avoid a decrease in accuracy. As a result, according to the present embodiment, it is possible to omit the mediator layer and simplify the configuration, and it is possible to reduce the number of steps required for manufacturing the sensor and to reduce the cost due to the omission of the step of forming the mediator layer. .. In addition, in this embodiment,
The electrodes and insulating substrate are made of inexpensive graphite and plastic (PE
Since it is created from T), the cost can be further reduced.

Further, using the biosensor 1 of the example in which Pt powder having a particle size of 2 μm and a content of 10 parts by weight is carried on the discriminating layer 9 together with glucose oxidase, glucose prepared at various glucose concentrations is prepared. When the reagent was measured, a linear relationship was obtained between the glucose preparation concentration and the measured concentration over the concentration range of 5 to 380 mg / dl. That is, according to this embodiment, a biosensor suitable for practical use can be easily obtained.

Next, another embodiment will be described. In the biosensor 1A in this embodiment, glucose oxidase and Pt are added to the identification layer 9 of the biosensor 1 described above.
It differs from the biosensor 1 in that potassium periodate (KIO ₄ ) having an oxidizing action is carried in addition to powder (particle size: 2 μm, content: 10 parts by weight). For this reason,
At the time of its production, electrodes and insulating layers were formed in the same manner as the biosensor 1, and the identification layer 9A was formed as follows.

First, like the biosensor 1, 90 wt%
Pt powder (particle size: 2) was added to an aqueous solution prepared by mixing 10 wt% glucose oxidase (GOD).
μm, content: 10 parts by weight) and potassium periodate are added to prepare a GOD-Pt paste. Then, the GOD-Pt is attached to the upper surface of the end of the working electrode 5 opposite to the terminal portion 13.
The paste was applied in a thickness of about 20 μm, and then naturally dried for 24 hours to be solidified (gelled) to form the identification layer 9. Thus, the biosensor 1A of the example in which glucose oxidase, Pt powder as a mediator, and potassium periodate are carried and immobilized is completed. In addition,
Biosensor 1A1 containing 2 parts by weight of potassium periodate in the discrimination layer and 4 parts by weight of biosensor 1A
Created 2 and.

The following experiment was conducted on the assumption that glucose in urine is measured using the biosensors 1A1 and 1A2. That is, uric acid was gradually added to a glucose reagent prepared to have a glucose concentration of 25 mg / dl to increase the uric acid concentration, and the relationship between the uric acid concentration in the glucose reagent and the glucose measurement value by each of the above sensors was investigated. The result is shown in FIG. Similarly, the relationship between the ascorbic acid concentration and the glucose measurement value by the sensor was examined. The result is shown in FIG.

As is clear from FIGS. 2 and 3, according to the biosensors 1A1 and 1A2 in which potassium periodate was also carried and immobilized on the discriminating layer, the concentration of uric acid in the glucose reagent was 0 to 80 mg / dl. Until the ascorbic acid concentration was 0 to 30 mg / dl, and the glucose concentration (25 mg / d) was accurately measured.
l) can be measured. In other words, even if uric acid or ascorbic acid that interferes with the electrode reaction accompanying the biochemical reaction due to the negative electrode being charged and having a reducing property is present in the solution to be measured, it is oxidized by potassium periodate carried on the discrimination layer. And make it harmless. Therefore, the influence of these interfering substances can be eliminated with a simple configuration in which potassium periodate is supported on the discrimination layer, and the glucose measurement accuracy can be improved. Particularly, in the biosensor 1A2 having 4 parts by weight of potassium periodate supported on the discrimination layer,
Accurately measure glucose levels over a wide range of uric acid concentrations up to 160 mg / dl and over a wide range of ascorbic acid concentrations up to 0-40 mg / dl by eliminating the effects of these interfering substances. be able to.

By the way, urine excreted from the human body contains 20-80 mg / dl of uric acid and ascorbic acid, respectively.
It is statistically known to be contained in a concentration range of 0 to 25 mg / dl. As a result, biosensor 1
According to A1 and 1A2, glucose in urine in which uric acid and ascorbic acid naturally exist can be accurately measured.

The present invention is not limited to the above-described embodiments, but can be carried out in various modes without departing from the scope of the invention, and the following modifications are possible. For example, it may be a sensor for glucose measurement, which uses an enzyme such as pyranose oxidase or mutarotase instead of glucose oxidase, or a microorganism such as Pseudomonas fluorescens.
Further, it may be a sensor for measuring glucuronic acid using glucuronic acid oxidase as a biological substance. Furthermore,
Sodium periodate (NaIO ₄ ), potassium perchlorate (KClO ₄ ), sodium perchlorate (NaClO ₄ ) is used instead of potassium periodate (KIO ₄ ) to be carried on the discrimination layer.
It is also possible to detoxify the negative electrode by oxidizing an interfering substance such as uric acid or ascorbic acid which has a reducing property by charging the negative electrode.

The noble metal powder to be carried on the discriminating layer is not limited to Pt, but Au, Ag, Pd, Rh, Ir or the like may be used. In this case, Pt, Au, and Ag are preferable because they are easily available. Each of these noble metals is common in that it belongs to Group 8 or Group 1B (transition elements) in the periodic table and exhibits a relatively inert property compared to other noble metals. Therefore, even with the above-mentioned sensor carrying each noble metal powder, it is possible to obtain the same result as that of the Pt powder carrying sensor of the above-mentioned embodiment.

In the above-mentioned embodiment, the biosensor provided with the two electrodes of the working electrode 5 and the counter electrode 7 as one set of electrodes has been described. However, the reference electrode is added to the two electrodes of the working electrode and the counter electrode. It may be a biosensor including one electrode as a set of electrodes. In this way, if three electrodes are used as a set of electrodes, the difference between the amount of change in the amount of electricity between the working electrode and the reference electrode and the amount of change in the amount of electricity between the counter electrode and the reference electrode is used as the change in the amount of electricity as a biosensor, It is possible to obtain a more accurate concentration of the substance to be measured excluding factors other than the substance to be measured. In addition,
In this case, the identification layer carrying the biological substance may be formed only on the surface of the working electrode.

[0031]

As described in detail above, according to the biosensor of the present invention in which a specific precious metal powder is supported with a biomaterial in a specific particle size and content, this precious metal powder is used as a mediator in the discrimination layer. Since it is made to act, it has the following effects. In other words, without forming an independent mediator layer, the electrode reaction accompanying the biochemical reaction is reflected as the response current in the working electrode by making the precious metal powder act as a mediator, and the decrease in measurement accuracy due to dissolved oxygen is avoided. can do. Therefore, it is possible to omit the mediator layer without reducing the measurement accuracy and simplify the configuration, and it is possible to reduce the number of steps required for manufacturing the sensor and to reduce the cost due to the omission of the step of forming the mediator layer. ..

In addition to the biological substance and the noble metal powder, the charge-eliminating substance is carried on the discriminating layer, so that the charged substance in the solution to be measured is electrically neutralized to remove the charged substance present in the solution to be measured. It is harmless to the electrode reaction that accompanies biochemical reactions. Therefore, according to the biosensor of the present invention, the influence on the electrode reaction accompanying the biochemical reaction exerted by the charged substance acting as an interfering substance is eliminated by a simple configuration in which the charge eliminating substance is carried on the discrimination layer, It is possible to improve the measurement accuracy of the measurement target substance in the measurement target solution in which the charged substance is present.

[Brief description of drawings]

FIG. 1 is a schematic perspective view used for explaining a configuration of a biosensor 1 of an example.

FIG. 2 is a graph for explaining the effect of the biosensor of the example and showing the relationship between sensor output (glucose concentration) and uric acid concentration.

FIG. 3 is a graph for explaining the effect of the biosensor of the example and showing the relationship between the sensor output (glucose concentration) and the ascorbic acid concentration.

FIG. 4 is a schematic perspective view used to describe the configuration of a conventional biosensor 100.

[Explanation of symbols]

 1 Biosensor 1A Biosensor 1A1 Biosensor 1A2 Biosensor 3 Insulating substrate 5 Working electrode 7 Counter electrode 9 Identification layer 9A Identification layer 11 Insulating layer 13, 15 Terminal part 17 Sensitive part 100 Biosensor 103 Working electrode 107 Identification layer

Claims (2)

[Claims] 1. A biosensor for detecting a substance to be measured by measuring the amount of electrical change associated with a biochemical reaction between the substance to be measured in a solution to be measured and a biological substance, which is formed on an insulating substrate. A pair of electrodes, and a discrimination layer formed on the surface of one electrode of the pair of electrodes carrying a biological substance having a discrimination function for the substance to be measured, wherein the discrimination layer is In addition to the biological material, Au, Ag, Pd,
One or more precious metal powders selected from Rh, Ir, and Pt are supported, and the supported precious metal powders have a particle size of 5 μm or less and are contained in an amount of 5 parts by weight or more based on 100 parts by weight of the biological material. Characteristic biosensor. 2. The identification layer, in addition to the biological substance and the noble metal powder, carries an antistatic substance having a property of electrically neutralizing the charged substance in the solution to be measured. 1. The biosensor according to 1.