JPH04343065A - Biosensor - Google Patents

Abstract

PURPOSE:To obtain a biosensor of a diaphragmed peroxide electrode type which is affected little by a water-soluble disturbing substance (ascorbic acid, uric acid or the like) and so can excellent accurate measurement and which is excellent in sensitivity. CONSTITUTION:A biosensor is of a diaphragmed peroxide electrode type having an internal electrolytic solution, and it has a hydro-phobic peroxide-permeable tubular body (formed of silicon rubber or the like) 3, rubber plugs 5 closing up the opposite ends of the tubular body 3, electrodes (a working electrode 1 and an opposite electrode 2) held in the tubular body, an electrolytic solution 6 and an enzyme fixing film (non woven fabric which bear glucose oxidase and the glucose oxidase is fixed) 4 disposed outside the tubular body 3.

Description

[Detailed description of the invention]

0001

[Field of Industrial Application] The present invention relates to a diaphragm-type hydrogen peroxide electrode type biosensor, and more specifically, a diaphragm made of a hydrophobic material such as silicone rubber, and an electrode covered with the diaphragm. The present invention relates to a biosensor that reduces the influence of water-soluble interfering substances and has excellent sensitivity by increasing the electrode area. INDUSTRIAL APPLICATION This invention is utilized for a clinical examination, chemistry, the food industry, environmental measurement, etc.

0002

[Prior art] In conventional biosensors, enzymes are immobilized on hydrogen peroxide electrodes, which have a structure in which an electrode is placed together with an electrolyte in an electrode support tube made of glass or plastic, and the end surface of the electrode is covered with a hydrophilic diaphragm. A type equipped with a membrane is known.

0003

[Problems to be solved by the invention] However, since the conventional biosensor uses a hydrophilic diaphragm,
Water-soluble interfering substances (ascorbic acid, uric acid, etc.) in the liquid to be measured penetrated into the diaphragm and were detected as error currents on the electrodes, making accurate measurements impossible. In addition, since only the end face of the electrode is used as the detection part, only a small electrode area can be obtained compared to the size of the electrode body, resulting in low sensitivity and making it impossible to miniaturize.

The present invention solves the above problems and provides a diaphragm-type hydrogen peroxide electrode type biosensor that is less affected by water-soluble interfering substances, enables accurate measurements, and has excellent sensitivity. The purpose is to provide.

[0005]

[Means for Solving the Problems] A diaphragm-type hydrogen peroxide electrode type biosensor having an internal electrolyte according to the first invention includes a hydrophobic hydrogen peroxide-permeable cylinder and a hydrogen peroxide-permeable cylinder held within the cylinder. The cylindrical body is characterized by having an electrode, an electrolytic solution, and an enzyme-immobilized membrane disposed outside the cylindrical body. Further, the biosensor of the third invention includes, for example, as shown in FIG. 6, an insulating substrate 7 having a recess, electrodes 1 and 2 disposed and filled in the recess, and an electrolyte (buffer). 6, a hydrophobic hydrogen peroxide permeable membrane 8 disposed on the recess and sealing the electrolyte;
It is also possible to have a laminated structure including an enzyme immobilization membrane 4 laminated on the hydrogen peroxide permeable membrane.

The hydrogen peroxide-permeable cylinder or hydrogen peroxide-permeable membrane may contain water-soluble interfering substances (
The material may be made of a hydrophobic material that does not allow hydrogen peroxide (such as ascorbic acid) to pass therethrough and allows hydrogen peroxide to pass through, such as silicone rubber or other Teflon membranes. Furthermore, various enzymes are selected based on the correlation with the substance to be measured (for example, glucose, etc.). The form of the enzyme-immobilized membrane is membranous (including layered).
It is sufficient if it becomes . Therefore, the substrate for supporting and immobilizing this enzyme is not particularly limited as long as it is in the form of a membrane. For example, it can be a nonwoven fabric, cellulose acetate, etc. It can also be supported on the surface of.

[0007]

[Operation] In the present invention, since the electrode is covered with a hydrophobic hydrogen peroxide permeable cylinder, the generated hydrogen peroxide passes through this cylinder or membrane and diffuses onto the electrode. Accurate measurement is possible because water-soluble interfering substances in the liquid to be measured cannot diffuse onto the electrode. In addition, since this hydrogen peroxide permeable cylinder etc. and the enzyme immobilization membrane cover the entire electrode,
The entire device is configured as a detection section, which increases the electrode area and improves sensitivity. Furthermore, when this hydrogen peroxide permeable cylinder is used, it also functions as an electrode support cylinder at the same time.

[0008]

[Effects of the Invention] As described above, by using the hydrogen peroxide electrode type biosensor of the present invention, accurate measurement can be performed with less influence of water-soluble interfering substances, and it can also be made with excellent sensitivity. can. In addition, when using this hydrogen peroxide permeable cylinder, it also functions as an electrode support cylinder, so it can stably hold the electrodes at the same time, and the entire structure can be used as a detection part. It becomes easy to downsize the sensor. Furthermore, when silicone rubber is used as the material for the hydrogen peroxide permeable cylinder or hydrogen peroxide permeable membrane, it has excellent flexibility and elasticity, so it can be designed into various shapes and can be freely adapted to the shape of the mating material. You can also match them.

[0009]

[Examples] The present invention will be explained in detail with reference to Examples below. Example 1 In this example, selectivity to interfering substances was investigated in comparison with Comparative Example 1. As shown in FIG. 1 (longitudinal cross-sectional view) and FIG. 2 (cross-sectional view), the diaphragm-type hydrogen peroxide electrode type glucose sensor of Example 1 includes a silicone rubber tube 3 and a silicone rubber tube 3 that closes both ends of the tube 1. Stopper 5 and
It consists of electrodes (working electrode 1 and counter electrode 2) held therein, an electrolyte 6, and an enzyme-immobilized nonwoven fabric (membrane) 4 disposed outside the tube 3. This sensor was manufactured as follows.

First, a silicone rubber tube (diameter: 2 m)
mφ, length: 15 mm, wall thickness: 50 μm) and buffer solution (
A phosphate buffer solution (pH=7.0) is added thereto, and one end is sealed with a silicone stopper 5. Next, prepare two platinum wires 1 and 2 with a diameter of 1 mm as a pair of electrodes, wrap a nylon net 9 around one platinum wire 2, and keep the platinum wires 1 and 2 from coming into direct contact with each other in a silicone rubber tube 3. Insert inside. Leads 11 and 21 are taken out from the other end of silicone rubber tube 3, and the other opening is similarly sealed with silicone plug 5. After that, a predetermined nonwoven fabric was immersed in a 5% glucose oxidase solution, and then immobilized with glutaraldehyde, and this enzyme-immobilized nonwoven fabric 4 was wrapped around the entire outside of the silicone rubber tube 3. A glucose sensor was constructed.

In order to investigate the selectivity of the present invention for interfering substances (ascorbic acid and uric acid), +600 m was applied to the working electrode.
A constant potential of V was applied and the current value was measured. In this case, the detected current value in the case of only glucose (100 mg/dl) (0.20 μA in Example 1, 0 in Comparative Example 1)
．． 18 μA) as 100, and ascorbic acid 10
When uric acid was added at 10 mg/dl, and when 10 mg/dl of uric acid was added, each was expressed as a percentage. In addition, as Comparative Example 1, a glucose sensor using a hydrophilic cellulose acetate membrane as a diaphragm was used. These results are shown in Table 1.

0012

[Table 1]

As shown in the results in Table 1, the sensor of Example 1 was not affected by ascorbic acid and uric acid at all, but in Comparative Example 1, the effects of ascorbic acid and uric acid were 40% and 45%, respectively.
also showed an impact.

Example 2 In this example, the sensitivity was compared between a sensor in which only the electrode end face was used as a detection part (Comparative Example 2) and the sensor of the present invention. The sensor used in this example is the same glucose sensor as in Example 1. As shown in FIG. 3, the sensor of Comparative Example 2 was made of two platinum wires (φ1 m
m, l10mm) 1 and 2 are glass tubes (diameter; 3mmφ,
length: 15 mm) 10, a silicone rubber membrane 8 is attached to the end face of the platinum wire 1, and an enzyme immobilization membrane (enzyme immobilization nonwoven fabric used in Example 1) 4 is placed on the outside of this membrane 8. A device with a similar configuration was used. Note that the size of the electrode body is almost the same in both Example 2 and Comparative Example 2. Using both the above sensors, glucose 100mg/dl and 300m
Table 2 shows the results of measuring the response current values to g/dl and comparing the sensitivities.

[0015]

[Table 2]

According to the results in Table 2, in this Example 2, the entire electrode was covered with a silicone rubber tube, and the entire surface of this tube was covered with an enzyme-immobilized membrane, so the entire electrode was used as a detection section. This enabled us to secure a large electrode area. On the other hand, in Comparative Example 2, only the end face of the electrode is used as a detection part. Therefore, the sensor of Example 2 is the same as that of Comparative Example 2.
Sensitivity has improved by 5 to 6 times compared to previous sensors. This silicone rubber tube also serves as an electrode support tube.

It should be noted that the present invention is not limited to those shown in the above-mentioned specific embodiments, but can be modified in various ways within the scope of the present invention depending on the purpose and use. That is,
Although platinum electrodes were used in the above embodiments, electrodes made of gold, carbon, metal oxides, etc. may also be used. Also, as mentioned above, when using a silicone rubber tube as an electrode support tube,
Due to its flexibility and elasticity, the biosensor can be designed in any shape. For example, it can be made into a long shape and wound into a spiral as shown in Figure 5, or it can be made into a U-shape as shown in Figure 5, or it can be made into various curved tube shapes or other shapes depending on the purpose and use. can. Further, the enzyme-immobilized membrane may be disposed on the entire surface of the cylindrical body, or may be placed on a desired portion thereof as shown in FIG. 4.

[Brief explanation of the drawing]

FIG. 1 is a longitudinal cross-sectional view of a glucose sensor of Example 1.

FIG. 2 is a cross-sectional view of the glucose sensor of Example 1.

FIG. 3 is a longitudinal cross-sectional view of a glucose sensor of Comparative Example 2.

FIG. 4 is an explanatory diagram of a biosensor in the shape of a long curved tube.

FIG. 5 is a longitudinal cross-sectional view of a U-shaped tube-shaped biosensor.

FIG. 6 is an explanatory cross-sectional view of a biosensor in which electrodes, an internal electrolyte, a hydrogen peroxide permeable membrane, and an enzyme immobilization membrane are laminated on an insulating substrate.

[Explanation of symbols]

1 Working electrode 2. Opposite 3 Hydrogen peroxide permeable cylinder 4 Enzyme immobilization membrane 5 Silicone stopper 6 Buffer solution 7 Insulating substrate 8 Hydrogen peroxide permeable membrane

Claims (4)

[Claims] Claim 1: A diaphragm-type hydrogen peroxide electrode type biosensor having an internal electrolyte, comprising: a hydrophobic hydrogen peroxide-permeable cylinder; an electrode and an electrolyte held within the cylinder; and the cylinder. An enzyme-immobilized membrane disposed outside the biosensor. 2. The biosensor according to claim 1, wherein the hydrogen peroxide permeable cylinder is made of silicone rubber. 3. A diaphragm-type hydrogen peroxide electrode type biosensor having an internal electrolyte, comprising: an insulating substrate having a recess, an electrode and an electrolyte arranged and filled in the recess, and an electrode and an electrolyte disposed on the recess. A biosensor comprising: a hydrophobic hydrogen peroxide-permeable membrane that is provided to seal the electrolytic solution; and an enzyme-immobilized membrane laminated on the hydrogen peroxide-permeable membrane. 4. The biosensor according to claim 3, wherein the hydrogen peroxide permeable membrane is made of silicone rubber.