JPH06324015A - Bio sensor and its manufacture - Google Patents

Abstract

PURPOSE:To provide a bio sensor which is capable of an accurate measurement and its manufacture by positively carrying an active enzyme, etc., by a specific amount. CONSTITUTION:In the bio sensor, a recessed part 12 for measurement is provided at an insulation substrate 14, a water-repellent layer 20 with an operation electrode 16, a counter electrode 18, and an opening 20a for introduction is formed on the upper part of the opening 12 for measurement, and further an identification layer 22 is formed inside the recessed part 12 for measurement. The identification layer 22 is formed by dropping a liquid solution containing an enzyme via the opening 22a for introduction of the water-repellent layer 20 and then drying and hardening it. The water-repellent layer 20 is filled by introducing a liquid solution to be measured to the recessed part 12 for measurement. A substance to be measured of the liquid solution to be measured reacts with a living organism substance carried by the identification layer 2 and the substance to be measured is measured based on the reaction. The enzyme carried into the identification layer 22 is simply contained in the liquid solution and then dried and hardened in the recessed part 12 for measurement, thus preventing activity from being lost since no chemical treatment is performed.

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 by measuring an amount of electrical change due to a biochemical reaction between the substance to be measured in a solution to be measured and a biological substance, and a method for producing the biosensor. Is.

[0002]

2. Description of the Related Art Conventionally, as this type of biosensor, for example, a flat plate type biosensor disclosed in JP-A-61-50262 is known. That is, as shown in FIG. 7, the flat plate type biosensor 100 includes a ceramic substrate 101, a working electrode 103 and a counter electrode 105 formed on the ceramic substrate 101, and a space between the working electrode 103 and the counter electrode 105. An insulating layer 108 for insulation and an identification layer 10 carrying a biological substance such as an enzyme on the working electrode 103.
7 and the terminal portion 10 of the working electrode 103 and the counter electrode 105.
9 and 111, each of which is provided with an electric measurement unit (not shown) for measuring a current value therebetween, and the identification layer 107 side serves as a sensitive unit 113.

Here, as a method for supporting the biological substance on the discrimination layer 107, an adsorption method, a chemical modification method, an encapsulation method and the like are known. The encapsulation method is a method of encapsulating a biological substance in a polymer matrix. For example, a carrier such as polyacrylamide gel as a polymer is formed on the working electrode 107, and the biological substance is chemically treated with glutamine aldehyde on the carrier. It is a method of supporting by. Also, the adsorption method is
A method of adsorbing and fixing a biological substance on an insoluble carrier film, for example, forming an adsorption film such as albumin or cellulose on an electrode, immersing the adsorption film in a solution in which the biological substance is dissolved, and further performing a cleaning treatment. This is a method of supporting the above-mentioned biological substance on the adsorbent by selecting the biological substance that is applied and surely attached to the working electrode 103.

To measure a solution to be measured using this biosensor 100, the sensitive part 113 is immersed in the solution to be measured. As a result, the biological substance carried on the identification layer 107 undergoes a biochemical reaction with the substance to be measured contained in the solution to be measured to generate oxygen or hydrogen peroxide. The substance to be measured is measured by measuring the electric current associated with the redox caused by the generation of oxygen and the like in the electric measuring unit.

[0005]

However, the biosensor in which the biological substance is supported on the carrier by the entrapment method or the adsorption method as described above has the following problems. That is, in the inclusion method, the biological substance is inactivated due to the chemical influence at the time of immobilization of the biological substance, or the biological substance is often clogged with the carrier and does not come into contact with the liquid to be measured. There is a problem that the usage efficiency decreases.

Further, in the adsorption method, a washing treatment is carried out after the immobilization, and a large amount of the biological substance is removed by this washing treatment. Therefore, in a small biosensor, a certain amount or more of the biological substance is maximally acting. There was a problem that it was difficult to fix to.

Further, the sensitive part 11 having different biological materials is used.
It has been studied to form a plurality of 3 on the same insulating substrate and simultaneously measure many substances to be measured in an insulating solution to be measured. However, when a large number of sensitive parts of different biological substances are provided on the same insulating substrate, the substance generated from one sensitive part interferes with other adjacent sensitive parts, and accurate measurement results cannot be obtained. There was also a problem.

The present invention solves the above-mentioned problems of the prior art. It stably comprises a predetermined amount or more of an active biological substance and simultaneously accurately measures a plurality of substances to be measured in a solution to be measured. It is an object of the present invention to provide a biosensor that can be manufactured and a method for manufacturing the same.

[0009]

In order to solve the above-mentioned problems, the invention of claim 1 is to measure the amount of electrical change due to the biochemical reaction between the substance to be measured and the biological substance in the solution to be measured. In a biosensor for measuring a substance to be measured by an insulating substrate, a measuring recess for storing a solution to be measured, which is formed on the insulating substrate, is provided in the measuring recess, the biological substance A discrimination layer having the above, a pair of electrodes provided near the opening of the measurement recess and provided so as to be immersed in the solution to be measured stored in the measurement recess, and an opening peripheral edge of the measurement recess And a water-repellent layer provided at least in the portion.

According to a second aspect of the present invention, in the biosensor according to the first aspect, a plurality of measurement portions including the measurement recess, the identification layer and a pair of electrodes are formed on an insulating substrate.

Further, claim 3 is a method for manufacturing a biosensor for measuring a substance to be measured by measuring an amount of electrical change caused by a biochemical reaction between the substance to be measured in a solution to be measured and a biological substance. Forming a pair of electrodes on the flexible substrate, forming a water repellent layer on the insulating substrate so that the regions facing the pair of electrodes are openings, and insulating through the openings. Forming a measuring recess for storing the solution to be measured by removing and forming a part of the flexible substrate; and inserting a solution containing a biological substance into the measuring recess through the opening, and drying the solution. And a step of forming an identification layer having the biological substance in the measurement recess.

[0012]

In the biosensor of the first aspect of the present invention, when the solution to be measured is dropped near the opening of the water-repellent layer, the solution to be measured is introduced into the measurement recess through the opening and comes into contact with the discrimination layer. The substance to be measured in the solution to be measured reacts with the biological substance carried on the discrimination layer to generate oxygen or hydrogen peroxide. The generation of oxygen or the like causes a redox reaction on the electrodes, and a current flows between the pair of electrodes. The substance to be measured in the solution to be measured is measured based on this current. At this time, the solution to be measured is guided from the opening to the measurement recess by the water-repellent layer,
Since it does not leak to other parts and is stored in the measuring recess, the measurement is performed accurately with a small amount of the solution to be measured.

Further, in the invention of claim 1, since the identification layer is formed in the space defined by the measurement recess, it can be formed accurately within a substantially fixed range.

Further, in the biosensor of the second aspect, a plurality of measuring portions including the measuring recess, the identification layer and a pair of electrodes are formed on the same insulating substrate. In these plural measuring sections, the solution to be measured is
It is introduced into each measuring recess through each opening and reacts with the biological material of the discriminating layer. At this time, since the measurement recesses of each measurement unit are formed independently of each other, the reaction product of each measurement unit does not affect the other measurement units. Therefore, a plurality of substances to be measured can be accurately measured at one time.

Further, in the method for manufacturing a biosensor according to claim 3, in order to hold the biological substance in the measuring recess, first, a solution containing the biological substance is prepared, and the solution is opened in the water repellent layer. Drop near. At this time, due to the water repellency of the water repellent layer, the solution to be measured is guided to fill the measurement recess through the opening. Then, the solution in the measurement recess is dried to form an identification layer carrying a biological substance in the measurement recess. At this time, the amount of the biological substance is adjusted to a predetermined amount that depends on the capacity of the measuring recess. Further, the water-repellent layer guides and fills the measurement target solution into the measurement recess through the opening when the measurement target solution is measured. The amount of the solution to be measured depends on the volume of the measuring recess as well as the amount of the biological substance in the discrimination layer, and has a correlation with the amount of the biological substance. That is, the volume of the measurement recess brings about a predetermined correlation between the biological substance and the amount of the solution to be measured, so the solution to be measured is:
Accurate measurement is possible regardless of the amount of biological material. In addition, since the biological substance is simply dried and fixed in the measurement recess and is not chemically treated, the biological substance is hardly deactivated.

[0016]

Preferred embodiments of the present invention will be described below in order to further clarify the structure and operation of the present invention described above.

FIG. 1 is a plan view of the sensitive portion of the biosensor, and FIG. 2 is a sectional view taken along line II-II of FIG.
The sensitive section 10 of the biosensor includes an insulating substrate 14 having a measurement recess 12, working electrodes 16 and counter electrodes 18 formed on the insulating substrate 14 and facing each other, the insulating substrate 14, the working electrode. The water repellent layer 20 is laminated on the counter electrode 16 and the counter electrode 18, and the discriminating layer 22 is formed on the bottom surface of the measuring recess 12.

The water-repellent layer 20 is a layer formed of an electrically insulating and hydrophobic material, and has a working electrode 16 and a counter electrode 18.
Has an introducing opening 20a in a region including the tip of the terminal and terminal openings 20c and 20d at the other ends. The introduction opening 20a is formed at a position where the measurement target solution can be introduced into the measurement recess 12.

The discrimination layer 22 is a layer containing a biological substance such as an enzyme that biochemically reacts with the substance to be measured in the solution to be measured. In addition, the working electrode 16 and the counter electrode 18 include detecting portions 16a and 18a located above the measuring recess 12, terminal portions 16b and 18b for connection, and detecting portions 16a and 18a and terminal portions 16b and 18b. Wiring unit 16 for connecting each
c, 18c. The terminal portions 16b and 18b of the working electrode 16 and the counter electrode 18 are exposed in the terminal openings 20c and 20d of the water repellent layer 20, and the connection lines of the electric measurement unit (not shown) are connected to each other. ing.

Next, a measuring method using the biosensor will be described. When the sensitive part 10 of the biosensor is immersed in the solution to be measured, the solution to be measured is difficult to wet this part because the water-repellent layer 20 is hydrophobic. Through the gap between the pole 16 and the counter electrode 18, it is guided to the measurement recess 12 and fills it. In this state, a constant voltage is applied between the working electrode 16 and the counter electrode 18. The substance to be measured in the solution to be measured undergoes a biochemical reaction by the action of the biological substance of the discrimination layer 22. A current flows along with this reaction, and this current is electrically processed to measure the substance to be measured. As the electrical treatment, a method of measuring the total amount of the substance to be measured in the solution to be measured based on the total amount of electricity obtained by integrating the current value, or a method of measuring from the amount of electricity when it is stable after a certain period of time is adopted. In addition, in order to store the solution to be measured in the measurement recess 12, as described above, the sensitive section 10 is used.
Alternatively, a small amount of the solution to be measured may be dropped near the introduction opening 20a of the water repellent layer 20.

Next, the manufacturing process of the biosensor will be described with reference to FIG. (1) Manufacturing process of insulating substrate 14 First, the insulating substrate 14 is formed. This insulating substrate 14
In the step of forming, a plate material may be prepared from a hydrophilic material such as glass or a composite material thereof, and cut into a predetermined shape. As another method, a method of firing a ceramic green sheet can be adopted.

(2) Step of forming working electrode 16 and counter electrode 18 (FIG. 3 (A)) The working electrode 16 and counter electrode 18 are formed on the insulating substrate 14. As a process of forming the working electrode 16 and the counter electrode 18, a known thick film printing method, vapor deposition method, sputtering method or the like can be adopted. For the working electrode 16, it is desirable to use a metal having a catalytic action for decomposing hydrogen peroxide, and for example, gold, platinum, palladium, titanium and the like and alloys thereof can also be used. Further, the counter electrode 18 may be any material that can take the potential of the solution to be measured, for example, gold, platinum,
Palladium, copper, iron, silver, titanium and the like and alloys thereof can be used.

(3) Step of forming the water repellent layer 20 (see FIG. 3)
(B)) Water repellent layer 2 on insulating substrate 14, working electrode 16 and counter electrode 18.
Stack 0. As the step of forming the water-repellent layer 20, a method of applying a water-repellent material to a predetermined thickness to form a layer, a method of depositing the water-repellent material to a predetermined thickness by an evaporation method, and forming a film from the water-repellent material. It is possible to employ a method of forming and adhering these, and the like. As a method for simplifying the process, it is desirable to use a photoresist method so that it may also serve as a mask for etching the measurement recess 12 as described later. In the case of the method of adhering the film, the introduction opening 20a is formed in the hydrophobic film, and 1 is formed on the film.
A pair of electrodes is formed and laminated and adhered to an insulating substrate.
As the water-repellent material, for example, novolac resin or isoprene rubber, polyimide / polyethylene, polyester or the like can be used. The introduction opening 20a of the water-repellent layer 20 is not limited to a square as shown in FIG. 1 but various shapes and sizes such as a circle and a polygon as long as the solution and the solution to be measured can be introduced easily. In the case of a square, for example, one side is formed with a thickness of 10 μm to 1 mm.

(4) Step of forming the measuring recess 12 (see FIG. 3)
(C)) Through the opening 20 a for introducing the water repellent layer 20, the insulating substrate 1
By removing the upper portion of 4 by etching, the measuring recess 1
Form 2. That is, when the etching liquid is applied from above the water repellent layer 20, the etching liquid has poor wettability at the portion of the water repellent layer 20, and therefore gathers in the introduction opening 20a. Therefore, the insulating substrate 14 on the lower side of the introduction opening 20a is
Is etched to form a measuring recess 12 in this portion.

(5) Step of Forming Discrimination Layer 22 A solution containing a biological substance such as an enzyme or immunity is prepared, and the solution is cut down toward the introduction opening 22a to make a measurement recess 12
Fill in. Then, by performing a drying process, the identification layer 22 is formed at the bottom of the measurement recess 12. In addition, it is desirable that the solution for dissolving the enzyme, the immunity and the like is adjusted to pH 4 to 5 by containing sodium phosphate or the like. As a result, the sodium phosphate in the solution is taken in as a salt when the identification layer 22 is solidified, but is dissolved when the solution to be measured enters the measurement recess 12. The dissolved sodium phosphate has an action of adjusting to a pH at which a biological substance such as an enzyme is activated by being dissolved in the solution to be measured even if the pH of the solution to be measured itself is relatively high. Therefore, conventionally, the solution to be measured has been used for the measurement after pH adjustment with a buffer solution so that the biological substance does not lose its activity, but this operation can be omitted. As the biological substance, in addition to various enzymes, microorganisms and the like can be used, and the substance to be measured corresponding thereto can be measured. The biological substance may be dissolved in an aqueous solution or may be supported by a gel substance.

In the above biosensor manufacturing method, an introduction opening 20a surrounded by the water-repellent layer 20 is formed in the upper part of the measurement recess 12, and a biological substance is contained through the introduction opening 20a. The solution is introduced. At this time, the water-repellent layer 20 guides the solution to the introduction opening 20a due to its hydrophobicity and acts so as to fill it. Therefore,
The identification layer is formed only in the measurement recess 12. Therefore, the amount of the biological material of the identification layer 22 depends on the amount of the solution stored in the measurement recess 12 and is a desired accurate amount.

When the solution to be measured is measured using the biosensor manufactured by such a method, the water-repellent layer 2
0 indicates that the solution to be measured is introduced into the measurement recess 12 through the introduction opening 20a, and only the solution to be measured stored in the measurement recess 12 is carried on the discrimination layer 22 and the biological substance and biochemistry. Perform a dynamic reaction. The amount of the liquid to be measured has a correlation with each other depending on the volume of the measuring recess 12 as with the amount of the biological substance. As a result, an accurate value can be obtained for the measured value of the solution to be measured without depending on the amount of the biological substance.

Further, in order to form the discrimination layer 22 having a biological substance, the solution containing the biological substance is simply dried in the measuring recess, and the chemical method is used as in the conventional comprehensive method. Since the treatment is unnecessary, the biological substance is not deactivated, and since the treatment such as washing as in the adsorption method is not necessary, the biological substance is not dropped off.

<Experimental example> Next, the biosensor according to the above-mentioned example was applied to a sensor for measuring glucose, and its evaluation test was conducted. Sensing part 1 of biosensor
0 was created by the following steps. First, the insulating substrate 1
Glass plate as No. 4 (manufactured by Corning: product name 7059)
Was processed into a size of 10 mm length × 10 mm width × 0.5 mm thickness. Next, a mask was formed with a photoresist, and Pt was evaporated to a thickness of 0.3 μm on the unmasked insulating substrate 14 by an evaporation method to form the working electrode 16 and the counter electrode 18.

Next, a photoresist (manufactured by Tokyo Ohka Kogyo Co., Ltd .: trade name OMR83) is applied to the insulating substrate 14 to a thickness of 20 μm, and further, an introduction opening 20a and terminal openings 20c and 20d are formed by photolithography. The water repellent layer 20 was formed. Then, a part of the insulating substrate 14 was etched through the unmasked introduction opening 20a to form the measurement recess 12. As an etching solution, a solution prepared by adding nitric acid to a mixed acid of hydrofluoric acid / ammonium hydrofluoride / acetic acid and water was adjusted to 40 ° C. and used. Then, a solution in which glucose oxidase was dissolved in water was dropped toward the introduction opening 20a and dried to form the identification layer 22.

Glucose was measured by connecting the sensitive section 10 of the biosensor prepared by the series of steps to the electric measuring section. That is, glucose at 100 m with water at 27 ° C
Prepared to a sample solution of g / dl, and the sensitive part 1 of the biosensor
A constant voltage of 0.8 V was applied between the working electrode 16 of 0 and the counter electrode 18. In this state, the introduction opening 20a of the sensitive section 10
The sample solution was dropped toward the.

Next, a second embodiment will be described with reference to FIGS. FIG. 4 is a plan view of the sensitive part of the biosensor, and FIG. 5 is a sectional view taken along line VV of FIG.

The sensitive section 50 of the biosensor is provided with a first measuring section 50C and a second measuring section 50D having substantially the same structure on the insulating substrate 14A. The first measurement unit 50C is
The measurement recess 12C, the discrimination layer 22C, the working electrode 16C, and a part of the counter electrode 18A are configured, while the second measurement unit 50D includes the measurement recess 12D, the discrimination layer 22D, the working electrode 16D, and the working electrode 16D. It is composed of a part of the counter electrode 18A. The counter electrode 18A includes the first measurement unit 50C and the second measurement unit 5
It is a common electrode for the working electrodes 16C and 16D of 0D. Further, the discrimination layer 22C and the discrimination layer 22D carry different biological substances such as glucose oxidase and cholesterol oxidase, respectively.

In the biosensor according to the second embodiment, the first measuring section 50C and the second measuring section 50D are formed on the same insulating substrate 14A, and a plurality of these measuring sections 5 are provided.
In 0C and 50D, at the time of measurement, the solution to be measured is introduced into the measurement recesses 12C and 12D through the introduction openings 20e and 20f.
And reacts with the biomaterials of the identification layers 20C and 20D, respectively. At this time, each measurement recess 12C,
Since 12D is formed as an independent section, the reaction products of the substance to be measured and the biological substance do not affect the reaction products of the other measurement recesses 12C and 12D. Therefore, it is possible to accurately obtain a plurality of types of measurement signals from the solution to be measured with one measurement.

Moreover, the first and second measuring units 50C, 50
As shown in FIGS. 6 (A) to 6 (C), D can be formed at the same time by the series of steps as described above, so that the manufacturing is easy. Note that FIG. 6A to FIG.
It corresponds to (A) to FIG.

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 can be made.

In the above embodiment, the insulating substrate 14 is made of hydrophilic glass. However, the insulating substrate 14 is not limited to this and may be made of a hydrophobic material such as resin or Si. In this case, it is preferable to prepare a solution dissolved in alcohol such as siloxane and immerse the hydrophobic insulating substrate in the solution so that the measurement recess has hydrophilicity.

[0038]

As described above, according to the biosensor of the first aspect of the present invention, the solution to be measured is guided from the opening of the water-repellent layer to the measurement recess, and the substance to be measured of the solution to be measured is measured. The substance to be measured is measured based on the reaction by reacting with the biological substance carried on the discrimination layer. Therefore, the solution to be measured is guided from the opening to the measurement recess by the water-repellent layer,
Since the substance to be measured is measured by being stored in the measuring recess without leaking to other portions, the amount of the measured solution can be accurately measured.

Further, in the biosensor of the second aspect, a plurality of measuring portions including the measuring recess, the identification layer and the pair of electrodes are formed on the same insulating substrate. In these multiple measurement units, during measurement, the water-repellent layer guides the measured solution from each opening to each measurement recess, and the plurality of measured substances in the measured solution are biological materials of the discrimination layer. Each reacts. At this time, since the measurement recesses of each measurement unit are formed independently of each other, the reaction product of each measurement unit does not affect the other measurement units. Therefore, a plurality of substances to be measured can be accurately measured at one time.

Furthermore, in the method for manufacturing a biosensor according to claim 3, a solution containing a biological substance is prepared for immobilizing the biological substance in the discrimination layer, and the solution is used for measurement through the opening of the water-repellent layer. It is performed by putting it in the recess and drying the solution in the measurement recess. Therefore, the water repellent layer is
From the water-repellent action, a solution containing a biological substance is introduced into the measurement recess to form an identification layer in the measurement recess. Therefore, the amount of the biological substance is adjusted to a predetermined amount that depends on the volume of the measuring recess. Further, the water-repellent layer guides and fills the measurement target solution into the measurement recess through the opening when the measurement target solution is measured. The amount of the solution to be measured depends on the volume of the measuring recess as well as the amount of the biological substance in the discrimination layer, and has a correlation with the amount of the biological substance. That is, since the capacity of the measuring recess brings about a predetermined correlation between the biological substance and the amount of the solution to be measured, the solution to be measured can be accurately measured without being influenced by the amount of the biological substance. Further, the biological substance is simply dried in the measuring recess and carried on the discriminating layer, and is not chemically treated. Therefore, the biological substance is hardly deactivated.

[Brief description of drawings]

FIG. 1 is a plan view showing a sensitive portion of a biosensor according to a first embodiment of the present invention.

FIG. 2 is a sectional view taken along line II-II of FIG.

FIG. 3 is an explanatory view illustrating a manufacturing process of a sensitive part of the biosensor according to the embodiment.

FIG. 4 is a plan view showing a sensitive portion of a biosensor according to a second embodiment of the present invention.

5 is a cross-sectional view taken along line VV of FIG.

FIG. 6 is an explanatory view illustrating a manufacturing process of a sensitive part of the biosensor according to the second embodiment.

FIG. 7 is a perspective view showing a sensitive part of a conventional biosensor.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 10 ... Sensitive part 12 ... Recessed part for measurement 14, 14A ... Insulating substrate 16, 16C, 16D ... Working electrode 16a ... Detecting part 16b ... Terminal part 16c ... Wiring part 18, 18C, 18D ... Counter electrode 18a ... Detecting part 18b ... Terminal part 18c ... Wiring part 20, 20A ... Water repellent layer 20a ... Introducing opening 20c, 20d ... Terminal opening 22, 22C, 22D ... Identification layer 50 ... Sensitive part 50C ... First measuring part 50D ... Second measuring part

Claims (3)

[Claims] 1. A biosensor for measuring a substance to be measured by measuring an 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, an insulating substrate, and the insulating substrate. And a measurement recess for storing a solution to be measured, provided in the measurement recess, and facing the vicinity of the opening of the measurement recess and the identification layer having the biological substance, and for the measurement A pair of electrodes provided so as to be dipped in the solution to be measured stored in the recess, and a water-repellent layer provided at least on the peripheral edge of the opening of the measurement recess. Sensor. 2. The biosensor according to claim 1, wherein a plurality of measurement portions each including the measurement recess, the identification layer, and a pair of electrodes are formed on an insulating substrate. 3. A method for producing a biosensor, which measures a substance to be measured by measuring an 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, the method comprising the steps of: A step of forming a pair of electrodes, a step of forming a water-repellent layer laminated on the insulating substrate so that areas facing the pair of electrodes are openings, and a part of the insulating substrate through the openings. Forming a measurement recess for storing the solution to be measured by removing and forming a solution containing a biological substance into the measurement recess through the opening, and drying the solution for measurement. And a step of forming an identification layer having the above-mentioned biological material in the recess, the manufacturing method of the biosensor.