JP2000146892A - Plainer type chemical sensor element, and manufacture thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To reduce deposit of a fouling substance on the outermost layer contacting with a test liquid upto a very low level, to stabilize thereby sensitivity even when used for a long period or repeatedly used, and to allow highly precise measurement. SOLUTION: This sensor element has an electrode system with at least a working electrode 3 and a counter electrode 4 formed on an insulating sustrate 1, and an immobilized enzyme membrane 5 provide on the electrode system. A pearmeability-restricting film 6 for restricting intrusion of a substarte, and a porous water repellant film 7 are laminated on the enzyme membrane 5 in order. Alternatively, the permeability-resricting film 6 and the porous water- repellant film 7 are laminated on the enzyme membrane 5 in order, in this chemical sensor element having an ion sensing type semiconductor electric field effect type transistor provided on the substarte 1 and the enzyme membrane 5 provided on the transistor.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a chemical sensor for measuring the concentration of a specific component contained in a solution using an enzymatic reaction, and more particularly, to a method for measuring the concentration of an electrode or a transistor formed on an insulating substrate. The present invention relates to an improvement of a planar type chemical sensor element provided with an immobilized enzyme film.

[0002]

2. Description of the Related Art Conventionally, as a chemical sensor for measuring the concentration of a specific component contained in a solution by utilizing an enzyme reaction, an immobilized enzyme film is provided on an electrode or a transistor formed on an insulating substrate. There is a planar type chemical sensor element.
In such chemical sensors, functional membranes (immobilized enzyme membranes)
Oxygen in solution and substrate (substance to be measured)
Are reacted to obtain an electrically measurable product, which is quantified. In this case, under a normal environment, the oxygen concentration in the solution takes a certain value depending on the temperature. for that reason,
As the amount of substrate increases, the reaction becomes limited due to lack of oxygen. Therefore, a sensor having only a functional film laminated on an electrode or a transistor cannot quantify a substrate having a certain concentration or more.

[0003] A limited permeation membrane is used to solve the above-mentioned problem. The limited permeation membrane is a porous membrane, a nonwoven fabric, or the like, and is laminated on the functional membrane to limit the amount of the substrate that invades the functional membrane. An ideal restricted-permeation membrane does not suppress the permeation of oxygen and other gases, and with such a membrane it is possible to relatively increase the oxygen concentration relative to the substrate and therefore measure up to high concentrations A sensor can be obtained. For example, when there is no restricted permeation membrane, the measurement range of the glucose sensor is 0 to 60 mg / dl.
However, if a restricted permeation membrane is used,
It can be expanded to a high concentration region such as 00 mg / dl or 0 to 5000 mg / dl.

[0004] A sensor element technology using a restricted transmission membrane is disclosed in, for example, Japanese Patent Application Laid-Open No. 9-21779. FIG. 5 is a schematic sectional view showing this sensor element. A reference electrode 2, a working electrode 3, and a counter electrode 4 are formed on an insulating substrate 1, and BSA (bovine serum albumin),
An immobilized enzyme membrane 5 formed by cross-linking GOD (glucose oxidase) and glutaraldehyde is provided. Further, on the immobilized enzyme membrane 5, a restricted permeation membrane 6 made of BSA and glutaraldehyde is provided.

The operation of the sensor element shown in FIG. 5 is as follows. First, immerse the sensor element in the test liquid,
The test liquid is brought into contact with the sensitive part 8 of the sensor element by dropping the test liquid onto the sensitive part 8 or the like. At this time, the sensor element is connected to the potentiostat, and the working electrode 3 is connected to the reference electrode 2
, A constant voltage (0.6 to 0.8 V) is applied. The intrusion of glucose in the test solution is restricted by the limiting permeable membrane 6, and the glucose concentration in the solution reaching the immobilized enzyme membrane 5 is tens to hundreds of the glucose concentration in the test solution (stock solution). It will be in a state of being diluted to a degree. In the immobilized enzyme membrane 5, glucose is oxidized by the action of GOD, and gluconic acid and hydrogen peroxide are generated. The generated hydrogen oxide is oxidized on the working electrode 3, and a Faraday current proportional to the glucose concentration flows. This current value is measured to determine the glucose concentration.

On the other hand, the test liquid contains a substrate other than the target substance to be measured. Among them, a substance that adheres to the surface of an electrode or a film and reduces or increases the output is called an interfering substance. If the outermost layer of the sensor element that comes in contact with the test liquid is BSA, which is not chemically inert, or silicone or acetylcellulose with large surface irregularities, contaminants easily adhere to this outermost layer, and the sensor element is repeatedly used. If used, the output will drop or rise, so
There was a problem that reproducibility of quantification was deteriorated. In addition, since the substance once adhered is not easily removed, the outermost layer of the sensor element must often be cleaned with a special chemical such as nitrous acid. Such chemical cleaning,
In addition to increasing the cost, there is also a risk that the washing waste liquid will have an adverse effect on the environment.

On the other hand, in order to solve the above-mentioned problem, a chemically stable material having excellent water repellency and oil repellency, such as a fluororesin or polyurethane, is used for the restricted permeation film, so that the sensor element of the sensor element is formed. Attempts have been made to prevent contaminants from adhering to the outermost layer. Such a technique is disclosed, for example, in US Pat. No. 5,696,314. FIG. 6 is a schematic sectional view showing the concept of this sensor. As the restricted permeation membrane 7 on the immobilized enzyme membrane 5, a membrane formed from powder of perfluorocarbon or polytetrafluoroethylene, which is a water-repellent fluororesin, using a similar resin as a binder is used. Its thickness is 2
0 μm.

However, the water-repellent fluororesin used as the material of the restricted permeation film in the technique of the above-mentioned US patent has an extremely high water-repellency. It was almost impossible to pass, and it was extremely difficult to make what actually worked as a sensor by this technique.

[0009]

SUMMARY OF THE INVENTION The present invention has been made in view of the above-mentioned circumstances, and has as its object to minimize the adhesion of contaminants to the outermost layer in contact with a test liquid, and It is an object of the present invention to provide a planar chemical sensor element which has stable sensitivity even in use or repeated use and can always perform highly accurate measurement, and a method for manufacturing the same.

[0010]

In order to achieve the above object, the present invention provides a planar chemical sensor element as described in the following (1) to (6), and a planar chemical sensor element as described in (7) and (8). Provided is a method for manufacturing a device. (1) In a chemical sensor element having an electrode system in which at least a working electrode and a counter electrode are formed on an insulating substrate, and an immobilized enzyme film provided on the electrode system, A planar-type chemical sensor element, wherein a restricted permeation film for restricting intrusion of a substrate and a porous water-repellent film are sequentially laminated. (2) Chemical sensor having an insulating substrate, an ion-sensitive semiconductor field-effect transistor provided on the insulating substrate, and an immobilized enzyme film provided on the ion-sensitive semiconductor field-effect transistor A planar-type chemical sensor element, wherein a restricted permeation membrane for restricting invasion of a substrate and a porous water-repellent membrane are sequentially laminated on the immobilized enzyme membrane. (3) The planar chemical sensor element according to (1) or (2), wherein the thickness of the water-repellent film is 0.1 μm or less. (4) The planar chemical sensor element according to any one of (1) to (3), wherein the water-repellent film is made of a fluororesin, polyurethane or polyxylene. (5) The water-repellent film has a surface tension of 30 dynes / cm or less and a water contact angle of 90 ° or more (1) to (4).
Planar chemical sensor element. (6) The planar chemical sensor element according to any one of (1) to (5), wherein the water-repellent film has a large number of microcracks formed thereon. (7) The method for producing a planar-type chemical sensor element according to any one of (1) and (3) to (6), wherein the limiting permeation membrane restricts invasion of a substrate onto an immobilized enzyme membrane provided on an electrode system. And applying a solution of the water-repellent material on the restricted permeation film obtained in the step, and then drying the solution by applying reduced pressure and heating to dry the porous water-repellent material on the restricted permeation film. Forming a film. 10. A method for manufacturing a planar type chemical sensor element, comprising: (8) The method for producing a planar-type chemical sensor element according to any one of (2) to (6), wherein a step of forming a restricted permeation membrane for restricting intrusion of a substrate on an immobilized enzyme membrane provided on an electrode system. And applying a solution of the water-repellent material on the restricted permeation film obtained in the step, forming a porous water-repellent film on the restricted permeation film by applying reduced pressure and heating to dry the solution. And a method of manufacturing a planar type chemical sensor element.

[0011]

Embodiments of the present invention will be described below with reference to the drawings. (Embodiment 1) FIG. 1 is a plan view showing an example of a chemical sensor element according to the present invention, and FIG. 2 is a schematic sectional view of the element. In the sensor element of this example, a reference electrode 2, a working electrode 3, and a counter electrode 4 are formed on an insulating substrate 1, and an immobilized enzyme film 5 is provided on these electrodes. Further, on the immobilized enzyme membrane 5, a limiting permeation membrane 6 for restricting the invasion of the substrate and a porous water-repellent membrane 7 are sequentially laminated.

Although the material of each electrode is not limited, for example, platinum or carbon is used for the working electrode 3 and the counter electrode 4, and a silver-silver chloride electrode, a gold electrode or the like is used as the reference electrode 2 in this case. Each electrode is connected to a detection circuit via a lead.

The immobilized enzyme membrane 5 can be appropriately selected depending on the purpose of the measurement. Usually, the enzyme is immobilized on a polymer membrane or the like by a covalent bonding method, a cross-linking method, an entrapment method, an adsorption method, or the like. Is used.

As the restricted permeation membrane 6, a substance capable of restricting the invasion of the substrate from the test solution is used. For example, a porous membrane made of polyorganosiloxane (silicone), acetylcellulose, albumin or the like is used.

The water-repellent film 7 has a function of preventing adsorption of contaminants contained in the test liquid such as blood and urine. The standard of water repellency of the water repellent film 7 is a surface tension of 30 dyn.
It is appropriate to use es / cm or less and a contact angle of water of 90 ° or more as a guide. In addition, the water-repellent film 7 is porous with a large number of microcracks formed, and hardly restricts the intrusion of the substrate. A microcrack is a crack whose size (width, length) is about several nm. The water-repellent film 7 is preferably formed of a fluorine resin, polyurethane or polyxylene.

Although not an essential condition of the present invention, a permselective membrane for blocking interfering substances (substrates other than the target substance and directly reacting with the working electrode) is provided between each electrode and the immobilized enzyme membrane. Then, the accuracy of the measurement is improved. The portion other than the sensitive portion 8 of the sensor element is usually covered with an insulating film 9 or placed in a housing so as not to come into contact with the test liquid.

When the sensor element of the present embodiment is configured as a glucose sensor, for example, the following can be suitably used as each component. -Substrate 1: glass substrate having a thickness of 0.5 to 1.5 mm-Working electrode 3 and counter electrode 4: platinum thin film-Reference electrode 2: silver-silver chloride electrode-Immobilized enzyme membrane 5: albumin and glutaraldehyde A membrane in which glucose oxidase is immobilized in a membrane obtained by cross-linking. ・ Limited permeation membrane 6: A membrane made of water-soluble silicone ・ A water-repellent membrane 7: A membrane made of a fluororesin The manufacturing method will be described. Here, a production example of the glucose sensor will be described. FIGS. 3A to 3E are schematic diagrams showing the manufacturing steps. The following manufacturing method can be applied to the two-pole method without using the reference electrode. (A) Reference electrode 2, working electrode 3 and counter electrode 4 on insulating substrate 1
Was formed. In this case, first, a Pt thin film having a thickness of 0.1 to 1.0 μm was formed on the substrate 1 by sputtering or vapor deposition, and the working electrode 3 and the counter electrode 4 were formed by photolithography. Next, similarly, a thickness of 0.1 to 1.0
An electrode was formed by forming and patterning an Ag thin film of μm. This electrode was subjected to constant current electrolysis in 0.1 mol hydrochloric acid, and silver chloride having a thickness of about 0.5 μm was deposited on the surface to form a reference electrode 2.
Was formed. (B) The immobilized enzyme membrane 5 was laminated on the electrode system obtained in the step (a). In this case, bovine serum albumin (BS
A), a mixed aqueous solution of glutaraldehyde (GA) and glucose oxidase (GOD) was spin-coated on the electrode system to form an immobilized enzyme membrane 5 having a thickness of 1 μm. (C) The restriction membrane 6 was laminated on the immobilized enzyme membrane 5 obtained in the step (b). In this case, a water-soluble silicone was spin-coated on the immobilized enzyme membrane 5, and this coating was dried to form a 0.5 μm-thick restricted permeation membrane 6. This restricted permeation membrane 6 was a porous membrane having pores having a pore diameter of 0.001 to 1 μm. (D) A fluorine-based resin solution was spin-coated on the restricted permeation film 6 obtained in the step (c) to form a 0.05-μm-thick fluorine-based resin layer 7a. Since the surface of the restricted permeable membrane 6 made of water-soluble silicone has irregularities with a height of about 0.01 μm, if the thickness of the fluororesin layer 7a is 0.1 μm or less, the fluororesin layer 7a has an infinite number of parts. Micro cracks are formed. For this reason, the fluorine-based resin layer 7a allows water and the substrate to permeate almost without limitation. (E) The fluorine-based resin layer 7a obtained in the step (d) was dried by heating to 50 to 100 ° C. in a reduced pressure atmosphere of several Pa. Thus, the water-repellent film 7 in which the microcracks 10 were uniformly formed was obtained.

Next, the operation of the glucose sensor will be described. First, the test liquid is brought into contact with the sensitive part 8 of the sensor element by immersing the sensor element in the test liquid or dropping the test liquid on the sensitive part 8. At this time, the sensor element is connected to a potentiostat, and a constant voltage (0.6 to 0.8 V) is applied to the working electrode 3 with respect to the reference electrode 2.

When the test solution comes into contact with the sensor element, water, glucose and other substrates pass through the microcracks 10 in the water-repellent film 7 and reach the restricted permeation film 6. Since the infiltration of glucose is suppressed in the restricted permeation membrane 6, the concentration of glucose diffused into the immobilized enzyme membrane 5 is reduced to about several tens to hundred times in the test solution.

In the immobilized enzyme membrane 5, glucose is oxidized to produce gluconic acid and hydrogen peroxide. The generated hydrogen peroxide is oxidized on the working electrode 3, and a Faraday current proportional to the glucose concentration flows. This current value is measured to determine the glucose concentration. When there is no restricted permeation membrane 6, the measurement range is about 0 to 60 mg / dl, but by using the restricted permeation membrane 6, the measurement range is 3000 mg / d.
1, 5000 mg / dl.

The fluororesin forming the water-repellent film 7 is chemically stable and has excellent water repellency and oil repellency.
Very little contaminant adheres, and even if it adheres, the contaminant can be easily removed by washing with water.
Further, since the microcracks have a very small width and length of several tens of nanometers, interfering substances and contaminants having a large molecular weight are prevented from entering the immobilized enzyme membrane 5. The same effect can be obtained even if the water-repellent film is formed of polyurethane or polyxylene.

As described above, since the sensor element of the present embodiment is provided with a chemically water-repellent film on the outermost layer that comes into contact with the test liquid, there is almost no adhesion of contaminants, and even if it adheres, it is washed with water. For example, contaminants can be easily removed.
In addition, since the water-repellent film has a sufficient number of microcracks than at the time of film formation and the size and number do not change, the transmittance of the entire laminated film does not change even when used for a long time. Therefore, the sensitivity of the sensor is always stable, and high-precision measurement can be performed even for a long period of time or repeated use. (Embodiment 2) FIG. 4 is a schematic sectional view showing another example of the chemical sensor element according to the present invention. This sensor element is called a field-effect biosensor, and an immobilized enzyme film 5 is formed on the gate of an ion-sensitive field-effect transistor (ISFET). I shown in this embodiment
In the SFET, an island-shaped semiconductor layer is formed on an insulating substrate 1, and the semiconductor layer includes a source region 11, a drain region 12, and a channel region 13. For example, when an npn-type transistor is used, the source region 11 and the drain region 12 are n ⁺ -type, and the channel region 13 is p-type.

The semiconductor layer is covered with an insulating film 14 and an ion-sensitive film 15. For example, silicon oxide, silicon nitride, or the like is used for the insulating film 14, and for example, silicon nitride, tantalum oxide, or the like is used for the ion-sensitive film 15. In an ISFET, an ion-sensitive film on a channel region corresponds to a gate electrode in a normal transistor. That is, the current flowing between the source and the drain is controlled by the potential generated in the ion-sensitive film. Therefore, if a hydrogen ion sensitive membrane is used, a change in pH can be observed as a change in current value. As an example of the ISFET, there is a configuration in which sapphire is used for a substrate, silicon is used for a semiconductor layer, silicon oxide is used for an insulating film, and silicon nitride is used for an ion-sensitive film.

In the present embodiment, the immobilized enzyme membrane 5, the restricted permeation membrane 6, and the water-repellent membrane 7 are provided on the ion-sensitive membrane as in the first embodiment. The thickness of the film and the like are the same as in the first embodiment. In the example of FIG. 4, all of these films are patterned on the gate portion. However, there is no problem if the film other than the immobilized enzyme film 5 is formed over the entire surface of the substrate.

Next, the operation of the sensor element of this embodiment will be described. First, a constant voltage (2 V) is applied between the source and the drain. When a test solution is brought into contact with the sensor element, hydrogen peroxide and gluconic acid are generated in exactly the same manner as in the first embodiment. Therefore, the pH of the lower surface of the immobilized enzyme membrane 5, that is, the surface of the ion-sensitive membrane changes. Therefore, the concentration of glucose can be quantified as a change in the value of the current flowing between the source and the drain.

In the present embodiment, for exactly the same reason as in the first embodiment, without being affected by contaminants,
Long-term measurement or repeated measurement can be performed with high accuracy. In addition, when an ISFET is used as in this example, no current flows through the immobilized enzyme film, so that the life of the sensor element is significantly increased, the sensor element is less susceptible to interfering substances and noise, and the sensor element is downsized. Various advantages can be obtained.

[0027]

As described above, in the planar sensor element of the present invention, since the porous water-repellent film is provided on the outermost layer in contact with the test solution, there is almost no adhesion of contaminants. Even in this case, contaminants can be easily removed by washing with water. Further, since the water repellent film is porous, it does not hinder the permeation of the substrate. Therefore, the sensitivity of the sensor is always stable, and high-precision measurement can be performed even for a long period of time or repeated use.

[Brief description of the drawings]

FIG. 1 is a plan view showing an example of a chemical sensor element according to the present invention.

FIG. 2 is a schematic sectional view of the element shown in FIG.

FIG. 3 is a schematic view illustrating an example of a manufacturing process of the chemical sensor element according to the present invention.

FIG. 4 is a schematic sectional view showing another example of the chemical sensor element according to the present invention.

FIG. 5 is a schematic sectional view showing an example of a conventional chemical sensor element.

FIG. 6 is a schematic sectional view showing an example of a conventional chemical sensor element.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Insulating substrate 2 Reference electrode 3 Working electrode 4 Counter electrode 5 Immobilized enzyme membrane 6 Restricted permeation membrane 7 Water-repellent membrane 10 Microcrack 11 Source region 12 Drain region 13 Channel region 14 Insulating film 15 Ion-sensitive film

Claims (8)

[Claims] 1. A chemical sensor element comprising: an electrode system having at least a working electrode and a counter electrode formed on an insulating substrate; and an immobilized enzyme film provided on the electrode system. A limited chemical permeation film for restricting invasion of a substrate and a porous water-repellent film are sequentially laminated. 2. An insulating substrate, an ion-sensitive semiconductor field-effect transistor provided on the insulating substrate, and an immobilized enzyme film provided on the ion-sensitive semiconductor field-effect transistor. In chemical sensor elements,
A planar-type chemical sensor element, wherein a restricted permeation membrane for restricting intrusion of a substrate and a porous water-repellent membrane are sequentially laminated on the immobilized enzyme membrane. 3. The planar chemical sensor element according to claim 1, wherein the thickness of the water-repellent film is 0.1 μm or less. 4. The planar chemical sensor element according to claim 1, wherein the water-repellent film is made of a fluorine resin, polyurethane or polyxylene. 5. The water-repellent film has a surface tension of 30 dynes /
2 cm or less and a contact angle of water of 90 ° or more.
The planar type chemical sensor element according to any one of claims 1 to 4. 6. The planar-type chemical sensor element according to claim 1, wherein the water-repellent film has a large number of microcracks formed thereon. 7. The method for producing a planar type chemical sensor element according to claim 1, wherein the intrusion of a substrate onto an immobilized enzyme membrane provided on an electrode system is restricted. Forming a restricted permeable membrane, and applying a solution of a water-repellent material on the restricted permeable membrane obtained in the step, and then performing pressure reduction and heating to dry the solution, thereby forming a porous layer on the restricted permeable membrane. Forming a hydrophobic water-repellent film. 8. The method for producing a planar type chemical sensor element according to claim 2, wherein the penetration of the substrate is restricted on an immobilized enzyme membrane provided on the electrode system. A step of forming a permeable membrane, and after applying a solution of a water-repellent material on the restricted permeable membrane obtained in the step, performing a reduced pressure and heating to dry the solution, thereby forming a porous layer on the restricted permeable membrane. Forming a water-repellent film.