JP3405405B2 - Limited permeation membrane, method for producing restricted permeation membrane, and chemical sensor and method for producing chemical sensor - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a restricted permeation film for restricting permeation of a substance to be measured contained in a liquid sample, a method for producing the same, and a specific substance contained in a liquid sample using the restricted permeation film. The present invention relates to a chemical sensor for identifying and measuring the concentration of such a specific substance, and a method for manufacturing the same.

[0002]

2. Description of the Related Art Recently, chemical sensors for measuring the concentration of a specific component in a liquid sample have been widely used. Such a chemical sensor is a site having a molecular identification function (hereinafter referred to as “receptor”).
And a signal conversion portion (hereinafter, referred to as “transducer”) that converts a change caused by the receptor into an electric signal.

Among chemical sensors, biological materials (microorganisms,
A biosensor uses a molecular recognition function of an enzyme, an antibody, a cell, etc., and uses such a biological material as a receptor. The biosensor is formed by using an enzyme / antibody / microorganism as a receptor and an electrode such as an oxygen electrode or a hydrogen peroxide electrode or a semiconductor device such as a transistor as a transducer. Since the receptor selectively reacts with a specific substance, even when various substances are mixed in the liquid sample, it only reacts with the specific substance.
The transducer converts a change obtained by the reaction between the receptor and a specific substance into an electric signal. For example, in a glucose sensor that measures glucose in an aqueous solution, glucose oxidase (glucose oxidase) is used as a receptor, and a Pt-made peroxide oxygen electrode is used as a transducer. Glucose oxidase is immobilized by being cross-linked using a cross-linking material or by being covered with a gel or semipermeable membrane of a natural or synthetic polymer (where glucose oxidase is immobilized). The converted membrane is referred to as “enzyme-immobilized membrane”). In this case, glucose in the aqueous solution is oxidized by glucose oxidase in the enzyme-immobilized membrane to produce gluconic acid and hydrogen peroxide. Glucose + O2 → Gluconic acid + H2O2 (Formula (1)) The hydrogen peroxide amount in the aqueous solution is converted into a current value by the hydrogen peroxide electrode, so that the concentration of glucose is changed from the difference between the hydrogen peroxide amount before and after the measurement. It is determined. By using a chemical sensor in addition to glucose by the same method, not only organic compounds but also various substances such as inorganic compounds and ions can be measured.

However, in the above-mentioned case, the glucose conversion reaction proceeds by consuming oxygen dissolved in the aqueous solution. Therefore, when the concentration of glucose exceeds a certain level, the current value converted by the peroxide oxygen electrode is saturated. As a result, the accurate concentration cannot be measured. That is, in the above-mentioned glucose sensor, the measurable concentration range of glucose is limited, and when the concentration of glucose exceeds a certain concentration, the accurate concentration cannot be detected.

A limited permeation membrane is used to solve such a problem. The limiting permeation membrane is provided on the outer layer of the enzyme-immobilized membrane and has a function of limiting the permeation of the substance to be measured. That is, by limiting the amount of the substance to be measured that reaches the enzyme-immobilized membrane by the limiting permeation membrane, it is possible to measure up to a higher concentration, and it is possible to expand the measurable concentration range of the substance to be measured. . Conventionally, the restricted permeation membrane has been formed by coating a porous substance such as albumin or cellulose on an enzyme-immobilized membrane or by adhering a porous film such as polycarbonate, Teflon or cellose.

[0006]

However, the conventional chemical sensor using the above-mentioned limited permeation membrane has the following problems. When the porous material is used as the restricted permeation film, reproducibility cannot be obtained because it is difficult to control the film thickness and film quality of the restricted permeation film. On the other hand, when the porous film is used as the restricted permeation membrane, the reproducibility is good, but the selection of the pore size and the density is limited,
It is difficult to obtain accurate measurement results within the target concentration range of the substance to be measured. Further, since the holes are cylindrical, particularly when the film is thick, it is difficult for the measurement target substance to reach the enzyme-immobilized membrane, and it takes time for the measurement target substance to reach the enzyme-immobilized membrane. It took a lot of time to obtain the measurement results. Further, in this case, it is necessary to attach an extremely thin porous film in order to reduce the thickness of the limited permeation film. However, since the thickness of the porous film to be applied is limited and cannot be set to several μm or less, it is difficult to shorten the time required to reach the transducer. That is, the time required to obtain the measurement result cannot be shortened. In addition, the thinner the porous film, the more difficult it is to stick it on the enzyme-immobilized membrane, resulting in a problem of reduced productivity.

The present invention has been made in view of the above problems in the prior art. An object of the present invention is to provide a limited permeation membrane and a chemical sensor which can be accurately measured in the concentration range of a target substance to be measured, and which can shorten the measurement time and can be obtained as a device with good yield. Is to provide. Another object of the present invention is to provide a method for manufacturing a limited permeation membrane and a method for manufacturing a chemical sensor, which can improve productivity.

[0008]

The first invention of the present application, which is provided to solve the above problems, provides a functional film containing a receptor which selectively reacts with a substance to be measured contained in a liquid sample. is formed on the outer layer, the hole penetrating is provided to the functional film, wherein the hole has an opening area of the bottom surface portion
It is a bowl-shaped limited permeation film having a large opening area on the upper surface .

The bottom part in the present application means, in the first place,
The part where the functional film is exposed due to the holes. Further, the upper surface portion means an opening portion formed on the surface of the limited permeation membrane by the hole. According to the limited permeation membrane of the first invention of the present application having the above-mentioned configuration, since the opening area of the upper surface portion is larger than the opening area of the bottom surface portion, the opening area of the bottom surface portion of the hole is Compared with a conventional limited permeation membrane that has pores whose opening area is almost the same as the upper surface of the pores, it becomes easier to take in the substance to be measured from the pores, which can shorten the time until the measurement results are obtained. it can.

In the second invention of the present application, a layer made of a hydrophobic resin containing a spacer is formed on the surface of the functional film, dried, and then the spacer is removed to penetrate the functional film. The method for producing a limited permeation film is characterized by forming a limited permeation film having holes.

According to the method of manufacturing a restricted permeation membrane of the second invention of the present application having the above-mentioned constitution, a layer made of a hydrophobic resin containing a spacer is formed on the surface of the functional membrane and dried, and then the spacer is removed. By forming a limiting permeation film having a hole penetrating to the functional film, the number, size, and density of the spacers to be dispersed can be arbitrarily adjusted, so that the aperture ratio in the limiting permeation film can be increased. Can be controlled. Further, since the limited permeation film is formed by applying the hydrophobic resin onto the functional film, the target film thickness can be easily obtained and the limited permeation film having a uniform film thickness can be obtained. The productivity can be improved by improving the yield. As described above, a restricted permeation membrane having an arbitrary measurable concentration range can be obtained with good reproducibility.

Further, in the third invention of the present application, after the spacers are charged, they are sprayed on the functional film, after which the surface of the functional film is coated with a hydrophobic resin and dried, and then the spacers are removed. By doing so, a limited permeation membrane having a hole penetrating to the functional membrane is formed, which is a method for producing a limited permeation membrane.

According to the method for producing a restricted permeation membrane of the third invention of the present application having the above-mentioned constitution, after applying a charge to the spacers, the spacers are dispersed on the functional membrane, and then a hydrophobic resin is applied to the surface of the functional membrane. The spacers can be evenly dispersed on the functional film by removing the spacers after coating and drying. As a result, the pores can be uniformly formed in the restricted permeation film, and a homogeneous restricted permeation film can be obtained.

Further, a fourth invention of the present application is that a transducer is provided on an insulating substrate, and a functional film is formed on the transducer, and the functional film is a substance to be measured contained in a liquid sample. And a transducer having a function of converting a change caused by the reaction of the receptor with the substance to be measured into an electric signal, and an external layer of the functional film. semi-permeable membrane is formed, is provided on the semi-permeable membrane pores penetrating the to the functional layer, wherein the hole is in the bottom portion opening
Do not use a bowl type with the opening area of the upper surface larger than the mouth area.
A chemical sensor, characterized in that to.

According to the chemical sensor of the fourth invention of the present application having the above structure, the restricted permeation film is formed in the outer layer of the functional film, and the restricted permeation film is provided with a hole penetrating to the functional film. By doing so, it is possible to limit the amount of the substance to be measured that reaches the functional film, and it is possible to measure up to a higher concentration, so the measurable concentration range of the substance to be measured can be expanded. .

According to a fifth aspect of the present invention, a transducer is formed on an insulating substrate, a functional film is subsequently formed on the transducer, and then a layer made of a hydrophobic resin containing spacers is formed. A method of manufacturing a chemical sensor, comprising forming a restricted permeation film having a hole penetrating into the functional film by removing the spacer after forming the film on the surface of the functional film and drying it.

According to the method of manufacturing a chemical sensor of the fifth invention of the present application having the above-mentioned constitution, a layer made of a hydrophobic resin containing a spacer is formed on the surface of the functional film and dried, and then the spacer is removed. By forming a limiting permeation membrane having a hole penetrating to the functional membrane, the number, size, and density of spacers to be dispersed can be arbitrarily adjusted, so that the aperture ratio in the limiting permeation membrane can be controlled. can do. Further, since the limited permeation film is formed by applying the hydrophobic resin onto the functional film, the target film thickness can be easily obtained and the limited permeation film having a uniform film thickness can be obtained. The productivity can be improved by improving the yield. As described above, a chemical sensor having an arbitrary measurable concentration range can be obtained with good reproducibility.

The chemical sensor according to the sixth invention of the present application is
A hole penetrating into the functional film by applying a charge to the spacer and then spraying it on the functional film, and then applying a hydrophobic resin to the surface of the functional film and drying it, and then removing the spacer. Is formed.

According to the method for manufacturing a chemical sensor of the sixth invention of the present application having the above-mentioned constitution, after applying a charge to the spacer, the spacer is sprayed on the functional film, and then the surface of the functional film is coated with a hydrophobic resin. Then, the spacers can be uniformly dispersed on the functional film by removing the spacers after drying. As a result, the holes can be formed uniformly in the restricted permeation film, so that a chemical sensor having a homogeneous restricted permeation film can be obtained.

[0020]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, a restricted permeation membrane and a chemical sensor according to an embodiment of the present invention, and a manufacturing method thereof will be described with reference to the drawings. FIG. 1 is a cross-sectional view showing a restricted permeation membrane and a chemical sensor according to an embodiment of the present invention. FIG. 2 is an enlarged schematic view of a hole portion of the restricted permeation membrane according to the embodiment of the present invention shown in FIG. Figure 3
FIG. 6 is a cross-sectional view showing a method of manufacturing a restricted permeation membrane and a chemical sensor according to an embodiment of the present invention. FIG. 4 is a diagram showing the relationship between the aperture ratio of the restricted permeation membrane and the current value obtained by the measurement in the chemical sensor according to the embodiment of the present invention.

The restricted permeation membrane 1 according to this embodiment is shown in FIG.
As shown in FIG. 3, the hole 4 is formed in the outer layer of the functional film 2 provided in the sensing part 0 of the chemical sensor, and has the hole 4 penetrating to the functional film 2. That is, FIG. 1 shows a restricted permeation film 1 according to the present embodiment and a sensing unit 0 of a chemical sensor provided with the restricted permeation film 1. Such a chemical sensor includes a functional film 2 containing a receptor that selectively reacts with a substance to be measured contained in a liquid sample, and a change generated by the reaction of the receptor with the substance to be measured, converted into an electric signal. The sensing unit 0 includes a transducer 3 that operates. The transducer 3 is provided on the insulating substrate 11, the functional film 2 is provided on the transducer 3, and is formed so as to cover the transducer 3.

The limiting permeation membrane 1 is formed on the outer layer of the functional membrane 2 and is provided with a hole 4 penetrating to the functional membrane 2 to limit the permeation of the substance to be measured into the functional membrane 2. Have. That is, since the pores 4 are provided in the restricted permeation film 1, the amount of the substance to be measured reaching the functional film 2 is restricted in the restricted permeation film 1, so that it is possible to measure up to a higher concentration. The measurable concentration range of the substance to be measured can be expanded. Further, when the thickness of the limiting permeation film 1 is less than 0.05 μm, the mechanical strength is low,
There is a high risk that the film will be broken when subjected to temperature changes or mechanical stress. If the film breaks, accurate concentration measurement cannot be performed. On the other hand, when the thickness of the limiting permeation film 1 is larger than 2 μm, the time taken for the substance to be measured to reach the functional film 2 from the outside of the limiting permeation film 1 through the holes 4 is long and the measurement time is long It takes. However, since the thickness of the limiting permeation membrane 1 is 0.05 to 2 μm, accurate concentration measurement can be performed in a short time. The thickness of the restricted permeation film 1 is more preferably 0.2 to
It is 0.5 μm. Further, the restricted permeation film 1 is made of silicone or a hydrophobic resin such as a fluororesin typified by perfluorocarbon. Here, the hydrophobic resin means a resin having a contact angle with water of 90 ° or more.

The pores 4 penetrate from the restricted permeation membrane 1 to the functional membrane 2 (see FIG. 2). The hole 4 is formed such that the opening area of the upper surface portion 10a of the hole 4 is larger than the opening area of the bottom surface portion 10b of the hole 4. Here, the bottom portion 1 of the hole 4
0b means a portion where the functional film 2 is exposed by the hole 4, and the upper surface portion 10a of the hole 4 means an opening portion formed by the hole 4 on the surface of the restricted permeation film 1. In the restricted permeation membrane 1 according to the present embodiment, the opening area of the top surface portion 10a of the hole 4 is larger than the opening area of the bottom surface portion 10b of the hole 4, so that the opening area of the bottom surface portion of the hole is Compared with a conventional limited permeation membrane that has pores whose opening area is almost the same as the upper surface of the pores, it becomes easier to take in the substance to be measured from the pores, which can shorten the time until the measurement results are obtained. it can.

By making the aperture ratio of the restricted permeation film 1 appropriately to prepare the restricted permeation film 1, the measurable concentration range of the substance to be measured can be set. The restricted permeation film 1 according to the present embodiment is installed so that the aperture ratio is 0.1 to 0.5%. Here, the aperture ratio means the ratio of the portion where the functional film 2 is exposed by the holes 4 to the surface area of the restricted permeation film 1. Since the measurement range is almost proportional to the aperture ratio, for example, if the aperture ratio is set to 1%, the measurement range will be expanded about 100 times.

The functional film 2 contains a receptor as described above. Examples of the substance used as a receptor include enzymes, antibodies, microorganisms and the like. For example, when the receptor is an enzyme, the functional membrane 2 is generally formed as a membrane (enzyme-immobilized membrane) with the receptor immobilized.

The transducer 3 is a portion for converting a change caused by the reaction of the receptor with the substance to be measured into an electric signal. The change caused by the reaction of the receptor with the measurement target substance is, for example, a change in current value caused by a substance generated by the reaction between the receptor and the measurement target substance, or light emission caused by the reaction. A change in current value. As the transducer 3, for example, various electrodes or semiconductor devices are used. In the present embodiment, as shown in FIG.
The case where the transducer 3 is composed of electrodes is shown. That is, in FIG. 1, the transducer 3 has a working electrode 31,
It is composed of a counter electrode 32 and a reference electrode 33. Although various metals can be used for these electrodes, platinum (Pt) is mainly used as the working electrode 31, and platinum (Pt), gold (Au), silver (as the counter electrode 32 and the reference electrode 33). Ag / AgCl) or the like is used. As the insulating substrate 11, for example, a glass substrate or a plastic substrate is used.

A buffer layer may be provided between the restricted permeation film 1 and the functional film 2. For example, when the restricted permeation film 1 is made of a fluororesin, the fluororesin is poor in flexibility. Therefore, for example, when the restricted permeation film is exposed to a rapid temperature change, the restricted permeation film 1 is cracked. It may occur. Therefore, by providing the buffer layer between the restricted permeation film 1 and the functional film 2, the stress generated on the restricted permeation film 1 can be relieved, and the risk of crack generation can be reduced. . As the buffer layer, for example, silicone, cellulose or the like is used.

Next, a method of manufacturing the restricted permeation film according to the present embodiment and a method of manufacturing a chemical sensor using the restricted permeation film will be described with reference to FIG. First, the working electrode 31 and the counter electrode 32 are formed on the insulating substrate 11 (see FIG. 3).
(See (a)). In this case, the reference electrode 33 may be provided as shown in FIG. Next, the functional film 2 is formed on these electrodes, and the limited permeation film 1 is further formed on the functional film 2. First, the hydrophobic resin 1a containing the spacer 5 is laminated on the functional film 2. Spacer 5
May be made of a material that is chemically stable and does not react with the hydrophobic resin 1a. For example, particles made of Teflon, glass, silica, epoxy resin, or the like are used. In this process, the hydrophobic resin 1a may be applied after the spacer 5 is charged and then sprayed on the functional film 2. As a result, the spacers 5 can be evenly distributed on the functional film 2, so that a uniform film can be obtained. Then, using an ultrasonic cleaner or the like, the device coated with the hydrophobic resin 1a is vibrated to remove the spacer 5, and the hole 4 is formed after the spacer 5 is removed. The limited permeation film 1 is obtained through the above steps (see FIG. 3B). As shown in FIG. 2, the hole 4 formed in the restricted permeation film 1 has a smaller area than the opening area of the bottom surface portion 10b of the hole 4, as shown in FIG.
The upper surface portion 10a of the hole 4 has a large opening area. As shown in FIG. 3B, when the spherical spacers 5 are used, the shape of the holes obtained is bowl-shaped. Further, if the diameter of the spacer 5 is about 5 times or more that of the limited permeation film 1, the spacer 5 can be almost completely removed.
The aperture ratio in the limiting permeation film 1 can be controlled by arbitrarily adjusting the number, size, and density of the spacers 5 to be scattered. As described above, the restricted permeation film 1 having an arbitrary dynamic range and the chemical sensor including the restricted permeation film 1 can be obtained with good reproducibility. Further, since the restricted permeation film 1 is formed by applying the hydrophobic resin 1a on the functional film 2, the restricted permeation film 1 having a uniform film thickness that can easily obtain a target film thickness is obtained. Therefore, the yield can be improved, and the productivity can be improved.

[0029]

EXAMPLE Next, an example of the restricted permeation film according to the present embodiment shown in FIG. 1 and a chemical sensor using the restricted permeation film will be described. In this example, among the chemical sensors, a glucose sensor for measuring the concentration of glucose in the aqueous solution was prepared. In such a glucose sensor,
An enzyme (glucose oxidase) was used as a receptor, an electrode was used as the transducer 3, an enzyme-immobilized film on which the enzyme was immobilized was used as the functional film 2, and a glass substrate was used as the insulating substrate 11. The electrodes are a Pt electrode as a working electrode 31 and a counter electrode 32, and a reference electrode 33.
An Ag / AgCl electrode was used as. The restricted permeation film 1 was made of perfluorocarbon. Next, the concentration measuring method and the concentration measuring mechanism of the glucose sensor according to the present embodiment will be described.

First, the glucose sensor is immersed in a liquid sample, or the liquid sample is dropped on a sensitive portion (a part of the sensor where sensing is performed). Next, a constant potential is applied to the working electrode with reference to the reference electrode. The potential is 0.6-0.8V. Glucose in the liquid sample enters the enzyme-immobilized membrane through the holes provided in the restricted permeation membrane,
Diffuses in the enzyme-immobilized membrane. That is, the same effect as that obtained by diluting glucose by the ratio of the total area of the pores to the cross-sectional area of the enzyme-immobilized membrane can be obtained. In the enzyme-immobilized membrane, glucose is converted by the enzyme (glucose oxidase)
It is converted into an equal amount of hydrogen peroxide together with gluconic acid. Of these, when hydrogen peroxide reaches the Pt electrode, which is the working electrode, it is oxidized on the electrode surface and gives electrons to the working electrode. That is, an oxidation current proportional to the amount of hydrogen peroxide produced flows. Further, since the amount of hydrogen peroxide is proportional to the amount of glucose in the liquid sample, the glucose concentration can be quantified by measuring this current value. The above-mentioned potential applied to the working electrode is set to efficiently oxidize hydrogen peroxide.

Subsequently, the glucose sensor according to this example was manufactured by the following steps. First, a Pt electrode and an Ag / AgCl reference electrode were formed on a glass substrate, and an enzyme-immobilized film containing glucose oxidase was formed thereon. Subsequently, a spacer having a diameter of 4 μm was coated on the enzyme-immobilized membrane together with a perfluorocarbon solution to give a film thickness of 0.
A thin film of 5 μm was formed. Subsequently, by applying a vibration to the sensor with an ultrasonic cleaner to remove the spacer,
A hole penetrating to the enzyme-immobilized membrane was formed in the thin film. Through the above steps, the glucose sensor according to the present example was prepared by forming a restricted permeation membrane made of perfluorocarbon and having a hole penetrating to the enzyme-immobilized membrane on the enzyme-immobilized membrane. Further, glucose sensors having restricted permeation membranes having different aperture ratios were prepared by changing the amount of spacers used, and the glucose concentration in the aqueous solution was measured using these glucose sensors.

FIG. 4 shows the relationship between the aperture ratio of the limited permeation film and the measured current value. The concentration of glucose in the measured aqueous solution was 2000 mg / dl in all cases. From FIG. 4, it was found that the measured current value approaches saturation as the aperture ratio increases, but the aperture ratio of the limiting permeation film and the current value are proportional while the aperture ratio is small.

[Brief description of drawings]

FIG. 1 is a cross-sectional view of a sensing portion of a restricted permeation membrane and a chemical sensor according to an embodiment of the present invention.

FIG. 2 is an enlarged schematic view of a hole portion of the restricted permeation membrane according to the embodiment of the present invention shown in FIG.

FIG. 3 is a cross-sectional view showing a method of manufacturing a restricted permeation membrane and a chemical sensor according to an embodiment of the present invention.

FIG. 4 is a diagram showing the relationship between the aperture ratio of the limiting permeation membrane and the current value obtained by measurement in the chemical sensor according to one example of the present invention.

[Explanation of symbols]

0 Sensing section 1 Restricted membrane 1a hydrophobic resin 2 Functional membrane 3 transducer 4 holes 5 spacers 10a upper surface 10b Bottom part 11 Insulating substrate 31 Working pole 32 opposite poles 33 reference pole

─────────────────────────────────────────────────── --Continued from the front page (58) Fields surveyed (Int.Cl. ⁷ , DB name) G01N 27/327

Claims (6)

(57) [Claims] 1. A hole formed in an outer layer of a functional film containing a receptor that selectively reacts with a substance to be measured contained in a liquid sample, the hole penetrating the functional film, The opening area of the upper surface of the hole is smaller than that of the bottom surface.
A restricted permeation membrane characterized by forming a large bowl shape . 2. A limited permeation film having a hole penetrating to the functional film is formed by forming a layer of a hydrophobic resin containing a spacer on the surface of the functional film, drying the layer, and then removing the spacer. A method for producing a restricted permeation film, which comprises forming the film. 3. The functional film is obtained by applying an electric charge to the spacer and then spraying the spacer on the functional film, then applying a hydrophobic resin on the surface of the functional film, drying the spacer, and then removing the spacer. A method of manufacturing a restricted permeation film, comprising forming a restricted permeation film having a hole penetrating therethrough. 4. A transducer is provided on an insulating substrate, and a functional film is formed on the transducer, and the functional film comprises a receptor that selectively reacts with a substance to be measured contained in a liquid sample. Containing, the transducer has a function of converting a change caused by the reaction of the receptor with the measurement target substance into an electric signal, a limited permeation film is formed on the outer layer of the functional film, hole penetrating to the functional film is provided on the semi-permeable membrane, wherein
The opening area of the top surface is larger than the opening area of the bottom surface.
A chemical sensor characterized by being formed into a bowl shape . 5. A transducer is formed on an insulating substrate, a functional film is subsequently formed on the transducer, and then a layer made of a hydrophobic resin containing a spacer is formed on the surface of the functional film and dried. After that, the spacer is removed to form a limited permeation film having a hole penetrating to the functional film. 6. The functional film is obtained by applying an electric charge to a spacer and then spraying the spacer on the functional film, then applying a hydrophobic resin to the surface of the functional film, drying the spacer, and then removing the spacer. A method of manufacturing a chemical sensor, comprising forming a restricted permeation film having a hole penetrating therethrough.