JP2009264920A - Sensor and biosensor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a sensor capable of detecting a substance to be detected in a proper concentration region in a simple constitution even if the concentration of the substance to be detected is unknown, and a biosensor. <P>SOLUTION: An enzyme sensor 1 is equipped with a plurality of sensor parts 10 each of which has a predetermined substrate 110, a permeable membrane 400 through which at least the substance T to be detected is permeated and the detection layer L1 formed between the substrate 110 and the permeable membrane 400 and containing enzyme 300 selectively reacted with the substance T to be detected permeated through the permeable membrane 400. The distance between the substrate 110 and the permeable membrane 400 at least in one sensor part 10 of a plurality of the sensor parts 10 is different from the distance between the substrate 110 and the permeable membrane 400 in another sensor part 10. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a sensor and a biosensor.

Conventionally, research and development of sensors such as biosensors for detecting the concentration of a specific substance in a gas sample or a liquid sample have been actively conducted.
By the way, in a sensor, since the density | concentration of the detection target substance in a gas sample or a liquid sample is often unknown, it is desired to have a wide detection density range.

Therefore, for example, in an enzyme electrode, a space for filling a sample is formed by arranging and forming an oxygen permeable membrane at a pre-set height in the measurement electrode group placement portion, and the sample is filled in the space. An enzyme electrode has been proposed which is designed to reduce the height between the air interface and the measurement electrode group so that a high-concentration specimen can be detected and to solve the reaction limitation due to the oxygen reaction rate limiting ( For example, see Patent Document 1).
In addition, for example, in an electrochemical sensor, an electrochemical sensor configured to detect a high-concentration specimen by increasing the number of semipermeable membranes covering the working electrode has been proposed (for example, a patent) Reference 2).
For example, in a chemical sensor provided in the order of at least a transducer, a functional membrane, and a restricted permeable membrane, detection can be performed by appropriately setting the uneven shape formed on the surface of the restricted permeable membrane facing the functional membrane. There has been proposed a chemical sensor capable of setting a possible concentration region of a detection target substance (for example, see Patent Document 3).
Japanese Patent No. 2536780 (FIG. 7) Japanese Patent Laid-Open No. 08-193969 (FIGS. 25 and 26) Japanese Patent No. 3683150

However, in the inventions described in Patent Documents 1 and 2, the concentration having a linear detection range can be obtained by reducing the height between the air interface of the specimen and the measurement electrode group or increasing the number of layers of the semipermeable membrane. Although the area is enlarged, the more the density area having the linear detection area becomes wider, the lower the accuracy when the density area in the wide area is partially viewed, and the detection error tends to increase.
In the invention described in Patent Document 3, the uneven shape of the restricted permeable membrane is set according to the concentration of the target detection target substance, but the target concentration of the detection target substance is unknown. In this case, there is a problem that a plurality of restricted permeable membranes having different concavo-convex shapes must be prepared.

  An object of the present invention is to provide a sensor and a biosensor that can detect a detection target substance in an appropriate concentration region even if the concentration of the detection target substance is unknown with a simple configuration.

In order to solve the above-mentioned problem, the invention described in claim 1
In the sensor
A plurality of sensor units having a predetermined substrate, at least a permeable film through which a detection target substance permeates, and a detection layer formed between the substrate and the permeable film;
A distance between the substrate and the permeable film in at least one sensor unit of the plurality of sensor units is different from a distance between the substrate and the permeable film in another sensor unit. .

The invention described in claim 2
The sensor according to claim 1, wherein
The sensor unit includes a spacer provided on the substrate,
The spacer supports the permeable membrane.

The invention according to claim 3
In the sensor
A sensor unit having a predetermined substrate, at least a transmission film through which a detection target substance permeates, and a detection layer formed between the substrate and the transmission film;
The distance between the substrate and the permeable membrane in the sensor unit is variable.

The invention according to claim 4
The sensor according to any one of claims 1 to 3,
The substrate comprises an electrode;
The detection target substance is detected by an electrochemical measurement method.

The invention described in claim 5
The sensor according to any one of claims 1 to 3,
The detection target substance is detected by an optical measurement method.

The invention described in claim 6
In biosensors,
Detection including a predetermined substrate, a permeable membrane through which at least a detection target substance permeates, and a receptor that is formed between the substrate and the permeable membrane and selectively reacts with the detection target substance that has passed through the permeable membrane. A plurality of sensor portions each having a layer and a spacer provided on the substrate;
The distance between the substrate and the permeable membrane in the sensor unit is different for each sensor unit,
The spacer supports the permeable membrane,
The substrate comprises an electrode;
The detection target substance is detected by an electrochemical measurement method.

The invention described in claim 7
In biosensors,
Detection comprising a predetermined substrate, at least a permeable membrane through which the detection target substance permeates, and a receptor that is formed between the substrate and the permeable membrane and selectively reacts with the detection target substance that has passed through the permeable membrane. A sensor unit having a layer,
The distance between the substrate and the permeable membrane in the sensor unit is variable,
The substrate comprises an electrode;
The detection target substance is detected by an electrochemical measurement method.

The invention according to claim 8 provides:
In biosensors,
Detection comprising a predetermined substrate, at least a permeable membrane through which the detection target substance permeates, and a receptor that is formed between the substrate and the permeable membrane and selectively reacts with the detection target substance that has passed through the permeable membrane. A plurality of sensor portions each having a layer and a spacer provided on the substrate;
The distance between the substrate and the permeable membrane in the sensor unit is different for each sensor unit,
The spacer supports the permeable membrane,
The detection target substance is detected by an optical measurement method.

The invention according to claim 9 is:
In biosensors,
Detection comprising a predetermined substrate, at least a permeable membrane through which the detection target substance permeates, and a receptor that is formed between the substrate and the permeable membrane and selectively reacts with the detection target substance that has passed through the permeable membrane. A sensor unit having a layer,
The distance between the substrate and the permeable membrane in the sensor unit is variable,
The detection target substance is detected by an optical measurement method.

According to the present invention, the sensor includes a plurality of sensor units each having a predetermined substrate, a permeable film through which at least the detection target substance permeates, and a detection layer formed between the substrate and the permeable film. The distance between the substrate and the permeable membrane in at least one of the sensor units is different from the distance between the substrate and the permeable membrane in the other sensor units.
Here, since the concentration region having the linear detection area depends on the distance between the substrate and the permeable membrane, the sensor includes a plurality of sensor units having different concentration regions having the linear detection area. . Therefore, with a simple configuration that includes a plurality of sensor units having different distances between the substrate and the permeable membrane, the entire sensor can have a wide detection concentration range, and the concentration of the detection target substance is unknown. In addition, the detection target substance can be detected by an appropriate sensor unit (a sensor unit having a linear detection region with an appropriate concentration region) among a plurality of sensor units included in the sensor.

According to the present invention, the sensor includes a sensor unit having a predetermined substrate, at least a permeable film through which the detection target substance permeates, and a detection layer formed between the substrate and the permeable film. The distance between the substrate and the permeable membrane is variable.
Here, since the concentration region having a linear detection area depends on the distance between the substrate and the permeable membrane, the sensor can detect the linearity by changing the distance between the substrate and the permeable membrane in the sensor unit. The concentration region having a region can be made variable. Therefore, with a simple configuration of changing the distance between the substrate and the permeable membrane, the entire sensor can have a wide detection concentration range, and even if the concentration of the substance to be detected is unknown, it can be transmitted through the substrate. The substance to be detected can be detected at an appropriate distance (a distance having an appropriate concentration region and a linear detection region) within a variable range of the distance to the membrane.

Hereinafter, the best mode for carrying out the present invention will be described in detail with reference to the drawings. The scope of the invention is not limited to the illustrated example.
In this embodiment, an enzyme sensor will be described as an example of the sensor (biosensor).

[First Embodiment]
First, the enzyme sensor 1 in the first embodiment will be described.
FIG. 1 is a diagram schematically showing a main part of the enzyme sensor 1. FIG. 2 is a plan perspective view of the enzyme sensor 1. FIG. 3 is a diagram schematically showing a cross section taken along line III-III in FIG. FIG. 4 is an exploded view of the sensor unit 10 included in the enzyme sensor 1.
Here, the detection layer L1 side in the enzyme sensor 1 is the lower side, and the supply layer L2 side is the upper side.

<Configuration of enzyme sensor>
First, the configuration of the enzyme sensor 1 will be described.
For example, as shown in FIG. 1, the enzyme sensor 1 is formed between a predetermined substrate 110, a permeable film 400 disposed above the substrate 110, and the substrate 110 and the permeable film 400. A plurality of sensor units 10 each including a detection layer L1 containing an enzyme 300 as a receptor that selectively reacts with the permeated detection target substance T and a spacer 200 disposed on the upper surface of the substrate 110 are provided.
The detection layer L1 formed between the substrate 110 and the permeable membrane 400 is filled with, for example, a liquid such as an electrolytic solution. The supply layer L2 above the permeable membrane 400 includes, for example, the detection target substance T. A gas sample or a liquid sample to be contained is supplied.

The substrate 110 includes an electrode 140 on the upper surface, and the enzyme sensor 1 detects the detection target substance T that has passed through the permeable membrane 400 and has moved from the supply layer L2 to the detection layer L1 by an electrochemical measurement method. It is like that.
Here, the distance d between the substrate 110 (the electrode 140 included in the substrate 110) and the permeable membrane 400 in each sensor unit 10 included in the enzyme sensor 1 is set to be different. That is, the enzyme sensor 1 has a plurality of detection layers L1 having different thicknesses (lengths in the vertical direction).

  Specifically, the sensor unit 10 included in the enzyme sensor 1 includes, for example, as shown in FIGS. 2 to 4, an electrode substrate unit 100, spacers 200 and 200 disposed on the upper surface of the electrode substrate unit 100, and electrodes The detection layer L1 disposed between the spacers 200 and 200 on the upper surface of the substrate unit 100, the enzyme 300 fixed to the carrier 310 disposed on the upper surface of the electrode substrate unit 100 in the detection layer L1, and the spacer 200 , 200 and the detection layer L1, a permeable membrane 400, a support member 500 disposed on the upper surface of the permeable membrane 400, a cover member 600 disposed on the upper surface of the support member 500, and the like. Is done.

  For example, as illustrated in FIG. 4, the electrode substrate unit 100 includes a substrate 110, a hydrophobic insulating film 130 having an opening (analysis unit 120) provided on the upper surface of the substrate 110, and analysis on the upper surface of the substrate 110. The working electrode (electrode 140), the counter electrode 150, and the reference electrode 160 disposed inside the portion 120, and the pads 170, 170 connected to the working electrode (electrode 140), the counter electrode 150, and the reference electrode 160 via wiring. , 170 and the like.

Specifically, for example, the spacer 200 is disposed between the substrate 110 (hydrophobic insulating film 130) and the permeable film 400 so as to expose the analysis unit 120, and supports the permeable film 400. That is, the region sandwiched between the spacers 200 and 200 in the analysis unit 120 and on the analysis unit 120 is the detection layer L1.
The spacer 200 is for keeping the distance d between the substrate 110 (electrode 140) and the permeable membrane 400 constant. Therefore, the thickness of the spacer 200 which each sensor part 10 with which the enzyme sensor 1 is provided is different.
The material constituting the spacer 200 is arbitrary as long as the distance d between the substrate 110 (electrode 140) and the permeable membrane 400 can be kept constant by the spacer 200. For example, Teflon (registered trademark) or the like can be used. Fluorine resin, other synthetic resins, ceramics such as silicon, metal and metal surface coated glass, glass and the like can be used.

Specifically, for example, the permeable membrane 400 is disposed on the upper surfaces of the spacers 200 and 200 so as to cover the analysis unit 120. That is, the detection layer L1 and the supply layer L2 are separated by the permeable membrane 400.
The detection target substance T supplied to the supply layer L2 passes through the permeable membrane 400, moves to the detection layer L1, and reacts with the enzyme 300 contained in the detection layer L1. Therefore, the permeable membrane 400 is arbitrary as long as it is a film through which at least the detection target substance T permeates, and can be appropriately changed depending on the type of the detection target substance T.
In particular, when a gas sample is supplied to the supply layer L2 and the detection target substance T contained in the gas sample is detected by the enzyme sensor 1, evaporation of a liquid such as an electrolyte in the detection layer L1 is prevented. From such a viewpoint, the permeable membrane 200 is preferably a gas permeable membrane that transmits the detection target substance T (detection target gas) but does not transmit a liquid such as an electrolytic solution.

Specifically, the support member 500 is disposed on the upper surface of the permeable membrane 400 so as to cover the analysis unit 120, for example.
The support member 500 is for supporting the permeable membrane 400 so that the permeable membrane 400 is not deformed during use of the enzyme sensor 1. Therefore, the support member 500 can more reliably keep the distance d between the substrate 110 (electrode 140) and the permeable membrane 400 constant.
The material of the support member 500 is arbitrary as long as the support member 500 can support the permeable membrane 400 so as not to be deformed. Specifically, for example, a mesh body such as a stainless mesh is used.
Here, as long as the support member 500 can support the permeable membrane 400 without being deformed, the support member 500 may be disposed on the lower surface of the permeable membrane 400, or the permeable membrane 400 may be sandwiched between the permeable membrane 400. You may arrange | position on the upper surface and the lower surface.

Specifically, the cover member 600 has, for example, a supply port 610 for supplying a gas sample or a liquid sample to the sensor unit 10, and is supported so that the supply port 610 corresponds to the analysis unit 120. It is arranged on the upper surface of the member 500. That is, the inside of the supply port 610 of the cover member 600 becomes the supply layer L <b> 2 of the sensor unit 10.
The cover member 600 is fixed to the electrode substrate unit 100 in a state where the spacers 200, 200, the permeable membrane 400 and the support member 500 are pressed against the electrode substrate unit 100 by the cover member 600.
The material of the cover member 600 is arbitrary as long as it can be fixed by sandwiching the spacers 200, 200, the permeable membrane 400, and the support member 500 between the cover member 600 and the electrode substrate portion 100. Specifically, for example, glass epoxy Examples thereof include a plate-like member such as a manufactured plate.

Specifically, the carrier 310 on which the enzyme 300 is fixed is disposed on the upper surfaces of the working electrode (electrode 140), the counter electrode 150, and the reference electrode 160 in the analysis unit 120, for example.
The enzyme 300 can be contained in the detection layer L1 simply by placing the carrier 310 on which the enzyme 300 is fixed in the analysis unit 120. Therefore, the operation of allowing the detection layer L1 to contain the enzyme 300 is simplified by the carrier 310.
The material of the carrier 310 is arbitrary as long as the enzyme 300 can be fixed (held) by the carrier 310. Specifically, for example, a hydrophilic Teflon membrane, a nylon membrane, or other materials (for example, cellulose mixed) Ester, polyvinylidene difluoride, polytetrafluoroethylene, polycarbonate, polypropylene, polyethylene terephthalate, etc.), porous bodies such as porous alumina, mesh bodies such as nylon mesh, and fibrous aggregates such as carbon nanotubes Examples include the body.
Here, the thickness of the carrier 310 on which the enzyme 300 is immobilized is equal to the thickness of the spacer 200 from the viewpoint of keeping the distance d between the substrate 110 (electrode 140) and the permeable membrane 400 constant by the spacer 200, or the like. It is preferable that it is less than that. Further, the carrier 310 is a spacer for preventing the permeable membrane 400 from being deformed during use of the enzyme sensor 1 and keeping the distance d between the substrate 110 (electrode 140) and the permeable membrane 400 constant. It also has a role.
The carrier 310 on which the enzyme 300 is fixed may be disposed at least on the upper surface of the working electrode (electrode 140) in the analysis unit 120.

The enzyme 300 may be any enzyme as long as it selectively reacts with the detection target substance T, and can be appropriately changed depending on the type of the detection target substance T.
Specifically, the enzyme 300 is an enzyme (enzyme protein) such as an oxidoreductase, a hydrolase, a transferase, and an isomerase.
In addition, the enzyme 300 may be, for example, a natural enzyme molecule or an enzyme fragment including an active site. The enzyme molecule or the fragment of the enzyme containing the active site is synthesized, for example, genetically or chemically, whether it is extracted from animals or plants or microorganisms, or is cleaved if desired. It may be a thing.

  Examples of the oxidoreductase include glucose oxidase, lactate oxidase, cholesterol oxidase, alcohol oxidase, formaldehyde oxidase, sorbitol oxidase, fructose oxidase, sarcosine oxidase, fructosylamine oxidase, pyruvate oxidase, xanthine oxidase, ascorbate oxidase, sarcosine. Oxidase, choline oxidase, amine oxidase, glucose dehydrogenase, lactate dehydrogenase, cholesterol dehydrogenase, alcohol dehydrogenase, formaldehyde dehydrogenase, sorbitol dehydrogenase, fructose dehydrogenase, hydroxybutyrate dehydrogenase, glycerol dehydrogenase Glutamate dehydrogenase, pyruvate dehydrogenase, malate dehydrogenase, glutamate dehydrogenase, can be used catalase, peroxidase, uricase, and the like. In addition, cholesterol esterase, creatininase, creatinase, DNA polymerase, mutants of these enzymes, and the like can be used.

  As the hydrolase, for example, protease, lipase, amylase, invertase, maltase, β-galactosidase, lysozyme, urease, esterase, nuclease group, phosphatase group and the like can be used.

  As the transferase, for example, various acyltransferases, kinase groups, aminotransferase groups and the like can be used.

  As the isomerase, for example, racemase group, phosphoglycerate phosphomutase, glucose 6-phosphate isomerase and the like can be used.

The enzyme 300 contained in the detection layer L1 may be one type of enzyme or two or more types of enzymes.
Specifically, the molecular weight of the enzyme 300 contained in the detection layer L1 may be, for example, one kind of enzyme or two or more kinds of enzymes having substantially the same molecular weight and / or size (diameter). And / or two or more enzymes having different sizes. Further, when there are two or more types of enzymes 300 contained in the detection layer L1, even if the enzymes 300 are two or more types of enzymes that act on the same type of detection target substance T (substrate), for example, different types of detection are possible. Even two or more types of enzymes that act on the target substance T may be two or more types of enzymes that act on the same and / or different types of detection target substances T.
Here, in particular, when there are two or more types of enzymes 300 contained in the detection layer L1 and the two or more types of enzymes act on different types of detection target substances T, for example, the detection potential is changed or analyzed. The enzyme sensor 1 can simultaneously detect the different types of detection target substances T (two or more types of detection target substances T) by arranging a plurality of electrodes in the unit 120.

<Manufacturing method of enzyme sensor>
Next, a method for manufacturing the enzyme sensor 1 will be described.

(1) Creation of electrode substrate part 100 First, the electrode substrate part 100 is created.

On the substrate 110, for example, as shown in FIG. 4, a plurality of patterns having a three-electrode structure including a working electrode (electrode 140), a counter electrode 150, and a reference electrode 160 are formed.
Specifically, for example, a plurality of patterns of a three-electrode structure including a working electrode (electrode 140), a counter electrode 150, and a reference electrode 160 are formed on the substrate 110 by a known photolithography method and lift-off or etching methods. .
More specifically, for example, an appropriate amount of photoresist is applied onto the substrate 110 using a spin coater or the like. Next, exposure is performed for several seconds with an ultraviolet exposure apparatus, and a photomask pattern having a tripolar structure of the working electrode (electrode 140), the counter electrode 150, and the reference electrode 160 is transferred. Next, post-baking is performed, and thereafter development is performed with a developer to form a photoresist pattern. Next, a metal thin film having a thickness of, for example, about several hundred nm is formed by sputtering, and then the resist is peeled off by lift-off to form a triode electrode.

Here, the substrate 110 is not particularly limited as long as it is an insulator, and specifically, for example, a glass epoxy substrate or the like can be used.
Moreover, as a metal thin film formed into a film, noble metals, such as gold | metal | money, platinum, copper, can be mentioned, for example.
Note that the creation of the tripolar structure pattern of the working electrode (electrode 140), the counter electrode 150, and the reference electrode 160 is not limited to the creation by the photolithography process described above. For example, a screen printing technique is used. It may be done simply. Further, the method of forming the metal thin film is not limited to the sputtering method, and for example, a vapor deposition method may be used.

  Next, a hydrophobic insulating film 130 is formed around the analysis unit 120 by forming a hydrophobic thin film such as SiO around the region corresponding to the analysis unit 120 by, for example, sputtering.

  Next, for example, a silver / silver chloride ink is applied to the pattern of the reference electrode 160 among the three electrode structure patterns of the working electrode (electrode 140), the counter electrode 150, and the reference electrode 160 that are created. The reference electrode 160, which is a silver / silver chloride electrode, is formed by baking.

(2) Preparation of enzyme-immobilized carrier Next, a carrier 310 (enzyme-immobilized carrier) on which the enzyme 300 is immobilized is prepared.

A plurality of carriers 310 are prepared, and the enzyme 300 is fixed to each.
Specifically, for example, a plurality of carriers 310 are prepared, and the same amount of the enzyme solution in which the enzyme 300 is dissolved is dropped on each carrier 310 with a pipette or a dispenser. As a result, the same amount of enzyme 300 is physically immobilized on each carrier 310.
The carrier 310 on which the enzyme 300 is immobilized is preferably subjected to a drying treatment after immobilization.

By the way, the enzyme 300 is a protein having a molecular weight of about 10,000 to 200,000, and it may be difficult for the active center in the enzyme molecule to perform fast electron transfer with the electrode 140. Therefore, it is preferable to introduce an electron carrier for promoting the transfer of electrons between the enzyme 300 and the electrode 140 into the detection layer L1. Even when the reaction is limited by the dissolved oxygen concentration and only a low-concentration sample can be measured, it is effective to introduce an electron carrier into the detection layer L1 for the purpose of expanding the detection concentration range.
Specifically, as the electron carrier, for example, potassium ferricyanide, ferrocene, ferrocene derivatives, benzoquinone, quinone derivatives, osmium complexes and the like are used.

It is also preferable to introduce a coenzyme that catalyzes the expression of the activity of the enzyme 300 into the detection layer L1, for example.
For example, in the case where the reaction between the enzyme 300 and the detection target substance T is a reaction that does not proceed easily by the catalysis of the amino acid side chain of the enzyme 300, such as a reaction via an unstable intermediate, the enzyme 300 has an appropriate structure. Coenzymes, which are low molecular weight organic compounds involved in the expression of enzyme reactions, are often used. In particular, when a coenzyme-dependent enzyme is used as the enzyme 300, the enzyme reaction can be efficiently performed by introducing the coenzyme of the detection layer L1.
The coenzyme can be appropriately selected according to the type of enzyme 300 (coenzyme-dependent enzyme). Specifically, examples of the coenzyme include nicotinamide adenine dinucleotide (NAD ⁺ ), nicotinamide adenine dinucleotide phosphate (NADP ⁺ ), coenzyme I, coenzyme II, flavin mononucleotide (FMN), and flavin. Examples thereof include one or a combination of two or more of adenine dinucleotide (FAD), lipoic acid, coenzyme Q and the like. Among these, NAD coenzymes such as nicotinamide adenine dinucleotide (NAD ⁺ ) and nicotinamide adenine dinucleotide phosphate (NADP ⁺ ) are used.

  When introducing an electron carrier or a coenzyme into the detection layer L1, the electron carrier or coenzyme may be fixed to the carrier 310 using a cross-linking agent such as glutaraldehyde or a photocrosslinkable resin, or an enzyme The electron carrier or coenzyme may be dissolved in the solution, and the electron carrier or coenzyme may be fixed to the carrier 310 together with the enzyme 300, or the electron carrier or coenzyme may be physically bonded to a conductive polymer or the like. Alternatively, a chemically bonded and fixed material may be disposed in the detection layer L1. Alternatively, an electron carrier or a coenzyme may be dissolved / dispersed in the electrolytic solution, and dropped into the detection layer L1 or the like.

(3) Creation of enzyme sensor Next, the enzyme sensor 1 is created.

A plurality of types of spacers 200 having different thicknesses are prepared, and each of them is arranged so as to expose the analysis unit 120, for example, as shown in FIG. 5, and is adhered and fixed on the substrate 110 (hydrophobic insulating film 130). To do.
Here, the spacer 200 may be an adhesive tape or the like, or may be bonded and fixed on the substrate 110 with an adhesive or the like.

  Next, each of the prepared enzyme-immobilized carriers (the carrier 310 on which the enzyme 300 is immobilized), for example, as shown in FIG. It is disposed on the upper surface of the electrode 160.

  Next, a plurality of permeable membranes 400 and a plurality of support members 500 are prepared. For example, as shown in FIG. 6, each analysis unit 120 is covered with each of the plurality of permeable membranes 400, and thereafter each of the plurality of support members 500. Each permeable membrane 400 is covered.

Next, a plurality of cover members 600 are prepared, and each is arranged on the upper surface of each support member 500 so that the supply port 610 corresponds to the analysis unit 140, for example, as shown in FIG. On the insulating insulating film 130).
The enzyme sensor 1 is manufactured as described above. In addition, the manufacturing method of the enzyme sensor 1 is not limited to the said manufacturing method.

  As the electrochemical measurement method using the enzyme sensor 1, for example, a known measurement method such as a chronoamperometry method, a coulometry method, or a cyclic voltammetry method for measuring an oxidation current or a reduction current can be used. The measurement method may be any of a disposable method, a batch method, a flow injection method, and the like.

  It is preferable that the measuring instrument main body to which the enzyme sensor 1 is attached when detecting the detection target substance T has a function of transmitting data to a personal computer in a wired or wireless manner, and can confirm the measurement value in real time. In addition, it is desirable to have a function of simultaneously measuring the detection results of the sensor units 10 included in the enzyme sensor 1 and comparing or examining the data.

  When detecting the detection target substance T, the voltage of the working electrode (electrode 140) with respect to the reference electrode 160 is set to a specific voltage, thereby avoiding the influence of the measurement interfering substance and selecting the detection target substance T with high sensitivity. Can be measured automatically. This set voltage differs depending on the detection target substance T.

  Further, in the enzyme sensor 1, it is necessary to change the type of the enzyme 300 depending on the detection target substance T. Specifically, as the enzyme 300, for example, when the detection target substance T is glucose, glucose oxidase or glucose dehydrogenase is used, and when the detection target substance T is ethanol, alcohol oxidase or alcohol dehydrogenase is used. When is formaldehyde, formaldehyde oxidase or formaldehyde dehydrogenase can be used, and when the detection target substance T is total cholesterol, a mixture of cholesterol esterase and cholesterol oxidase can be used.

  Hereinafter, the present invention will be described with reference to specific examples, but the present invention is not limited thereto.

  An enzyme sensor 1 including five sensor units 10 having different distances d between the substrate 110 (electrode 140) and the permeable membrane 400 was prepared. Then, the enzyme sensor 1 was evaluated.

<1> Production of enzyme sensor 1 First, an enzyme sensor 1 was produced. In this example, an enzyme sensor 1 for detecting formaldehyde gas was prepared. As the enzyme 300, formaldehyde dehydrogenase (formaldehyde dehydrogenase), which is a coenzyme (NAD ⁺ ) -dependent enzyme, was used.

<1-1> Creation of Electrode Substrate 100 First, five patterns of a three-electrode structure including a working electrode (electrode 140), a counter electrode 150, and a reference electrode 150 were created on a substrate 110.
Specifically, a glass epoxy substrate 110 (thickness 0.8 mm, manufactured by Matsushita Electric Works) formed in a substantially rectangular shape was pre-baked at 95 ° C. for 90 seconds using a hot plate. Thereafter, 100 μL of negative resist was applied using a spin coater, and a tripolar photomask pattern of a working electrode (electrode 140), a counter electrode 150 and a reference electrode 160 was transferred using an ultraviolet exposure apparatus. Subsequently, it was post-baked at 120 ° C. for 60 seconds, then developed with a developer for 70 seconds, and washed with distilled water. Next, a metal thin film (platinum thin film) having a film thickness of 800 nm is formed by sputtering, and then the substrate 110 is immersed in acetone and washed with ultrasonic waves for 30 minutes by lift-off, and the resist is peeled off to remove the platinum electrode. Formed. The deposition conditions for the platinum layer were a vacuum of 10 ⁻⁵ Pa, a substrate temperature of 60 ° C., and an argon gas flow rate of 40 sccm.

Next, a hydrophobic insulating film 130 was formed around the analysis unit 120, and a reference electrode 160 that was a silver / silver chloride electrode was formed.
Specifically, the hydrophobic insulating film 130 was formed around the analysis unit 120 by forming a 500 nm-thickness SiO thin film around the region corresponding to the analysis unit 120 by sputtering. Thereafter, a silver / silver chloride ink was applied to the pattern of the reference electrode 160 and sintered at 120 degrees to create a reference electrode 160 which is a silver / silver chloride electrode.

<1-2> Preparation of enzyme-immobilized membrane First, an enzyme solution was prepared.
Specifically, 0.5 U of formaldehyde dehydrogenase, 0.25 μmol of NAD ⁺ , and 10 μmol of naphthaquinone were dissolved in 1000 μL of phosphate buffer (pH 7.5) to prepare an enzyme solution.

Next, an enzyme-immobilized membrane was prepared as an enzyme-immobilized carrier.
Specifically, as the carrier 310, five types of hydrophilic Teflon membranes having a substantially rectangular shape and different thicknesses (pore diameter: 0.1 μm, thickness: 50 μm, 100 μm, 200 μm, 500 μm, 1000 μm, manufactured by Nihon Millipore) ) Were prepared one by one, 20 μL each of the prepared enzyme solution was collected with a micropipette and dropped onto each of these hydrophilic Teflon membranes. Then, it naturally dried at room temperature (25 degreeC) for 3 hours, and produced five types of enzyme fixed films | membranes from which thickness differs, respectively.

<1-3> Preparation of enzyme sensor 1 First, the spacer 200 was installed.
Specifically, as the spacer 200, a hydrophobic Teflon tape having a substantially L-shape and having five types of different adhesive properties (thickness: 50 μm, 100 μm, 200 μm, 500 μm, 1000 μm, manufactured by Nitto Denko) Two pieces of each of these hydrophobic Teflon tapes were prepared so as to expose the analysis part 120 and adhered and fixed on the hydrophobic insulating film 130.
More specifically, for example, each of the five analysis units 120 formed above is referred to as “analysis unit a”, “analysis unit b”, “analysis unit c”, “analysis unit d”, and “analysis unit e”. In this case, a hydrophobic Teflon tape having a thickness of 50 μm is disposed so as to surround the “analysis portion a”, and is adhered and fixed on the hydrophobic insulating film 130, and a hydrophobic Teflon tape having a thickness of 100 μm is attached to the “analysis portion b”. Is placed on the hydrophobic insulating film 130 and is fixed on the hydrophobic insulating film 130. A hydrophobic Teflon tape having a thickness of 200 μm is placed on the hydrophobic insulating film 130 so as to surround the “analysis part c”. A hydrophobic Teflon tape having a thickness of 500 μm is disposed so as to surround the “analysis portion d” and is fixedly adhered to the hydrophobic insulating film 130, and a hydrophobic Teflon tape having a thickness of 1000 μm is surrounded by the “analysis portion e”. Placed on the hydrophobic insulating film 130 Bonded and fixed.

Next, an enzyme-immobilized membrane was installed.
Specifically, each of the prepared enzyme-immobilized membranes is associated with the thickness of the hydrophobic Teflon tape on the upper surface of the working electrode (electrode 140), counter electrode 150, and reference electrode 160 in each analysis unit 120. Arranged.
More specifically, for example, an enzyme-immobilized membrane made of a hydrophilic Teflon membrane having a thickness of 50 μm is disposed in the “analysis part a” surrounded by a hydrophobic Teflon tape having a thickness of 50 μm, and the hydrophobic Teflon having a thickness of 100 μm is arranged. An enzyme-immobilized membrane made of a hydrophilic Teflon membrane having a thickness of 100 μm is disposed in the “analysis portion b” surrounded by the tape, and the thickness is placed in the “analysis portion c” surrounded by the hydrophobic Teflon tape having a thickness of 200 μm. An enzyme-immobilized membrane consisting of a 200 μm hydrophilic Teflon membrane is placed, and an enzyme-immobilized membrane consisting of a 500 μm thick hydrophilic Teflon membrane is placed in the “analysis part d” surrounded by a hydrophobic Teflon tape having a thickness of 500 μm. Then, an enzyme-immobilized membrane made of a hydrophilic Teflon membrane having a thickness of 1000 μm was disposed in the “analysis part e” surrounded by a hydrophobic Teflon tape having a thickness of 1000 μm.

Subsequently, the permeable membrane 400, the support member 500, and the cover member 600 were installed.
Specifically, five gas permeable membranes (thickness: 100 μm, manufactured by Gore-Tex) formed in a substantially rectangular shape were prepared as the permeable membrane 400, and each analysis unit 120 was covered with each. Next, five stainless steel meshes (aperture 100 μm, manufactured by Semi-Tech) were prepared as support members 500 and each permeable membrane 400 was covered with each. Next, as the cover member 500, five glass epoxy plates having an opening (supply port 610) formed in a substantially rectangular shape are prepared, and each of the openings corresponds to the analysis unit 140. As described above, the support member 500 was disposed on the upper surface and fixed on the hydrophobic insulating film 130.

<2> Evaluation of enzyme sensor 1 Next, the enzyme sensor 1 was evaluated.

First, the measuring device M for evaluating the enzyme sensor 1 will be described.
For example, as shown in FIG. 8, the measuring apparatus M includes a standard air generator M1, a formaldehyde storage unit M2, a gas generator M3, a sensor chamber M4, a potentiostat M5, a signal processing unit M6, and a computer. M7 and the like.

  The sensor chamber M4 has a support part M41 for supporting the enzyme sensor 1. Each sensor unit 10 included in the enzyme sensor 1 is configured such that the supply port 610 of the cover member 600 is connected to the support unit M41 via an O-ring, and the supply layer L2 of the enzyme sensor 1 (in the supply port 610). A formaldehyde gas having a specified concentration is introduced from the gas generator M3 through the support M41. Thereby, formaldehyde (detection target substance T), which is a substrate of enzyme 300 (formaldehyde dehydrogenase), passes through permeable membrane 400, moves from supply layer L2 to detection layer L1, and is detected in detection layer L1. It will be.

  The working electrode (electrode 140), the counter electrode 150, and the reference electrode 160 are connected to the potentiostat M5 (manufactured by BAS) from the corresponding pad 170 by a lead wire.

Next, the enzyme sensor 1 was evaluated using the measuring device M.
Specifically, 30 μL of a phosphate buffer solution (30 μL) is added to each enzyme-immobilized membrane (the portion of the enzyme-immobilized membrane that protrudes from the permeable membrane 400 (see FIG. 6)) of each sensor unit 10 included in the enzyme sensor 1. pH 7.5) is dropped as an electrolytic solution, and the electrolytic solution is sucked into the detection layer L1 by capillary action from the end of the enzyme-immobilized membrane made of a hydrophilic Teflon membrane. In addition, the inside of the detection layer L1 was filled with an electrolyte containing an electron carrier.

  Then, a voltage of +650 mV is applied to the working electrode (electrode 140) with respect to the reference electrode 160, and formaldehyde gas whose concentration is continuously changed by the gas generator M3 at room temperature (25 ° C.) is introduced into the sensor chamber M4. Then, measurement was performed by current measurement by the amperometry method. The result is shown in FIG.

FIG. 9 shows a change in response current of each sensor unit 10 when the concentration of formaldehyde gas to be introduced is changed.
In FIG. 9, the horizontal axis indicates the formaldehyde gas concentration, the vertical axis indicates the response current (equilibrium value), and the rhombus (◇) plot indicates the spacer 200 and the enzyme-immobilized membrane (enzyme comprising the carrier 310 serving as a spacer). The result of the sensor part 10 with the thickness of the immobilization membrane) of 50 μm is shown, the result of the spacer 200 and the sensor part 10 with the thickness of the enzyme immobilization film of 100 μm are shown by the square (□) plot, and the spacer by the triangle (Δ) plot 200 and the results of the sensor part 10 with the enzyme-immobilized membrane thickness of 200 μm are shown, and the circle (◯) plots show the results of the spacer 200 and the sensor part 10 with the enzyme-immobilized membrane thickness of 500 μm, and an inverted triangle (▽) The result of the sensor unit 10 in which the thickness of the spacer 200 and the enzyme-immobilized film is 1000 μm is shown in the plot.

According to FIG. 9, the sensor unit 10 having the spacer 200 and the enzyme-immobilized membrane having a thickness of 50 μm maintains good linearity in the concentration region of 0.2 ppb to 100 ppb, and is saturated in the concentration region exceeding 100 ppb. Yes.
In addition, the sensor unit 10 having the spacer 200 and the enzyme-immobilized membrane having a thickness of 100 μm maintains good linearity in a concentration region of 1 ppb to 900 ppb, and is saturated in a concentration region exceeding 900 ppb.
In addition, the sensor unit 10 having the spacer 200 and the enzyme-immobilized membrane having a thickness of 200 μm maintains good linearity in a concentration region of 10 ppb to 3000 ppb, and is saturated in a concentration region exceeding 3000 ppb.
In addition, the sensor unit 10 having the spacer 200 and the enzyme-immobilized membrane having a thickness of 500 μm maintains good linearity in a concentration region of 100 ppb to 10,000 ppb, and is saturated in a concentration region exceeding 10,000 ppb.
In addition, the sensor unit 10 having the spacer 200 and the enzyme-immobilized membrane having a thickness of 1000 μm maintains good linearity in a concentration region of 100 ppb to 100,000 ppb.

From the results of FIG. 9, it was found that each sensor unit 10 has a narrow range of density regions each having a linear detection range, but has a different density region having a linear detection range. Therefore, the enzyme sensor 1 as a whole has a wide detection concentration range and an appropriate sensor unit 10 (a sensor unit 10 having a linear detection range in an appropriate concentration region) among the plurality of sensor units 10 provided in the enzyme sensor 1. It was found that the detection target substance T can be detected.
Here, formaldehyde in the formaldehyde gas supplied to the supply layer L2 passes through the gas permeable membrane 400, moves from the supply layer L2 to the detection layer L1, dissolves in the electrolytic solution, and reacts with the enzyme 300. Finally, the electron carrier is oxidized on the electrode 140. That is, since the diffusion in the detection layer L1 depends on the distance d between the substrate 110 (electrode 140) and the transmission film 400, the sensitivity of the sensor unit 10 is as follows. It is thought that it changes with the distance d between.

According to the enzyme sensor 1 of the first embodiment described above, the substrate 110 including the electrode 140, the permeable membrane 400 through which at least the detection target substance T is transmitted, and the substrate 110 and the permeable membrane 400 are formed. A plurality of sensor portions 10 each having a detection layer L1 containing an enzyme 300 as a receptor that selectively reacts with the detection target substance T that has passed through the permeable membrane 400, and the substrate 110 and the permeable membrane 400 in the sensor portion 10 Is different for each sensor unit 10.
Here, since the concentration region having a linear detection region depends on the distance d between the substrate 110 (electrode 140) and the permeable membrane 400, the enzyme sensor 1 is a sensor having a different concentration region having a linear detection region. A plurality of parts 10 are provided. Therefore, the enzyme sensor 1 as a whole can have a wide detection concentration range with a simple configuration that includes a plurality of sensor units 10 having different distances d between the substrate 110 (electrode 140) and the permeable membrane 400, and Even if the concentration of the detection target substance T is unknown, the detection is performed by an appropriate sensor unit 10 (a sensor unit 10 having an appropriate concentration region and a linear detection region) among the plurality of sensor units 10 included in the enzyme sensor 1. The target substance T can be detected.

Further, according to the enzyme sensor 1 of the present invention described above, the sensor unit 10 includes the spacer 200 provided on the substrate 110 and the enzyme immobilization film (enzyme immobilization film composed of the carrier 310 serving as a spacer). The spacer 200 and the enzyme-immobilized membrane support the permeable membrane 400.
Therefore, since the distance d between the substrate 110 (electrode 140) and the permeable membrane 400 can be kept constant by the spacer 200 and the enzyme-immobilized membrane, the detection target substance T can be detected stably. .

[Second Embodiment]
Next, the enzyme sensor 1A according to the second embodiment will be described.
FIG. 10 is a diagram schematically showing a main part of the enzyme sensor 1A. FIG. 11 is a plan perspective view of the enzyme sensor 1A. 12 is a diagram schematically showing a cross section taken along line XII-XII of FIG. FIG. 13 is an exploded view of the enzyme sensor 1A.

<Configuration of enzyme sensor>
First, the configuration of the enzyme sensor 1A will be described.
Note that the enzyme sensor 1A of the second embodiment is configured to include one sensor unit 10A, and between the substrate 110 (the electrode 140 included in the substrate 110) and the permeable membrane 400 in the sensor unit 10A. The difference from the enzyme sensor 1 of the first embodiment is that the distance d is variable. Therefore, only different parts will be described, and other common parts will be described with the same reference numerals.

For example, as shown in FIG. 10, the enzyme sensor 1 </ b> A includes a predetermined substrate 110 placed on a lower case body 1100 </ b> A (described later), a permeable membrane 400 disposed above the substrate 110, and the substrate 110 and the transmission. A detection layer L1 containing an enzyme 300 as a receptor that is formed between the membrane 400 and selectively reacts with the detection target substance T that has passed through the permeable membrane 400; a spacer 200A disposed on the upper surface of the substrate 110; A sensor unit 10A having
The detection layer L1 formed between the substrate 110 and the permeable membrane 400 is filled with, for example, a liquid such as an electrolytic solution. The supply layer L2 above the permeable membrane 400 includes, for example, the detection target substance T. A gas sample or a liquid sample to be contained is supplied.

The substrate 110 includes an electrode 140 on the upper surface, and the enzyme sensor 1A detects the detection target substance T that has passed through the permeable membrane 400 and has moved from the supply layer L2 to the detection layer L1 by an electrochemical measurement method. It is like that.
Here, the distance d between the substrate 110 (the electrode 140 included in the substrate 110) and the permeable membrane 400 in the sensor unit 10A included in the enzyme sensor 1A can be changed.

  Specifically, for example, as shown in FIGS. 11 to 13, the enzyme sensor 1 </ b> A includes a sensor unit 10 </ b> A, a case body 1000 </ b> A for housing the sensor unit 10 </ b> A, and the like.

  The case body 1000A provided in the enzyme sensor 1A has, for example, a lower case body 1100A and an upper case whose outer shape is substantially cylindrical and which is divided in a direction (horizontal direction) perpendicular to the vertical direction at a substantially central position in the vertical direction. A body 1200A, a connecting member 1300A for connecting the lower case body 1100A and the upper case body 1200A, and the like are configured.

In the lower case body 1100A, for example, a liquid reservoir 1110A for storing a liquid such as an electrolyte injected into the enzyme sensor 1A, and a liquid such as an electrolyte from the outside to the inside of the enzyme sensor 1A are injected. An inlet 1120A for discharging, an outlet 1130A for discharging a liquid such as an electrolyte from the inside to the outside of the enzyme sensor 1A, a mounting portion 1140A for mounting the electrode substrate unit 100, A recess 1150A, 1150A,... That can be inserted into the connecting member 1300A is provided at the upper edge of the side case body 1100A.
Here, the depths of the liquid storage unit 1110A and the mounting unit 1140A are set to be substantially the same as the thickness of the electrode substrate unit 100 (substrate 110), for example.

  For example, the upper case body 1200A is supplied with a gas sample or a liquid sample containing the detection target substance T in the sensor unit 10A, which is formed at a position corresponding to the liquid storage unit 1110A on the upper surface of the upper case body 1200A. The opening 1210A and holes 1220A, 1220A,... That can be penetrated by the connecting member 1300A are provided at positions corresponding to the recesses 1150A, 1150A,... . That is, the inside of the supply port 1210A of the upper case body 1200A is the supply layer L2 of the sensor unit 10A.

  For example, the connecting member 1300A can be inserted into the hole 1220A and can be inserted into the recess 1150A. The lower case body 1100A and the upper case body 1200A can be connected by penetrating from the upper side to the lower side of the hole 1220A and inserted into the recess 1150A, and the sensor unit 10A for the case body 1000A. (Spacer 200A, permeable membrane 400 and support member 500) can be positioned.

  Here, since the recess 1150A, the hole 1220A, and the connecting member 1300A are not threaded, the lower case body 1100A and the upper case body 1200A are connected to each other by the connecting member 1300A. The position of the lower case body 1100A (the distance between the lower case body 1100A and the upper case body 1200A) can be changed. Thereby, the distance d between the board | substrate 110 (electrode 140) and the permeable film 400 in 10 A of sensor parts can be changed now.

The sensor unit 10A included in the enzyme sensor 1A is provided, for example, at a position corresponding to the liquid storage unit 1110A disposed on the electrode substrate unit 100 mounted on the mounting unit 1140A and the upper surface of the lower case body 1100A. The spacer 200A having the opening 210A, the detection layer L1 disposed in the liquid reservoir 1110A and the opening 210A, the enzyme 300 contained in the detection layer L1, and the upper surfaces of the spacer 200A and the detection layer L1 The permeable membrane 400 is disposed, the support member 500 is disposed on the upper surface of the permeable membrane 400, and the like.
Here, the electrode substrate unit 100 included in the sensor unit 10A is, for example, one cut out from the electrode substrate unit group in which the plurality of electrode substrate units 100 are formed in the first embodiment.

The spacer 200 </ b> A can change the distance d between the substrate 110 (electrode 140) and the permeable membrane 400 without leaking a liquid such as an electrolyte injected into the enzyme sensor 1 </ b> A to the outside of the enzyme sensor 1 </ b> A. It is for making. Therefore, the spacer 200A can be expanded and contracted at least in the thickness direction.
The material of the spacer 200A is such that the distance d between the substrate 110 (electrode 140) and the permeable membrane 400 is not leaked to the outside of the enzyme sensor 1A, such as an electrolyte solution injected into the enzyme sensor 1A by the spacer 200A. Any material can be used as long as it can be changed, and specific examples include elastic members such as a silicone rubber sheet.

  Specifically, the spacer 200A is formed in a substantially ring shape having an opening 210A, for example. The spacer 200A is provided with holes 220A, 220A,... Through which the connecting member 1300A can pass, for example, at positions corresponding to the recesses 1150A, 1150A,. The spacer 200A can be positioned with respect to the case body 1000A by allowing the connecting member 1300A to pass therethrough.

  Hereinafter, the present invention will be described with reference to specific examples, but the present invention is not limited thereto.

  An enzyme sensor 1A was created and the enzyme sensor 1A was evaluated.

<1> Production of enzyme sensor 1A First, an enzyme sensor 1A was produced. In this example, an enzyme sensor 1 for detecting formaldehyde gas was prepared. As the enzyme 300, formaldehyde dehydrogenase (formaldehyde dehydrogenase), which is a coenzyme (NAD ⁺ ) -dependent enzyme, was used.

<1-1> Creation of electrode substrate unit 100 By cutting out one electrode substrate unit 100 from the electrode substrate unit group of five electrode substrate units 100 created in the example of the first embodiment. The electrode substrate part 100 was created.

<1-2> Creation of Enzyme Sensor 1A First, a case body 1000A was created.
Specifically, the lower case body 1100A and the upper case body 1200A were created by processing a peak material, which is an insulator, using a lathe or a milling machine.
The depths of the liquid storage unit 1110A and the mounting unit 1140A were set to be substantially the same as the thickness (0.8 mm) of the electrode substrate unit 100.
The material of the case body 1000A is not limited to the peak material as long as it is insulative. Specifically, for example, ceramic, glass, plastic, Teflon, or the like may be used.

Subsequently, the electrode substrate part 100 was installed in the lower case body 1100A.
Specifically, the prepared lower case is arranged so that the analysis unit 120 is arranged in the liquid storage unit 1110A and the pad 170 is arranged outside the enzyme sensor 1A. It was mounted on the mounting portion 1140A of the body 1100A and fixed to the lower case body 1100A.
The method of fixing the electrode substrate unit 100 to the lower case body 1100A may be a method of sealing and fixing using a tape member such as Teflon tape, or a method of fixing using an adhesive or the like. Also good.

Next, the lower case body 1100A was placed on the precision Z stage N1.
Specifically, for example, as shown in FIG. 14, the lower case body 1100A on which the electrode substrate unit 100 is mounted is fixed on the precision Z stage N1 using screws or the like (not shown).

Next, a spacer 200A was installed.
Specifically, as the spacer 200A, one silicon rubber sheet (thickness: 500 μm) having a hole portion 220A formed in a substantially ring shape is prepared, and the hole portion 220A has the lower case body. It arrange | positioned corresponding to the recessed part 1150A of 1100A.

Next, the permeable membrane 400 and the support member 500 were installed.
Specifically, one gas permeable membrane (made by Gore-Tex) formed in a substantially circular shape is prepared as the permeable membrane 400, and one stainless mesh formed in a substantially circular shape is prepared as the support member 500. The opening 210A of the spacer 200A was covered with the gas permeable membrane and the stainless mesh.
Here, in order to improve the strength of the gas permeable membrane, the gas permeable membrane and the stainless steel mesh are overlapped, and portions of the gas permeable membrane and the stainless mesh other than the portion corresponding to the analysis unit 120 of the electrode substrate unit 100 are arranged. The gas permeable membrane and the stainless mesh were integrated by bonding. The gas permeable membrane and the stainless steel mesh are integrated to cover the opening 210A of the spacer 200A.

Next, the upper case body 1200A was installed.
Specifically, the created upper case body 1200A was arranged such that the hole 1220A corresponds to the recess 1150A of the lower case body 1100A, and was connected to the lower case body 1100A by the connecting member 1300A. Then, for example, as shown in FIG. 14, the position of the upper case body 1200A is fixed by the holder N2.

<2> Evaluation of enzyme sensor 1A Next, the enzyme sensor 1A was evaluated.

First, the measuring device N for evaluating the enzyme sensor 1A will be described.
As shown in FIG. 15, for example, the measuring device N includes a standard air generator M1, a formaldehyde storage unit M2, a gas generator M3, a precision Z stage N1, a holder N2, a potentiostat M5, a signal, A processing unit M6, a computer M7, and the like are provided.

  A specified concentration of formaldehyde gas is directly introduced from the gas generator M3 into the supply layer L2 (inside the supply port 1210A) of the enzyme sensor 1A. Thereby, formaldehyde (detection target substance T), which is a substrate of enzyme 300 (formaldehyde dehydrogenase), passes through permeable membrane 400, moves from supply layer L2 to detection layer L1, and is detected in detection layer L1. It will be.

The precision Z stage N1 can be moved in the Z-axis direction (vertical direction) in units of 10 μm, for example.
The lower case body 1100A of the enzyme sensor 1A is fixed to the precision Z stage N1, and the position of the upper case body 1200A is fixed to the holder N2, and the lower case body 1100A and the upper case body 1100A are connected by the connecting member 1300A. Since the position of the lower case body 1100A relative to the upper case body 1200A can be changed with the case body 1200A connected, the lower case body 1100A is moved in the vertical direction by the precision Z stage N1. Thus, the distance d between the substrate 110 (electrode 140) and the permeable membrane 400 in the sensor unit 10A can be changed by changing the thickness of the spacer 200A by deforming the spacer 200A.

  The working electrode (electrode 140), the counter electrode 150, and the reference electrode 160 are connected to the potentiostat M5 (manufactured by BAS) from the corresponding pad 170 by a lead wire.

Next, the enzyme sensor 1A was evaluated using the measuring device N.
Specifically, 1.0 U formaldehyde dehydrogenase, 0.5 μmol NAD ⁺ , and 20 μmol naphthaquinone were dissolved in 2000 μL phosphate buffer (pH 7.5) to prepare an enzyme solution. By injecting the enzyme solution into the enzyme sensor 1A adjusted so that the thickness of the spacer 200A is 500 μm from the injection port 1120A using a syringe, and continuing to inject until it leaks out from the discharge port 1130A, the enzyme solution Filled the detection layer L1. At this time, about 1000 μL of the enzyme solution was injected into the enzyme sensor 1A.
Thereafter, the discharge port 1130A was capped with a silicon seal or the like while the syringe containing the enzyme solution was still connected to the injection port 1120A.

  Then, a voltage of +650 mV is applied to the working electrode (electrode 140) with respect to the reference electrode 160, and formaldehyde gas whose concentration is continuously changed by the gas generator M3 at room temperature (25 ° C.) is supplied to the enzyme sensor 1A. It introduce | transduced into the layer L2 (inside the supply port 1210), and measured by the current measurement by the amperometry method. The results are shown in FIGS.

FIG. 16 shows a change in the response current of the sensor unit 10A when the concentration of formaldehyde gas to be introduced is constant (100 ppb) and the thickness of the spacer 200A is changed in the range of 50 μm to 500 μm using the precision Z stage N1. Indicates.
In FIG. 16, the horizontal axis represents the thickness of the spacer 200A, and the vertical axis represents the response current (value in the equilibrium state).
According to FIG. 16, it was found that the response current increases as the thickness of the spacer 200A decreases (that is, as the distance d between the substrate 110 (electrode 140) and the permeable membrane 400 decreases).
Here, formaldehyde in the formaldehyde gas supplied to the supply layer L2 passes through the gas permeable membrane 400, moves from the supply layer L2 to the detection layer L1, dissolves in the electrolytic solution, and reacts with the enzyme 300. Finally, the electron carrier is oxidized on the electrode 140. That is, since the diffusion in the detection layer L1 depends on the distance d between the substrate 110 (electrode 140) and the permeable membrane 400, the sensitivity of the sensor unit 10A is that of the substrate 110 (electrode 140) and the permeable membrane 400. It is thought that it changes with the distance d between.

FIG. 17 shows changes in the response current of the sensor 10A when the thickness of the spacer 200A is adjusted using the precision Z stage N1 and the concentration of formaldehyde gas to be introduced is changed at each thickness.
In FIG. 17, the horizontal axis represents the formaldehyde gas concentration, the vertical axis represents the response current (equilibrium value), the rhombus (◇) plot shows the results when the thickness of the spacer 200A is adjusted to 50 μm, and the square (□ ) The plot shows the results when the spacer 200A thickness is adjusted to 100 μm, the triangle (Δ) plot shows the results when the spacer 200A thickness is adjusted to 200 μm, and the circle (◯) plot shows the spacer thickness 500 μm. The results when adjusted are shown.

According to FIG. 17, when the thickness of the spacer 200A is adjusted to about 50 μm, good linearity is maintained in the concentration region of 0.2 ppb to 100 ppb, and saturation is achieved in the concentration region exceeding 100 ppb.
Further, when the thickness of the spacer 200A is adjusted to about 100 μm, good linearity is maintained in a concentration region of 10 ppb to 300 ppb, and saturation is achieved in a concentration region exceeding 300 ppb.
Further, when the thickness of the spacer 200A is adjusted to about 200 μm, good linearity is maintained in a concentration region of 10 ppb to 1000 ppb, and saturation is achieved in a concentration region exceeding 1000 ppb.
Further, when the thickness of the spacer 200A is adjusted to about 500 μm, good linearity is maintained in a concentration region of 100 ppb to 10,000 ppb, and saturation is achieved in a concentration region exceeding 100000 ppb.

  From the result of FIG. 17, when the thickness of the spacer 200A (that is, the distance d between the substrate 110 (electrode 140) and the permeable membrane 400) is constant, the range of the concentration region having a linear detection region is narrow. It was found that changing the thickness of A (that is, the distance d between the substrate 110 (electrode 140) and the permeable membrane 400) changes the concentration region having a linear detection region. Accordingly, the enzyme sensor 1A as a whole has a wide detection concentration range, and an appropriate distance d (variable linearly in an appropriate concentration region) in a variable range of the distance between the substrate 110 (electrode 140) and the permeable membrane 400. It was found that the detection target substance T can be detected at a distance d) having a detection area.

According to the enzyme sensor 1A of the second embodiment described above, the substrate 110 including the electrode 140, the permeable membrane 400 through which at least the detection target substance T is transmitted, and the substrate 110 and the permeable membrane 400 are formed. And a detection layer L1 containing an enzyme 300 as a receptor that selectively reacts with the detection target substance T that has passed through the permeable membrane 400, and is transmitted through the substrate 110 (electrode 140) in the sensor unit 10A. The distance d between the film 300 is variable.
Here, since the concentration region having a linear detection range depends on the distance between the substrate 110 (electrode 140) and the permeable membrane 400, the enzyme sensor 1A transmits the substrate 110 (electrode 140) and the transmission in the sensor unit 10A. By changing the distance d between the membrane 400, the concentration region having a linear detection region can be made variable. Therefore, the enzyme sensor 1A as a whole can have a wide detection concentration range and the concentration of the detection target substance T with a simple configuration in which the distance d between the substrate 110 (electrode 140) and the permeable membrane 400 is changed. Is unknown at an appropriate distance d (distance d having a linear detection range in an appropriate concentration region) out of the variable range of the distance d between the substrate 110 (electrode 140) and the permeable membrane 400. The detection target substance T can be detected.

  The present invention is not limited to the above-described embodiment, and can be appropriately changed without departing from the gist thereof.

<Modification 1>
In the first embodiment, the detection target substance T that has passed through the permeable membrane 400 and has moved from the supply layer L2 to the detection layer L1 is detected by the electrochemical measurement method. For example, FIG. As in the enzyme sensor 1B shown in FIG. 19, the detection target substance T that has passed through the permeable membrane 400 and has moved from the supply layer L2 to the detection layer L1 may be detected by an optical measurement method.
The enzyme sensor 1B according to the first modification differs from the enzyme sensor 1 according to the first embodiment in that the material of the substrate 110 and the permeable membrane 400 and the substrate 110 are not provided with the electrode 140 or the like.
Moreover, in the enzyme sensor 1B of the modification 1, it is preferable that the enzyme contained in the detection layer L1 is directly fixed on the substrate 110.

The substrate 110 is, for example, a transparent substrate that transmits at least light having a desired wavelength, and the transmission film 400 is, for example, a transparent transmission film that transmits at least light having a desired wavelength.
As an optical measurement method using the enzyme sensor 1B, for example, a method for observing a change in absorbance of a product, a method for observing a change in absorbance of an enzyme-labeled one, and coating a metal on the surface and utilizing SPR (surface plasmon resonance) Examples thereof include a method, a method of producing a fluorescent substance as a product, and measuring the fluorescence.

A method for observing a change in absorbance of a product will be described by taking, as an example, a case where formaldehyde dehydrogenase is used as the enzyme 300 and formaldehyde is detected as a substrate. Formaldehyde dehydrogenase is immobilized on a substrate 110 (for example, a transparent glass substrate) by a known immobilization method, and in the presence of coenzyme (NAD ⁺ ), a catalytic reaction is performed to oxidize formaldehyde to formic acid. Then, for example, light is emitted from the lower surface side of the substrate 110 using a light source such as a laser, and a wavelength (340 nm) characteristic of NADH (reduced type) generated by reduction of NAD ⁺ (oxidized type) by reflection or transmission. ) To measure the change in absorbance. Thereby, the density | concentration of produced | generated NADH, ie, a substrate concentration, can be determined.

  A method for observing the change in absorbance of the enzyme-labeled product will be described by taking, for example, a case where alcohol oxidase is used as the enzyme 300 and alcohol is detected as a substrate. A labeling enzyme (peroxidase) is immobilized on a substrate 110 (for example, a transparent glass substrate) by a known immobilization method. As an electrolytic solution, a mixture of alcohol oxidase, 4-aminoantipyrine (4-AA), N- (3-sulfopropyl) -3-methoxy-5-methylaniline (HMMPS) is prepared, and in the presence of oxygen Thus, the alcohol oxidase oxidizes the alcohol to generate hydrogen peroxide. The generated hydrogen peroxide oxidizes and condenses 4-AA and HMMPS by the action of peroxidase. As a result, a blue pigment is generated. For example, light is emitted from the lower surface side of the substrate 110 using a light source such as a laser. The substrate concentration can be determined by measuring the absorbance of the blue dye produced, by reflection or transmission. In this case, both the alcohol oxidase and the peroxidase may be fixed to the substrate 110, only the alcohol oxidase may be used, or only the peroxidase may be used as described above.

  As a method of using SPR by coating the surface with metal, for example, an SPR sensor substrate is prepared as the substrate 110, an antibody (receptor) is fixed on the surface of the substrate 110, and the detection layer L1 is transmitted through the permeable membrane 400. And a method of measuring the increase in the resonance angle of SPR according to the concentration of the antigen by binding the antigen (substance to be detected) introduced into the antibody to the antibody immobilized on the substrate 110. Here, as the substrate 110, for example, a glass substrate formed by sputtering a gold thin film can be used. Examples of the method for immobilizing the antibody on the substrate 110 include a chemical bonding method using a thiol bond and a physical adsorption method.

  As a method for producing a fluorescent substance as a product and measuring the fluorescence, for example, an antigen-antibody reaction is performed by a sandwich method, and a secondary antibody is previously enzyme-labeled to produce a fluorescent substance as a reaction product. And a method of measuring the fluorescence.

<Modification 2>
In the second embodiment, the detection target substance T that has passed through the permeable membrane 400 and moved from the supply layer L2 to the detection layer L1 is detected by the electrochemical measurement method. For example, FIG. Like the enzyme sensor 1C shown in FIG. 21, the detection target substance T that has passed through the permeable membrane 400 and has moved from the supply layer L2 to the detection layer L1 may be detected by an optical measurement method.
The enzyme sensor 1C according to the second modification differs from the enzyme sensor 1A according to the second embodiment in that the material of the substrate 110 and the permeable membrane 400 and the electrode 110 and the like are not provided on the substrate 110.
Moreover, in the enzyme sensor 1 </ b> C of the second modification, it is preferable that the enzyme contained in the detection layer L <b> 1 is directly fixed on the substrate 110.

The substrate 110 is, for example, a transparent substrate that transmits at least light having a desired wavelength, and the transmission film 400 is, for example, a transparent transmission film that transmits at least light having a desired wavelength.
As an optical measurement method using the enzyme sensor 1C, for example, a method for observing a change in the absorbance of a product, a method for observing a change in the absorbance of an enzyme-labeled product, or coating a metal on the surface and utilizing SPR (surface plasmon resonance). Examples thereof include a method, a method of producing a fluorescent substance as a product, and measuring the fluorescence.
When optical measurement is performed from the lower surface side of the substrate 110 (that is, for example, measurement is performed by irradiating light using a light source such as a laser from the lower surface side of the substrate 110), the substrate 110 is at least It is made of a transparent substrate such as glass that transmits light having a desired wavelength, and the lower case body 1100A (at least the bottom surface of the liquid storage portion 1110A) or the lower case body 1100A (at least the bottom surface of the liquid storage portion 1110A), The transmissive film 400 and the support member 500 need to be formed of a transparent material that transmits at least light having a desired wavelength.

In the first embodiment, the carrier 300 on which the enzyme 300 is fixed is arranged in the detection layer L1 so that the enzyme 300 is contained in the detection layer L1, but the enzyme 300 is contained in the detection layer L1. The method of inclusion is not limited to this, and may be a method of directly fixing on the electrode 140 by a known fixing method, or an enzyme solution in which the enzyme 300 is dissolved is directly introduced into the detection layer L1. It may be a method to do. In addition, when the enzyme 300 is contained in the detection layer L1 by these methods, the carrier 310 (the carrier 310 on which the enzyme 300 is not fixed) is set to a distance d between the substrate 110 (electrode 140) and the permeable membrane 400. As a spacer for keeping it constant, it may be disposed between the substrate 110 (electrode 140) and the permeable membrane 400.
In the second embodiment, the enzyme solution in which the enzyme 300 is dissolved is directly introduced into the detection layer L1 so that the enzyme 300 is contained in the detection layer L1, but the enzyme 300 is contained in the detection layer L1. The method is not limited to this, and may be a method of directly fixing on the electrode 140 or a method of arranging the carrier 310 on which the enzyme 300 is fixed in the detection layer L1.
In the first embodiment and the second embodiment, when the enzyme 300 is immobilized on the carrier 310 or the electrode 140, the enzyme 300 may be immobilized using a crosslinking agent such as glutaraldehyde or a photocrosslinkable resin. good.

  In the example of the first embodiment and the example of the second embodiment, a gas sample is supplied to the supply layer L2, and the detection target substance T in the gas sample is changed using the enzyme sensors 100 and 100A. Although detected, when a liquid sample is supplied to the supply layer L2, the detection target substance T in the liquid sample can be detected using the enzyme sensors 100 and 100A.

In the second embodiment, the precision d stage N1 and the holder N2 are used to change the distance d between the substrate (electrode 140) and the permeable membrane 400 in the sensor unit 10A. The means for changing the distance d between the substrate (electrode 140) and the permeable membrane 400 is not limited to this, and other electrical or mechanical means such as a piezo element can be used for the distance d. May be changed.
The same applies to the second modification.

In the first embodiment, the distance d between the substrate 110 and the permeable membrane 400 in the sensor unit 10 is set to be different for each sensor unit 10, but the present invention is not limited to this. The distance d in at least one sensor unit 10 among the units 10 may be different from the distance d in other sensor units 10. That is, the thicknesses of the plurality of detection layers L1 included in the enzyme sensor 1 may not be the same.
The same applies to the first modification.

In the first embodiment and the second embodiment, the receptor is not limited to the enzyme 300 but may be a biological substance (a molecular identification element derived from a living body) that selectively reacts with the detection target substance T. For example, an antibody or a microorganism may be used.
The same applies to Modification 1 and Modification 2.

In the first embodiment and the second embodiment, as an example of the working electrode (electrode 140), the counter electrode 150, and the reference electrode 160, for example, as shown in FIG. 4 and FIG. Although microelectrodes are used, these electrodes are not particularly limited in size, shape, and configuration.
Specifically, for example, these electrodes may be large electrodes used in commercially available electrolytic cells, measurement cells, etc., or may be disk electrodes, rotating ring disk electrodes, fiber electrodes, etc. For example, microelectrodes (disc electrodes, cylindrical electrodes, strip electrodes, array strip electrodes, array disc electrodes, ring electrodes, spherical electrodes, comb electrodes, pair electrodes, etc.) created by known microfabrication techniques such as photolithography. May be.
In the first embodiment and the second embodiment, the tripolar electrode is used. However, a bipolar electrode may be used.
The same applies to Modification 1 and Modification 2.

In the first embodiment, a photoresist can be used as the carrier 310. For example, a photocrosslinkable photoresist can be photocrosslinked and solidified on the electrode 140 as the carrier 310, and also has a transmissive film 400. In the initial application of the photoresist, the transmissive film 400 (or a gas permeable film including a spacer) can be obtained by changing the lamination conditions (for example, the number of revolutions and time for a spin coater) and changing the thickness. The thickness can be changed.
The same applies to the first modification.

In the first and second embodiments, the sensor is a biosensor containing a biocatalyst (receptor) that selectively reacts with the detection target substance in the detection layer L1, but the sensor of the present invention is However, the sensor does not necessarily have to include a biocatalyst in the detection layer L1.
Specifically, for example, a sensor in which a metal catalyst is contained in the detection layer L1 may be used.
Examples of the sensor in which the detection layer L1 contains a metal catalyst include an electrolyte type sensor (constant potential electrolytic sensor) in which a metal catalyst such as gold or platinum is supported on the electrode 140. A constant potential electrolytic sensor is a sensor that electrolyzes gas on a working electrode (electrode 140) maintained at a constant potential and detects a current generated as a gas concentration.
The same applies to Modification 1 and Modification 2.

It is a figure which shows typically the principal part of the enzyme sensor of 1st Embodiment. It is a top perspective view of the enzyme sensor of a 1st embodiment. It is a figure which shows typically the cross section in the III-III line of FIG. It is an exploded view of the sensor part of 1st Embodiment. It is a 1st figure for demonstrating the structure of the sensor part with which the enzyme sensor of 1st Embodiment is provided. It is a 2nd figure for demonstrating the structure of the sensor part with which the enzyme sensor of 1st Embodiment is provided. It is a 3rd figure for demonstrating the structure of the sensor part with which the enzyme sensor of 1st Embodiment is provided. It is a figure which shows typically the measuring apparatus for evaluating the enzyme sensor of the Example of 1st Embodiment. It is a figure which shows the evaluation result of the enzyme sensor of the Example of 1st Embodiment. It is a figure which shows typically the principal part of the enzyme sensor of 2nd Embodiment. It is a top perspective view of the enzyme sensor of a 2nd embodiment. It is a figure which shows typically the cross section in the XII-XII line | wire of FIG. It is an exploded view of the enzyme sensor of 2nd Embodiment. It is a figure for demonstrating how to change the distance between the board | substrate and the permeable film in the sensor part of 2nd Embodiment. It is a figure which shows typically the measuring apparatus for evaluating the enzyme sensor of the Example of 2nd Embodiment. It is a figure which shows the 1st evaluation result of the enzyme sensor of the Example of 2nd Embodiment. It is a figure which shows the 2nd evaluation result of the enzyme sensor of the Example of 2nd Embodiment. It is a figure which shows typically the principal part of the enzyme sensor of the modification 1. It is a top perspective view of the enzyme sensor of the modification 1. It is a figure which shows typically the principal part of the enzyme sensor of the modification 2. It is a top perspective view of the enzyme sensor of the modification 2.

Explanation of symbols

1,1A, 1B, 1C Enzyme sensor (sensor, biosensor)
10, 10A, 10B, 10C Sensor part 110 Substrate 140 Electrode 200 Spacer 300 Enzyme (Receptor)
310 Carrier (spacer)
400 Permeation membrane L1 Detection layer T Substance to be detected

Claims (9)

A plurality of sensor units having a predetermined substrate, at least a permeable film through which a detection target substance permeates, and a detection layer formed between the substrate and the permeable film;
A distance between the substrate and the permeable film in at least one sensor unit of the plurality of sensor units is different from a distance between the substrate and the permeable film in another sensor unit. Sensor. The sensor according to claim 1, wherein
The sensor unit includes a spacer provided on the substrate,
The sensor, wherein the spacer supports the permeable membrane. A sensor unit having a predetermined substrate, at least a permeable film through which a detection target substance permeates, and a detection layer formed between the substrate and the permeable film;
The sensor is characterized in that the distance between the substrate and the permeable membrane in the sensor unit is variable. The sensor according to any one of claims 1 to 3,
The substrate comprises an electrode;
A sensor for detecting the detection target substance by an electrochemical measurement method. The sensor according to any one of claims 1 to 3,
A sensor for detecting the substance to be detected by an optical measurement method. Detection including a predetermined substrate, a permeable membrane through which at least a detection target substance permeates, and a receptor that is formed between the substrate and the permeable membrane and selectively reacts with the detection target substance that has passed through the permeable membrane. A plurality of sensor portions each having a layer and a spacer provided on the substrate;
The distance between the substrate and the permeable membrane in the sensor unit is different for each sensor unit,
The spacer supports the permeable membrane,
The substrate comprises an electrode;
A biosensor, wherein the detection target substance is detected by an electrochemical measurement method. Detection including a predetermined substrate, a permeable membrane through which at least a detection target substance permeates, and a receptor that is formed between the substrate and the permeable membrane and selectively reacts with the detection target substance that has passed through the permeable membrane. A sensor unit having a layer,
The distance between the substrate and the permeable membrane in the sensor unit is variable,
The substrate comprises an electrode;
A biosensor, wherein the detection target substance is detected by an electrochemical measurement method. Detection including a predetermined substrate, a permeable membrane through which at least a detection target substance permeates, and a receptor that is formed between the substrate and the permeable membrane and selectively reacts with the detection target substance that has passed through the permeable membrane. A plurality of sensor portions each having a layer and a spacer provided on the substrate;
The distance between the substrate and the permeable membrane in the sensor unit is different for each sensor unit,
The spacer supports the permeable membrane,
A biosensor, wherein the detection target substance is detected by an optical measurement method. Detection including a predetermined substrate, a permeable membrane through which at least a detection target substance permeates, and a receptor that is formed between the substrate and the permeable membrane and selectively reacts with the detection target substance that has passed through the permeable membrane. A sensor unit having a layer,
The distance between the substrate and the permeable membrane in the sensor unit is variable,
A biosensor, wherein the detection target substance is detected by an optical measurement method.

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