Chemical and biosensor elements

Abstract

To solve the problem that it has been impossible to simultaneously detect multiple detection targets while maintaining high spatial resolution at the micron level, even though it is effective to have means that enables simultaneous observation of behavior of electrolytic ions, bioactive substances, and neurotransmitters in a microscopic region in order to determine efficacy of therapeutic drugs by observing behavior of biological cells in the medical and drug discovery fields.SOLUTION: A chemical / bio sensor element that enables observation of behavior of microscopic biological cells is provided by laminating multiple sensitive films on a CMOS ion image sensor, and then etching portions of the sensitive film to form openings.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] This invention relates to the multi-functionalization of chemical and biosensor elements. [Background technology]

[0002] A CMOS-type ion image sensor (Non-Patent Literature 1) has a structure that detects changes in pH or hydrogen ion concentration, and by further forming another sensitive film on the surface of the sensor, it is possible to detect changes in the concentration of various substances. As a sensitive film, for the detection of physiologically active substances and neurotransmitters such as lactic acid, ATP, and glucose, an enzyme-added film is formed by adding the oxidoreductase of the target substance to an organic resin film (Non-Patent Literature 2). Oxidases are used as oxidizing enzymes, and reductases are used as reducing enzymes. On the other hand, electrolyte ions (K + Ca 2+ kaNa + Cl - Mg 2+ ) uses an ionophore-supported thin film, in which an ionophore is supported on an organic resin film, as the sensitive film (Non-Patent Literature 3). Here, an ionophore is a general term for a lipid-soluble low-molecular-weight compound that selectively allows specific ions to pass through. In semiconductor biosensors, K + kaNa + Ca 2+ A crown ether suitable for detecting electrolyte ions such as these is used. A solvent, plasticizer, and ion exchange agent are mixed onto a polyvinyl chloride (sometimes abbreviated as "PVC" in this specification) substrate and formed as a detection film on the sensor surface. Note that because PVC substrates have poor biocompatibility, a sol-gel glass membrane may be used as the substrate when measuring living cells.

[0003] In the fields of medicine and drug discovery, methods that can simultaneously observe the behavior of electrolyte ions and physiologically active substances / neurotransmitters in a micro-region are effective in determining the efficacy of therapeutic drugs by observing the behavior of iPS cells and other micro-cells. This invention realizes a chemical / biosensor element that can simultaneously observe electrolyte ions and physiologically active substances / neurotransmitters in a micro-region by forming multiple different sensitive membranes on the same semiconductor chip. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Japanese Patent Publication No. 51-139289 [Patent Document 2] Japanese Patent Publication No. 2019-039734 [Non-patent literature]

[0005] [Non-Patent Document 1] You-Na Lee and et al., "High-density 2-um-picth pH image sensor with high-speed operation up to 1933 fps," IEEE Trans. on Biomedical Circuits aand Systems, vol.13, issue2, pp. 352-363, (2019). [Non-Patent Document 2] Hideo Doi, et al., "Fabrication of an Enzyme-Type High-Resolution Label-Free ATP Image Sensor and Extracellular Imaging of the Hippocampus," 35th Symposium on Sensors, Micromachines and Applied Systems, 31am2-C-2 (2018). [Non-Patent Document 3] H. Doi and et al., "Label-free real-time imaging of extracellular Ca2+ uptake in the hippocampal slice using Ca-PVC membrane based on charge-transfer-type potentiometric sensor arrays," IEEE SENSORS 2019, 1289, (2019). [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] Patent Document 1 shows the configuration of an ion-sensitive field-effect transistor in which the gate electrode is removed, and discloses that a chemically selective film is formed directly on the gate insulating film of the transistor. Examples of chemically selective films include polyvinyl chloride films supporting ion exchange materials. Patent Document 1 further discloses that multiple ion-sensitive field-effect transistors (sometimes abbreviated as "ISFET" in this specification) are arranged on a semiconductor substrate, and different chemically selective films are formed on each of them.

[0007] However, while ISFETs can be formed simultaneously using semiconductor processes, the chemically selective films are formed in separate steps for each element. If the chemically selective film is made of an organic resin-based material, heat treatment or etching is required, and this processing history can affect the properties of the chemically selective film, potentially leading to deactivation. Therefore, the degree of freedom for each chemically selective film is low, posing a challenge in practicality.

[0008] Patent Document 2 aims to measure the creatinine and sodium ion concentrations in urine. It includes an ISFET with a channel through which a liquid sample passes on a single substrate, a polyvinyl chloride (PVC) resin film supporting the ionophore Bis(12-crown-4), and a temperature sensor that detects temperature changes due to the reaction heat of catalytic action between the creatinine and an enzyme (creatinase) based on creatinine. In this configuration, sodium (Na) ion concentration and creatinine concentration are detected simultaneously by passing a liquid sample through it.

[0009] However, while this configuration is suitable for liquid samples, observing living cells or iPS cells derived from living organisms requires introducing the living cells into each respective channel. Therefore, it is not possible to simultaneously observe the behavior of living cells or other substances in relation to drugs.

[0010] As disclosed in Patent Documents 1 and 2, configurations in which multiple sensing regions are arranged on a planar surface of a substrate or semiconductor substrate are unsuitable for shortening the spacing between sensors, and it is not possible to arrange multiple sensors in a two-dimensional manner with fine spacing. In drug discovery screening, research is also being conducted on performing drug efficacy evaluation in single cells, and there is a need to provide chemical and biosensor elements suitable for miniaturization of sensor arrays. Here, the size of a single cell is assumed to be about 10 μm to 100 μm. [Means for solving the problem]

[0011] In view of the above circumstances, the inventors have invented a chemical / biosensor element that can observe minute living cells by layering multiple sensitive films on a CMOS-type ion image sensor and then etching it.

[0012] The first aspect of this invention is defined as follows: an array of chemical and physical phenomenon measuring units comprising: a plurality of sensing regions that change the depth of a potential well according to the surface potential; electrodes that define the sensing regions via a first insulating layer covering the sensing regions; a first sensitive film formed via a second insulating layer covering the electrodes defining the sensing regions; and through holes connecting the electrodes defining the sensing regions and the first sensitive film; A chemical / biosensor element characterized by having an ionophore supported directly above a first sensitive membrane constituting an array, a second sensitive membrane formed whose potential changes upon capture of electrolyte ions, a third sensitive membrane made of metal formed on the second sensitive membrane at a predetermined interval, and the second sensitive membrane transmitting the potential change detected by the third sensitive membrane to the first sensitive membrane.

[0013] A second aspect of this invention is defined as follows: A chemical / biosensor element characterized in that, in the first aspect, a portion of the second sensitive film is removed and the first sensitive film comes into contact with the object to be measured.

[0014] The third aspect of this invention is defined as follows. In the first and second aspects, a method for identifying the concentration of a substance to be detected by permeating a chemical and biosensor element into a solution to which a substance having redox activity, an oxidase substance for detecting the substance to be detected, and a hydrogen peroxide-decomposing enzyme are added, detecting the redox potential of the solution by the third sensitive film. Here, redox activity is a property that releases electrons and becomes a +1-valent cation when oxidized, and returns to its original neutral state by receiving electrons when reduced. Ferrocene (chemical formula: Fe(C5H5)2) is an iron cyclopentadienyl complex and is a typical substance having redox activity. It is insoluble in water, and usually, powdered ferrocene methanol (chemical formula: Fe(C5H5)2-OH) is dissolved in methanol for use.

[0015] Ferrocene has extremely stable redox characteristics, and the redox potential of a mixed solution of Fe(III) / Fe(II) is used as a reference in cyclic voltammetry measurement, which is a basic electrochemical measurement. Here, Fe(III) represents potassium ferricyanide, a reagent containing trivalent iron ions, and Fe(II) represents potassium ferrocyanide, a reagent containing divalent iron ions.

Effects of the Invention

[0016] The invention of the present application can acquire image images of pH, electrolyte ion concentration, and biomaterial concentration while maintaining the resolution required for observing a minute detection target such as a single cell by the element structure and concentration identification means shown above.

Brief Description of the Drawings

[0017] [Figure 1] It is a cross-sectional view of a chemical and biosensor element showing the structure of Example 1. [Figure 2] It is a cross-sectional view of a chemical and biosensor element showing the structure of Example 2. [Figure 3] It is a cross-sectional view showing the structure of a measurement unit according to this embodiment. [Figure 4]This is a potential distribution diagram illustrating the operation of the measurement unit according to this embodiment. [Modes for carrying out the invention]

[0018] (Embodiment) In this invention, a chemical / biosensor element 1 is disclosed that detects multiple different detection targets by laminating a second sensitive membrane 30 for detecting electrolyte ions and a third sensitive membrane (metal electrode) for detecting solution potential on a measuring unit 100 having a first sensitive membrane 19 for detecting pH or surface potential, and removing a portion of the third sensitive membrane 31 and the second sensitive membrane 30 to create an opening.

[0019] The first sensitive film 19 is a tantalum pentoxide (Ta2O5) film. It was deposited to a thickness of 150 nm using an active sputtering method utilizing O2+Ar plasma, and then heat-treated at 460°C for 60 minutes. Alternatively, the first sensitive film 19 may be a silicon nitride (Si3N4) film. In this case, it can be deposited using plasma CVD or thermal CVD. If thermal CVD is used, it must be performed before the metal wiring process.

[0020] (Example 1) A second sensitive membrane 30 is provided directly above and in contact with the first sensitive membrane 19. The second sensitive membrane 30 is made of a material that captures electrolyte ions and whose membrane potential changes according to the concentration. The potential detected by the second sensitive membrane 30 is transmitted to the first sensitive membrane 19, thereby detecting the concentration of the substance that the second sensitive membrane 30 is intended to detect.

[0021] The method for forming the second sensitive film 30 is described below. The second sensitive film 30 is a film that supports an ionophore that captures electrolyte ions, and uses a sol-gel glass produced by hydrolyzing tetraethyl orthosilicate (TEOS) and diethoxydimethylsilane (DEDMS) as a substrate. For film formation, 100 mg of TEOS (manufactured by Tokyo Chemical Industry Co., Ltd.) and 330 mg of DEDMS (manufactured by Tokyo Chemical Industry Co., Ltd.) were mixed with 1.5 mg of Valinomycin (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) as an ionophore, 0.9 mg of K-TCPB (manufactured by Dojin Chemical Laboratories, Ltd.) as an anion exclusion agent, and 400 μL of ethanol and 50 μL of 0.1 M HCl as solvents. Subsequently, heat treatment was performed at 70°C for 24 hours to form a sol. Unwanted precipitates should preferably be filtered out using a filter at the level of a few microns. The film was deposited by spin-coating a sol-formed solution, followed by heat treatment at 80°C for 12 hours and 150°C for 12 hours. The thickness of the sol-gel glass sensitive film was 100 nm. Here, we have described the method for depositing a K-ion sensitive film, but for Na ions, Bis(12-crown-4) (manufactured by Dojin Chemical Laboratories) etc. can be used as the ionophore, and for Ca ions, HDOPP-Ca (manufactured by Dojin Chemical Laboratories) etc. can be used as the ionophore, and the appropriate choice can be made depending on the target to be detected.

[0022] The second sensitive film 30 may use PVC resin as its base material. Alternatively, the second sensitive film 30 may be an enzyme-containing film that detects hydrogen ions generated by an enzymatic reaction with the target substance. At this stage, the second sensitive film 30 may be processed to partially expose the first sensitive film 19. In this case, it can be operated as a sensor element that simultaneously detects pH and electrolyte ions, or pH and physiologically active substances / neurotransmitters. In this embodiment, the second sensitive film 30 is patterned using a laser microfabrication device (manufactured by Hisol). To ensure selectivity with the underlying first sensitive film, a YAG laser (wavelength 266 nm) was used, with a 10 μm × 10 μm rectangular beam spot and an irradiation energy of 130 mJ. The third sensitive film 31 is a thin metal film, with Ti 5 nm and Au 20 nm deposited by Ar sputtering.

[0023] (Example 2) In order to detect physiologically active substances and neurotransmitters, it has a third sensitive membrane 31 that further contacts the second sensitive membrane 30. Here, it is suitable to select a metal thin film as the third sensitive membrane 31. The potential detected by the third sensitive membrane 31 is detected as a potential by the first sensitive membrane 19 through the second sensitive membrane 30. The second sensitive membrane 30 transmits the change in the potential in the membrane or on the membrane surface to the first sensitive membrane 19. In Example 2, the third sensitive membrane 31 and the second sensitive membrane 30 are removed in two steps by a laser microfabrication apparatus. The third sensitive membrane 31 is a metal film. Using a YAG laser (wavelength 532 nm), the irradiation energy with a 10 μm × 10 μm rectangular beam spot is 15 mJ. A part is selected from the location where the third sensitive membrane 31 has been removed to remove the second sensitive membrane 30. The conditions for laser processing of the second sensitive membrane 30 are a YAG laser (wavelength 266 nm), and the irradiation energy with a 10 μm × 10 μm rectangular beam spot is 130 mJ. A laser microfabrication apparatus is used for the processing of the second sensitive membrane 30 and the third sensitive membrane 31, but etching processing by photolithography may also be used. Further, since the third sensitive membrane 31 is a metal thin film, a lift-off method using a photoresist may also be used.

[0024] The measurement principle is described taking lactic acid detection as an example. In a basic electrochemical measurement method such as cyclic voltammetry, an electrode is penetrated into a measurement solution to measure the potential of the solution. The measurement solution is composed of a detection substance, a supporting salt, and a buffer. Here, the detection substance in the measurement solution may be any substance that serves as a substrate for an oxidation-reduction reaction, may be a substance that undergoes an oxidation-reduction reaction alone, or may be a substance that undergoes an oxidation-reduction reaction in the presence of a catalyst or cocatalyst of an enzyme or coenzyme. Examples of the supporting salt used as an electrolyte to increase the conductivity of the measurement solution (and auxiliary solution) include metal halides such as potassium chloride, and metal nitrates such as silver nitrate and potassium nitrate. Examples of the buffer used for the purpose of adjusting the pH of the measurement solution include HEPES (manufactured by Dojindo Laboratories).

[0025] The process of lactic acid detection is described. The detection target is penetrated into a pH buffer solution or a culture solution, and ferrocenemethanol (chemical formula: C 11 H12 FeO (sometimes referred to as "2FcMeOH" in this specification), oxidases, and hydrogen peroxide-degrading enzyme (HRP) are added or immobilized on the sensor surface. Detection is performed via a two-step enzymatic reaction. In the case of lactic acid detection, if lactate oxidase (LOX) is used as the degrading enzyme, hydrogen peroxide is generated. Lactate + O2 → Pyruvate + H2O2(1) H2O2 + 2H + +2FcMeOH → 2H2O+2FcMeOH + (2) During the decomposition of the generated hydrogen peroxide using HRP as a catalyst, FcMeOH is oxidized, and the solution potential changes. The change in solution potential is detected by a third sensitive film 31 (Ti / Au), and the potential is measured via the second sensitive film 30 and then through the first sensitive film 19. The second sensitive film is a dielectric, and the potential detected by the third sensitive film can be transmitted to the first sensitive film by capacitive coupling.

[0026] The measurement solution for detecting lactate release from cells contained 135 mM NaCl, 5 mM KCl, 22 mM CaCl, 21 mM MgCl, 10 mM D-glucose, and HEPES-NaOH as supporting salts and buffers, and mixed 5 units of lactate oxidase (LOX), 10.5 units of horseradish peroxidase (HRP), and 500 μM ferrocenemethanol as the detection substances. Here, 1 unit is defined as the amount of enzyme (1 micromol / min) that can convert 1 micromol / min of substrate per minute under optimal conditions (at a temperature of 30°C and the acidity at which the chemical reaction proceeds most effectively).

[0027] (Configuration and operation of the measurement unit) Figure 3 shows the fundamental configuration of the measurement unit 100 that constitutes the chemical / biosensor element 1 of this embodiment. Figure 4 shows the potential distribution within the semiconductor. The measurement unit 100 is divided on a silicon substrate 2 into a sensing (Sen) region 6, a first floating diffusion (FD1) region 7, a charge transfer (TG) region 10, a second floating diffusion (FD2) region 5, a charge transfer control (AG) region 11, a charge storage (FD) region 9, a reset (RG) region 12, and a second charge discharge (D2) region 5, in order from the first charge discharge (D1) region 4 in the direction of charge transfer.

[0028] The division of each region is defined by the difference in the conduction type of the silicon semiconductor on the surface of the silicon substrate 2. When electrons are used as the charge, the first charge discharge (D1) region 4, the first floating diffusion (FD1) region 7, the second floating diffusion (FD2) region 8, the charge accumulation (FD) region 9, and the second charge discharge (D2) region 5 are n+ type regions, while the sensing (Sen) region 6, the charge transfer (TG) region 10, the charge transfer control (AG) region 11, and the reset (RG) region 13 are p type regions.

[0029] A silicon oxide insulating film 3 is laminated on the surface of the silicon substrate 2. A sensing region defining electrode 13 is formed on the sensing (Sen) region 6. Furthermore, a silicon oxide layer 18 is laminated, and a silicon nitride film or a tantalum pentoxide film is laminated on the surface of the silicon oxide layer 18 as the first sensitive film 19. Potential changes on the surface of the first sensitive film 19 are transmitted to the sensing region defining electrode 13 via a conductive layer 17 embedded in the silicon oxide layer 18. A charge transfer electrode 14 is formed on the charge transfer (TG) region 10 via the silicon oxide insulating film 3, a charge transfer control electrode 15 is formed on the charge transfer control (AG) region 11 via the silicon oxide insulating film 3, and a reset electrode 16 is formed on the reset (RG) region 12 via the silicon oxide insulating film 3.

[0030] The first floating diffusion (FD1) region 7 is positioned in close proximity to the sensing (Sen) region 6 and accumulates a charge amount that reflects the potential of the sensing (Sen) region 6. The potential of the charge transfer (TG) region 10 is appropriately set to a sufficiently low voltage or sufficiently high voltage, such as the ground potential (GND) or the power supply voltage (VDD), and the charge transfer (TG) region 10 performs charge transfer between the first floating diffusion (FD1) region 7 and the second floating diffusion (FD2) region 8.

[0031] The charge transfer control (AG) region 11 is positioned in close proximity between the second floating diffusion (FD2) region 8 and the charge storage (FD) region 9, and a predetermined potential, either ground potential or between the power supply voltage and the potential of the sensing (Sen) region 6, is applied to it. The charge transfer control (AG) region 11 controls the amount of charge transferred from the second floating diffusion (FD2) region 8 to the charge storage (FD) region 9.

[0032] The reset (RG) region 12 is located in close proximity between the charge storage (FD) region 9 and the second charge discharge (D2) region 5, and is to which the ground potential or power supply voltage is applied. The reset (RG) region 12 controls the charge transfer from the charge storage (FD) region 9 to the second charge discharge (D2) region 5.

[0033] The operation of the measurement unit 100 will be explained with reference to Figure 4. The operation of the measurement unit 100 consists of four steps, from Figure 4A to Figure 4D. The potential height is indicated by the arrow, with the lower arrow indicating a higher potential. Here, we assume that the charge is an electron. In the following explanation, we will use electrons, which are negatively charged, as the charge.

[0034] The first charge discharge (D1) region 4 and the second charge discharge (D2) region 5 are subjected to a sufficiently high voltage throughout the entire step, such as the power supply voltage, to constantly discharge charge.

[0035] Figure 4A shows the initial state. When the potential of the reset (RG) region 12 is set to the power supply voltage, the potentials of the second charge discharge (D2) region 5 and the charge storage (FD) region 9 become equal, and the charge in the charge storage (FD) region 9 is discharged. However, an unspecified amount of charge remains in the first floating diffusion (FD1) region 7 and the second floating diffusion (FD2) region 8. At the end of this step, the potential of the reset (RG) region 12 must be set to the ground potential, and charge movement between the charge storage (FD) region 9 and the second charge discharge (D2) region 5 must be blocked.

[0036] In Figure 4B, charge is injected into the second floating diffusion (FD2) region 8. The potentials of the charge transfer (TG) region 10 and the charge transfer control (AG) region 11 are temporarily set to ground potential, and charge is injected from the charge injection circuit 20. The minimum potential of the charge held in the second floating diffusion (FD2) region 8 is equal to ground potential. At the end of this step, charge injection from the charge injection circuit 20 is terminated.

[0037] In Figure 4C, the potential of the charge transfer (TG) region 10 is set to the power supply voltage, and a portion of the charge in the second floating diffusion (FD2) region 8 is transferred to the first charge discharge (D1) region 4 via the first floating diffusion (FD1) region 7. The minimum potential of the charge remaining in the second floating diffusion (FD2) region 8 is determined by the potential of the sensing (Sen) region 6.

[0038] In Figure 4D, the potential of the charge transfer (TG) region 10 is set to the ground potential, blocking the movement of charge between the first floating diffusion (FD1) region 7 and the second floating diffusion (FD2) region 8. In this step, the lowest potential of the charge held in the first floating diffusion (FD1) region 7 is the potential detected by the sensing (Sen) region 6, and the lowest potential of the charge held in the second floating diffusion (FD2) region 8 is held at the potential of the charge transfer control (AG) region 11. At this time, by making the potential of the charge transfer control (AG) region 11 higher than the potential of the sensing (Sen) region 6, an amount of charge corresponding to the potential difference is transferred to the charge storage (FD) region 9.

[0039] Since the amount of charge accumulated in the charge storage (FD) region 9 reflects the potential height in the sensing (Sen) region 6, the potential can be measured using a buffer circuit 21 or similar device with high input impedance.

[0040] By repeating the steps shown in Figures 4B to 4D, the amount of charge accumulated in the charge accumulation (FD) region 9 increases by the number of repetitions.

[0041] Here, the configuration and operation of the measurement unit 100 used in this embodiment are described. However, the measurement unit 100 only needs to detect the potential induced in the sensing region, and may use the ion sensor structure described in Non-Patent Literature 1, or an ion-sensitive field-effect transistor (ISFET) structure that changes the channel conductance in response to the potential on the surface of the sensitive film.

[0042] The structure and operation of the measurement unit 100 described in the above embodiments allow for the detection of the pH or hydrogen ion concentration of the solution to be detected, and the detection of the surface potential of the first sensitive film 19. [Explanation of Symbols]

[0043] 1. Chemical and Biosensor Elements 2. Silicon substrate 3. First insulating film (silicon oxide film) 4. First charge discharge (D1) region 5. Second charge discharge (D2) region 6. Sensing (Sen) area 7. First floating-diffusion (FD1) region 8. Second floating-diffusion (FD2) region 9 Charge storage (FD) region 10 Charge Transfer (TG) Region 11 Charge Transfer Control (AG) Region 12 Reset (RG) area 13 Sensing area defining electrode 14 Charge transfer electrodes 15 Charge transfer control electrodes 16 Reset electrode 17. Conductive layer 18. Second insulating film (silicon oxide film) 19 Sensitive films (tantalum pentoxide film, silicon nitride film) 20 Charge injection circuit 21 Output voltage detection circuit (buffer circuit) 30. The second sensitive membrane 31. The third sensitive membrane 100, 101, 102 Measurement Units

Claims

1. An array of chemical and physical phenomenon measurement units comprising: multiple sensing regions that change the depth of a potential well according to the surface potential; electrodes that define the sensing regions via a first insulating layer covering the sensing regions; a first sensitive film that detects pH or surface potential formed via a second insulating layer covering the electrodes defining the sensing regions; and a conductive layer connecting the electrodes defining the sensing regions and the first sensitive film; A second sensitive film carrying an ionophore is formed directly above the first sensitive film in a specific measuring unit constituting the array, and a third sensitive film made of metal for detecting the solution potential formed at predetermined intervals with the measuring unit as the unit is formed on the second sensitive film. In the measuring unit where the second sensitive film is formed, the surface of the first sensitive film is laminated with the second sensitive film, and in the measuring unit where the third sensitive film is formed, the surface of the second sensitive film is laminated with the third sensitive film. A chemical / biosensor element characterized in that the potential change detected by the third sensitive film is transmitted to the first sensitive film on which the second sensitive film is laminated, via the second sensitive film on which the third sensitive film is laminated, and then transmitted to the sensing region of the measurement unit.

2. The chemical / biosensor element according to Claim 1, characterized in that the second sensitive film is removed in a part of the measurement unit in which the third sensitive film is not laminated, and the first sensitive film is configured to come into contact with the object to be measured.

3. A method for determining the concentration of a target substance by impregnating a chemical / biosensor element according to claim 1 or 2 into a solution containing a substance having redox activity, an oxidase substance for the substance to be detected, and a hydrogen peroxide decomposing enzyme, detecting the redox potential of the solution with the third sensitive membrane, and determining the concentration of the substance to be detected.

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