Apparatus for detecting target substances and method for detecting target substances
The target substance detection device and method simplify the detection process by immersing a substrate with capture molecules in an evaluation solution, directly detecting reaction products to suppress noise signals and ensure accurate target substance detection.
Patent Information
- Authority / Receiving Office
- JP · JP
- Patent Type
- Patents
- Current Assignee / Owner
- MURATA MFG CO LTD
- Filing Date
- 2023-07-12
- Publication Date
- 2026-06-30
AI Technical Summary
Existing target substance detection methods suffer from noise signals due to solution handling and require complex processes such as washing and introducing substrates, complicating the detection process.
A target substance detection device and method that involves a substrate coated with capture molecules and an enzyme-labeled second capture molecule, where the substrate is immersed in an evaluation solution containing a modified substrate, allowing for direct detection of reaction products without introducing additional substrates, thereby suppressing noise signals.
The method effectively suppresses noise signals and simplifies the detection process by directly detecting reaction products, providing accurate and sensitive target substance detection without the need for additional solution handling steps.
Smart Images
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Abstract
Description
[Technical Field]
[0001] The present invention relates to a target substance detection device and a target substance detection method. [Background technology]
[0002] There is a need for highly sensitive detection devices and methods that can rapidly detect target substances such as viruses and proteins.
[0003] Patent Document 1 discloses a method for measuring electrical changes for the detection or quantification of a target substance in a sample, comprising the steps of: 1) binding a compound A capable of selectively binding to the target substance and generating a charged substance to the target substance; 2) generating a charged substance in compound A; and 3) measuring the electrical changes caused by the charged substance in a required volume using a field-effect transistor.
[0004] In the method described in Figure 1 of Patent Document 1, the target substance is first immobilized on a field-effect transistor (FET). Compound A is a urease modified to selectively bind to a specific site, and compound A generates ammonia in an aqueous reaction field into which a predetermined amount of urea has been introduced. This ammonia becomes ammonium ions, which are charged substances, in water, and the generation of these ammonium ions is detected by the FET. The amount of ammonium ions can then be quantified based on the detected amount of current. Since the amount of ammonium ions generated is governed by the amount of urease and introduced urea, the reaction rate between urease and urea is measured in advance, and the amount of selectively bound urease can be determined by tracking the change in the detected amount of electricity over a predetermined time. Since the amount of selectively bound urease is proportional to the amount of the target substance, the amount of the target substance can be quantified as a result.
[0005] Patent Document 2 discloses a measuring device comprising: a container having an antibody against a substance to be measured; a sample solution supply means for supplying a sample solution containing the substance to be measured into the container; an enzyme-labeled antibody supply means for supplying an enzyme-labeled antibody conjugated with an enzyme that produces a thiol compound and an antibody against the substance to be measured into the container; a substrate supply means for supplying a substrate for the enzyme; a field-effect transistor; an electrode connected to the gate of the field-effect transistor by wiring and in contact with the solution in the container; a reference electrode in contact with the solution in the container; a power supply for applying a voltage between the electrode and the reference electrode; and a detection unit for detecting the output of the field-effect transistor.
[0006] In the method described in Figure 1 of Patent Document 2, first, a sample solution is injected into the reaction solution in the measurement cell using a sample solution injector to bind the antigen and antibody in the sample solution. After a certain period of time, an enzyme-labeled antibody solution is injected into the reaction solution using an enzyme-labeled antibody solution injector to cause an antigen-antibody reaction and form an antibody-antigen-enzyme-labeled antibody. Subsequently, the bound enzyme-labeled antibody and the free enzyme-labeled antibody are separated by washing the measurement cell and replacing the reaction solution in the measurement cell. After washing the measurement cell and replacing the solution, the substrate of the labeled enzyme is injected using a substrate solution injector, and the substrate is decomposed by the enzyme, generating a thiol compound. The generated thiol compound is adsorbed onto a gold electrode formed on an insulated gate field-effect transistor (FET), forming a self-assembled film. As a result, the potential on the gold electrode changes. The measurement is performed by monitoring the source-drain current in the insulated gate field-effect transistor, which changes before and after substrate injection using the substrate solution injector, in real time, and recording it with a signal processing circuit and a data processing device. The adsorption rate of thiol compounds to the gold electrode is proportional to the rate of thiol compound formation, i.e., the amount of antibody-antigen-enzyme-labeled antibody. Therefore, by measuring the adsorption rate of thiol compounds to the gold electrode, the amount of bound labeling enzyme, i.e., the amount of antigen in the sample solution, can be obtained. In this process, to reduce the influence of external fluctuations during measurement, a high-frequency voltage is applied to the reference electrode by a power supply during measurement. [Prior art documents] [Patent Documents]
[0007] [Patent Document 1] Japanese Patent Publication No. 2018-36154 [Patent Document 2] Japanese Patent Publication No. 2007-263914 [Overview of the Initiative] [Problems that the invention aims to solve]
[0008] The method described in Patent Document 1 is characterized by detecting a charged substance generated from compound A, which selectively binds to the target substance and generates a charged substance through a predetermined reaction, rather than detecting the target substance itself. Patent Document 1 describes a preferred example of compound A as a substance having a part that functions as an enzyme and a part that selectively binds to the target substance (for example, an antibody modified with urease). In this case, the ammonium ion produced by the reaction of urease and urea is detected.
[0009] However, the method described in Patent Document 1 requires the introduction of a substrate such as urea in order to react with an enzyme such as urease. Therefore, there is a risk of detecting noise signals other than ammonium ions due to the solution handling when introducing the substrate. In addition, in order to remove enzymes that are not bound to the target substance, it is necessary to perform operations such as washing the inside of the container or adding a new solution to the container before introducing the substrate. As a result, the overall process may become complicated.
[0010] Although the method described in Patent Document 2 uses different types of enzymes and substrates, it detects the reaction products generated by the enzymatic reaction, rather than the target substance itself, similar to the method described in Patent Document 1.
[0011] However, even in the method described in Patent Document 2, there is a risk of detecting noise signals due to the handling of the solution when injecting the enzyme substrate. Furthermore, since it is necessary to perform operations such as cleaning the inside of the measurement cell and changing the solution inside the cell before injecting the substrate, the overall process may become complicated.
[0012] The present invention was made to solve the above problems and aims to provide a target substance detection device capable of suppressing noise signals. Furthermore, the present invention aims to provide a target substance detection method capable of suppressing noise signals. [Means for solving the problem]
[0013] The target substance detection device of the present invention comprises a target substance-coated substrate and a sensor body. The target substance-coated substrate includes a substrate on which a first capture molecule is immobilized on at least a portion of its surface, a target substance bound to the first capture molecule, and an enzyme-labeled second capture molecule on which a second capture molecule and an enzyme are bound, with the second capture molecule bound to the target substance. The sensor body includes an evaluation solution containing a substrate modified by the enzyme, a container for containing the evaluation solution, and an electrical sensor provided to be in contact with the evaluation solution for sensing components in the evaluation solution. The target substance-coated substrate is separated independently from the sensor body. The portion of the target substance-coated substrate on which the first capture molecule is immobilized on the surface of the substrate is immersed in the evaluation solution.
[0014] The present invention provides a method for detecting a target substance, comprising the steps of: preparing a target substance-attached substrate comprising a substrate on which a first capture molecule is immobilized on at least a portion of its surface; a target substance bound to the first capture molecule; and an enzyme-labeled second capture molecule on which a second capture molecule and an enzyme are bound, with the second capture molecule bound to the target substance; immersing the portion of the target substance-attached substrate on which the first capture molecule is immobilized on the surface of the substrate into an evaluation solution containing a substrate modified by the enzyme; and sensing the components in the evaluation solution using an electrical sensor provided in contact with the evaluation solution. [Advantages of the Invention]
[0015] According to the present invention, it is possible to provide a detection device for a target substance capable of suppressing noise signals. Further, according to the present invention, it is possible to provide a detection method for a target substance capable of suppressing noise signals. [Brief Description of the Drawings]
[0016] [Figure 1] FIG. 1 is a schematic diagram schematically showing an example of a detection device for a target substance of the present invention. [Figure 2] FIG. 2 is a schematic diagram of a state in which a substrate with a target substance and a sensor body constituting the detection device for the target substance shown in FIG. 1 are separated. [Figure 3] FIG. 3 is a graph showing an example of the relationship between the gate-source voltage Vgs and the source-drain current Ids. [Figure 4] FIG. 4 is a graph showing an example of the change over time of the charge neutral point CNP. [Figure 5] FIG. 5 is a schematic diagram schematically showing a detection device for a target substance and a detection method for a target substance according to Example 1. [Figure 6] FIG. 6 is a schematic diagram schematically showing a detection device for a target substance and a detection method for a target substance according to Example 2. [Figure 7] FIG. 7 is a schematic diagram schematically showing a detection device for a target substance and a detection method for a target substance according to Example 3. [Figure 8] FIG. 8 is a schematic diagram schematically showing a detection device for a target substance and a detection method for a target substance according to Example 4. [Figure 9] FIG. 9 is a schematic diagram schematically showing a detection device for a target substance and a detection method for a target substance according to Example 5. [Figure 10] FIG. 10 is a schematic diagram schematically showing a detection device for a target substance and a detection method for a target substance according to Example 6. [Figure 11] FIG. 11 is a schematic diagram schematically showing another example of a substrate with a target substance. [Modes for carrying out the invention]
[0017] The following describes the target substance detection apparatus and target substance detection method of the present invention. However, the present invention is not limited to the following configurations and may be modified as appropriate without altering the essence of the invention. Furthermore, a combination of several of the preferred configurations described below also constitutes the present invention.
[0018] The drawings shown below are schematic representations, and their dimensions, aspect ratios, and scales may differ from those of the actual product.
[0019] Figure 1 is a schematic diagram illustrating an example of a target substance detection device according to the present invention.
[0020] The target substance detection device 1 shown in Figure 1 comprises a substrate 10 coated with the target substance and a sensor body 20. The substrate 10 coated with the target substance is separated independently from the sensor body 20.
[0021] Figure 2 is a schematic diagram showing the target substance-coated substrate and sensor body, which constitute the target substance detection device shown in Figure 1, in a separated state.
[0022] As shown in Figures 1 and 2, the target substance-attached substrate 10 includes a substrate 12 on which a first capture molecule 11A is immobilized on at least a portion of its surface, a target substance 13 bound to the first capture molecule 11A, and an enzyme-labeled second capture molecule 15 to which a second capture molecule 11B and an enzyme 14 are bound, with the second capture molecule 11B bound to the target substance 13. The target substance-attached substrate 10 has a structure in which the target substance 13 is sandwiched between the first capture molecule 11A and the enzyme-labeled second capture molecule 15.
[0023] The first capture molecule 11A is a molecule capable of capturing the target substance 13. Examples of the first capture molecule 11A include antibodies, aptamers, peptides, glycans, receptors, molecular templates, antigens for antibodies, and secondary antibodies for antibodies. The first capture molecule 11A may be fixed to or in close contact with the surface of the substrate 12, or it may be movable with a certain degree of freedom, as long as it remains on the surface of the substrate 12.
[0024] The material of the base material 12 is not particularly limited, and examples include glass, resin, ceramic, metal, etc. The size, thickness, shape, etc. of the base material 12 are also not particularly limited, but it is preferable that the surface has an uneven structure in order to increase the surface area.
[0025] Examples of target substances 13 include viruses, proteins, nucleic acids, and low-molecular-weight biomolecules.
[0026] The enzyme-labeled second capture molecule 15 is formed by the binding of the second capture molecule 11B and the enzyme 14. The second capture molecule 11B is a molecule capable of capturing the target substance 13. Examples of the second capture molecule 11B include antibodies, aptamers, peptides, glycans, receptors, molecular templates, and secondary antibodies against antibodies. The second capture molecule 11B may be of the same type as the first capture molecule 11A, or it may be of a different type.
[0027] Examples of enzyme 14 include urease and L-amino acid oxidase, but are not limited to these as long as they are enzymes that produce charged substances.
[0028] As shown in Figures 1 and 2, the sensor body 20 includes an evaluation solution 21 containing a substrate 24 modified by an enzyme 14, a container 22 for containing the evaluation solution 21, and an electrical sensor 23 provided in contact with the evaluation solution 21 to sense the components in the evaluation solution 21.
[0029] The electrical sensor 23 differs from both optical and chemical sensors. In the examples shown in Figures 1 and 2, the electrical sensor 23 is a field-effect transistor (FET) sensor with a graphene semiconductor channel layer.
[0030] Although not shown in Figures 1 and 2, the sensor body 20 may be provided with multiple electrical sensors 23.
[0031] The electrical sensor 23 includes, for example, a semiconductor layer 25 and a source electrode 26 and a drain electrode 27 electrically connected to the semiconductor layer 25, as shown in Figures 1 and 2. The semiconductor layer 25 between the source electrode 26 and the drain electrode 27 constitutes the channel of the electrical sensor 23.
[0032] Although not shown in Figures 1 and 2, the electrical sensor 23 may further include a gate electrode for applying an external electric field to the semiconductor layer 25. In that case, the gate electrode is immersed in the evaluation solution 21.
[0033] The number of semiconductor layers 25 is not limited to one layer, but may be two or three or more layers. Preferably, the number of semiconductor layers 25 is 10 or less, and more preferably 5 or less. Furthermore, the number of layers does not need to be uniform throughout the semiconductor layer 25; for example, a mixture of one-layer and two or more-layer portions may be present. The number of semiconductor layers 25 can be measured, for example, by Raman spectroscopy or by cross-sectional observation using a transmission electron microscope (TEM).
[0034] In Figures 1 and 2, the source electrode 26 and the drain electrode 27 are arranged on the insulating substrate 28, separated from each other, with the insulating substrate 28 exposed between the source electrode 26 and the drain electrode 27. The semiconductor layer 25 is arranged on the insulating substrate 28 so as to cover the exposed portion of the insulating substrate 28. The semiconductor layer 25 may also be arranged to cover the end of the source electrode 26 or to cover the end of the drain electrode 27.
[0035] The source electrode 26 and drain electrode 27 are, for example, multilayer electrodes with a titanium (Ti) layer and a gold (Au) layer stacked on top of each other. In addition to titanium and gold, other metals such as gold, platinum, titanium, and palladium may be used as electrode materials in single layers, or two or more metals may be combined to form a multilayer structure.
[0036] If the electrical sensor 23 includes a gate electrode, the gate electrode applies a potential to the source electrode 26 and the drain electrode 27, and generally a reference electrode such as a silver or silver chloride electrode or a precious metal is used. The gate electrode is provided at a location other than where the source electrode 26 and the drain electrode 27 are formed. It is usually provided on or outside the insulating substrate 28, but it is preferable to provide it above the source electrode 26 or the drain electrode 27.
[0037] The evaluation solution 21 contains a substrate 24 that is modified by the enzyme 14. The evaluation solution 21 is, for example, an electrolyte solution containing an electrolyte.
[0038] Examples of substrate 24 include urea and L-amino acids.
[0039] The container 22 includes, for example, an insulating substrate 28 and a partition wall 29 provided on the insulating substrate 28.
[0040] In Figures 1 and 2, the insulating substrate 28 can be, for example, a thermal silicon oxide substrate or a boron nitride (BN) substrate, which is formed by oxidizing the surface of a silicon (Si) substrate to create a silicon oxide (SiO2) layer. The material of the insulating substrate 28 is not particularly limited, and can be, for example, an inorganic compound such as silicon oxide, silicon nitride, aluminum oxide, titanium oxide, or calcium fluoride, or an organic compound such as acrylic resin, polyimide, or fluororesin. The shape of the insulating substrate 28 is not particularly limited, and may be flat or curved. The insulating substrate 28 may also be flexible.
[0041] Examples of the partition wall 29 include, but are not limited to, silicone sheets (also known as rubber pools), epoxy resin, acrylic resin, etc.
[0042] As shown in Figure 2, the substrate 10 with the target substance is immersed in the evaluation liquid 21 of the sensor body 20. As a result, in the target substance detection device 1 shown in Figure 1, the area where the first capture molecule 11A of the substrate 10 with the target substance is fixed to the surface of the substrate 12 is immersed in the evaluation liquid 21.
[0043] An example of the method for detecting the target substance of the present invention will be described below with reference to Figures 1 and 2.
[0044] First, a substrate 10 with a target substance is prepared. As described above, the substrate 10 with a target substance includes a substrate 12 on which a first capture molecule 11A is immobilized on at least a portion of the surface, a target substance 13 bound to the first capture molecule 11A, and an enzyme-labeled second capture molecule 15 to which a second capture molecule 11B and an enzyme 14 are bound, with the second capture molecule 11B bound to the target substance 13.
[0045] Next, the portion of the target substance-attached substrate 10 where the first capture molecule 11A is immobilized on the surface of the substrate 12 is immersed in the evaluation solution 21 containing the substrate 24.
[0046] The substrate 24 is modified by the enzyme 14. For example, if the substrate 24 is urea and the enzyme 14 is urease, the reaction between urea and water produces ammonia and carbon dioxide. Ammonia and carbon dioxide exist in water as ammonium ions and carbonate ions. Therefore, the total amount of ions in the evaluation solution 21 changes before and after immersing the substrate 10 with the target substance in the evaluation solution 21.
[0047] Therefore, components in the evaluation liquid 21 are sensed using an electrical sensor 23 provided to contact the evaluation liquid 21. For example, by observing the change in the components in the evaluation liquid 21 before and after immersing the substrate 10 with the target substance into the evaluation liquid 21, the presence or concentration of the reaction product generated by the enzymatic reaction can be sensed. Since the amount of the reaction product is proportional to the amount of the target substance 13, as a result, the presence or concentration of the target substance 13 can be sensed.
[0048] As an example, when the electrical sensor 23 is a FET sensor, first, by applying a voltage between the source electrode 26 and the drain electrode 27, the current flowing between the source electrode 26 and the drain electrode 27 (hereinafter referred to as the source-drain current I ds ) is measured.
[0049] While keeping the voltage applied between the source electrode 26 and the drain electrode 27 constant, the voltage applied between the gate electrode (not shown) and the source electrode 26 (hereinafter referred to as the gate-source voltage V gs ) is repeatedly swept, and the value of the gate-source voltage V ds when the source-drain current I gs becomes minimum is acquired. In the following description, the value of the gate-source voltage V ds when the source-drain current I gs becomes minimum is called the charge neutrality point (CNP; Charge Neutrality Point).
[0050] FIG. 3 is a graph showing an example of the relationship between the gate-source voltage V gs and the source-drain current I ds .
[0051] For example, when the total amount of ions in the evaluation liquid 21 changes before and after immersing the substrate 10 with the target substance into the evaluation liquid 21, as shown in FIG. 3, the value of the gate-source voltage V ds when the source-drain current I gs becomes minimum, that is, the charge neutrality point CNP changes.
[0052] Figure 4 is a graph showing an example of the change in the charge neutral point (CNP) over time.
[0053] Figure 4 shows the change in the charge neutral point CNP over time when the target substance-coated substrate 10 is immersed in the evaluation solution 21 containing the substrate 24, indicated by circles (Positive Control), and the change in the charge neutral point CNP over time when the substrate without the immobilized first capture molecule 11A is immersed, indicated by squares (Negative Control). As shown by the arrows in Figure 4, the charge neutral point CNP changes before and after immersion of the target substance-coated substrate 10 in the evaluation solution 21. The change in the charge neutral point CNP corresponds to the amount of reaction product produced by the enzymatic reaction. Therefore, by evaluating the change in the charge neutral point CNP, the target substance 13 can be detected.
[0054] As described above, the target substance detection device and target substance detection method of the present invention, similar to Patent Documents 1 and 2, do not detect the target substance itself, but rather detect the reaction products generated by the enzymatic reaction. Therefore, any target substance can be detected regardless of the presence or absence of charge, the magnitude of the charge, or the size of the target substance.
[0055] On the other hand, in the target substance detection device and target substance detection method of the present invention, unlike Patent Documents 1 and 2, a substrate modified by an enzyme is not introduced; instead, a substrate with the target substance is immersed in an evaluation solution that already contains the substrate. As a result, the solute components contained in the evaluation solution do not change before and after sensing, and noise signals caused by such changes can be suppressed. Furthermore, operations such as changing or washing the solution for introducing the substrate, or adding solution, are unnecessary.
[0056] In the target substance detection device of the present invention, it is preferable that a portion of the substrate coated with the target substance protrudes from the evaluation solution. Similarly, in the target substance detection method of the present invention, it is preferable that the substrate coated with the target substance is immersed in the evaluation solution such that a portion of the substrate coated with the target substance protrudes from the evaluation solution. Specifically, it is preferable that a portion of the substrate in which the first capture molecules are not immobilized protrudes from the evaluation solution. Since the area of the portion on the surface of the substrate to which the first capture molecules are immobilized can be controlled, the dynamic range of the sensor can be improved. Furthermore, since the location in which the substrate coated with the target substance is immersed in the evaluation solution can be controlled, for example, the portion on the surface of the substrate to which the first capture molecules are immobilized can be placed near the sensing unit, or near the semiconductor layer in Figure 1.
[0057] In the target substance detection apparatus and method of the present invention, for example, the substrate is urea and the enzyme is urease. When nonionic urea is hydrolyzed, it decomposes into ionic ammonia and carbon dioxide, so the target substance can be detected by evaluating the ionic components in the evaluation solution.
[0058] In the target substance detection apparatus and target substance detection method of the present invention, the electrical sensor is preferably one of an FET sensor, a conductivity sensor, an ion electrode, or an impedance sensor. By using these electrical sensors, the components in the evaluation solution can be evaluated with high sensitivity.
[0059] In the target substance detection apparatus and target substance detection method of the present invention, the electrical sensor is preferably an FET sensor, and is particularly preferably an FET sensor using graphene as a semiconductor channel.
[0060] Graphene is a two-dimensional material composed of carbon atoms bonded together in a hexagonal network. Graphene has a very large specific surface area (surface area per unit volume) and also possesses very high electrical mobility.
[0061] In the target substance detection device and target substance detection method of the present invention, if the electrical sensor is an FET sensor, it may be an FET sensor with a semiconductor channel made of a material other than graphene. Examples of materials other than graphene include carbon nanotubes, molybdenum disulfide, boron nitride, silicon, oxide semiconductors, organic semiconductors, and the like.
[0062] In the target substance detection device and target substance detection method of the present invention, if the electrical sensor is an FET sensor, an ion-sensitive film may be provided on the surface of the FET sensor. The ion-sensitive film allows for highly sensitive sensing of ionic components.
[0063] Examples of materials for ion-sensitive membranes include vinyl chloride containing ammonium ionophores. Nonactin is a typical example of an ammonia ionophore.
[0064] In the target substance detection device and target substance detection method of the present invention, it is preferable that the conductivity of the evaluation solution before the substrate coated with the target substance is immersed is 1 mS / cm or less. In this case, the rate of change before and after the substrate coated with the target substance is immersed in the evaluation solution becomes large, so sensing can be performed with high sensitivity.
[0065] The conductivity of the evaluation solution before the substrate with the target substance is immersed is, for example, 1 μS / cm or higher.
[0066] Furthermore, if the conductivity of the evaluation solution before the substrate with the target substance is immersed is 1 mS / cm or less, the conductivity of the evaluation solution during sensing may also be 1 mS / cm or less, or it may exceed 1 mS / cm.
[0067] In this specification, the conductivity of the evaluation solution means the conductivity measured by the AC two-electrode method.
[0068] The following are examples that more specifically disclose the target substance detection apparatus and target substance detection method of the present invention. However, the present invention is not limited to these examples.
[0069] [Example 1] Figure 5 is a schematic diagram illustrating the target substance detection apparatus and target substance detection method according to Example 1.
[0070] In Example 1, the target substance-attached substrate 10 is prepared by immersing a substrate 12, on which the first capture molecule 11A is immobilized on at least a portion of its surface, in a preparation solution 30 containing an enzyme-labeled second capture molecule 15 and the target substance 13, as shown in Figure 5.
[0071] For example, a preparation solution 30 can be prepared by supplying a sample containing the target substance 13 to an electrolyte containing an enzyme-labeled second capture molecule 15. If the sample containing the target substance 13 is a liquid, the sample may be added dropwise using a dropper or the like, or introduced using a flow channel.
[0072] Examples of samples containing the target substance 13 include bodily fluids such as saliva, nasal swabs, pharyngeal swabs, tears, and blood from a subject, biological samples such as urine and feces, suspensions of cells or viruses themselves, drinking water, sewage, and breath. The sample containing the target substance 13 does not have to be a liquid.
[0073] After preparing the target substance-coated substrate 10, the portion of the target substance-coated substrate 10 where the first capture molecule 11A is immobilized on the surface of the substrate 12 is immersed in the evaluation solution 21 containing the substrate 24.
[0074] [Example 2] Figure 6 is a schematic diagram illustrating the target substance detection apparatus and target substance detection method according to Example 2.
[0075] In Example 2, the target substance-attached substrate 10 is prepared by immersing a substrate 12 in a preparation solution 30 containing an enzyme-labeled second capture molecule 15, to which the first capture molecule 11A is immobilized on at least a portion of the surface, and to which the target substance 13 is bound, as shown in Figure 6.
[0076] For example, by supplying a sample containing the target substance 13 to a substrate 12 on which the first capture molecule 11A is immobilized on at least a portion of its surface, the target substance 13 can be bound to the first capture molecule 11A immobilized on the surface of the substrate 12.
[0077] After preparing the target substance-coated substrate 10, the portion of the target substance-coated substrate 10 where the first capture molecule 11A is immobilized on the surface of the substrate 12 is immersed in the evaluation solution 21 containing the substrate 24.
[0078] [Example 3] Figure 7 is a schematic diagram illustrating the target substance detection apparatus and target substance detection method according to Example 3.
[0079] In Example 3, the substrate on which the first capture molecule is immobilized is composed of magnetic particles 31 on which the first capture molecule 11A is immobilized, and a magnetic substrate 32 that holds the magnetic particles 31 by magnetism, as shown in Figure 7.
[0080] By using the magnetic substrate 32 and magnetic particles 31, more target substances 13 can be captured. As a result, sensitivity can be increased.
[0081] The method for binding the target substance 13 to the first capture molecule 11A may be the same as in Example 1, or the same as in Example 2.
[0082] Examples of materials for the magnetic particles 31 include ferrite, iron oxide, iron, nickel, and cobalt. The average particle size of the magnetic particles 31 is, for example, 50 nm or more and 5 μm or less.
[0083] Examples of materials for the magnetic substrate 32 include ferrite, iron oxide, iron, nickel, and cobalt. The material of the magnetic substrate 32 may be the same as or different from the material of the magnetic particles 31.
[0084] [Example 4] Figure 8 is a schematic diagram illustrating the target substance detection apparatus and target substance detection method according to Example 4.
[0085] In Example 4, as shown in Figure 8, the container 22 is divided into multiple wells 33, each containing the evaluation solution 21 in a volume of 1 pL or less.
[0086] Since the target substance 13 is confined and detected in the tiny space of the well 33, if we assign a value of 1 to the well 33 in which the target substance 13 is detected and 0 to the well 33 in which it is not detected, the signal can be digitized and sensed with high sensitivity.
[0087] The container 22 is preferably divided into 100 or more wells 33, more preferably into 1,000 or more wells 33, and even more preferably into 10,000 or more wells 33. On the other hand, the container 22 is preferably divided into 1,000,000 or fewer wells 33, and even more preferably into 500,000 or fewer wells 33.
[0088] Each well 33 is preferably filled with 1 pL or less of the evaluation solution 21, and more preferably with 100 fL or less. On the other hand, each well 33 is preferably filled with 1 fL or more of the evaluation solution 21.
[0089] The form of the substrate 12 may be, for example, particles or beads. In that case, the average particle size of the substrate 12 may be, for example, 50 nm or more and 5 μm or less. The substrate 12 may also be magnetic.
[0090] [Example 5] Figure 9 is a schematic diagram illustrating the target substance detection apparatus and target substance detection method according to Example 5.
[0091] In Example 5, as shown in Figure 9, the end of the base material 12 is divided into two or more parts. In the example shown in Figure 9, the end of the base material 12 is divided into two parts.
[0092] In Figure 9, the first capture molecule 11A is immobilized on one end of the substrate 12, while the first capture molecule 11A is not immobilized on the other end of the substrate 12. Each end of the substrate 12 is immersed in a different evaluation solution 21.
[0093] In Example 5, the possibility of a false positive can be reduced by using the side on which the first capture molecule 11A is not immobilized as a negative control.
[0094] The number of ends to which the first capture molecule 11A is immobilized may be two or more. Similarly, the number of ends to which the first capture molecule 11A is not immobilized may be two or more. The number of ends to which the first capture molecule 11A is not immobilized may be the same as or different from the number of ends to which the first capture molecule 11A is immobilized.
[0095] The method for binding the target substance 13 to the first capture molecule 11A may be the same as in Example 1, or the same as in Example 2.
[0096] [Example 6] Figure 10 is a schematic diagram illustrating the target substance detection apparatus and target substance detection method according to Example 6.
[0097] In Example 6, as shown in Figure 10, the target substance 13 includes a first target substance 13A and a second target substance 13B. Furthermore, the end of the substrate 12 is divided into two or more parts. In the example shown in Figure 10, the end of the substrate 12 is divided into two parts.
[0098] In Figure 10, a first capture molecule 11A is immobilized on one end of the substrate 12, and a third capture molecule 11C, which has a different binding target than the first capture molecule 11A, is immobilized on the other end of the substrate 12. The first target substance 13A binds to the first capture molecule 11A, and the second target substance 13B binds to the third capture molecule 11C.
[0099] The target substance-coated substrate 10 further contains an enzyme-labeled fourth capture molecule 16, to which a fourth capture molecule 11D is bound to an enzyme 14, and the fourth capture molecule 11D is bound to a second target substance 13B. The ends of the substrate 12 are each immersed in a separate evaluation solution 21.
[0100] In Example 6, multiple types of target substances 13 can be detected simultaneously.
[0101] The first capture molecule 11A may have two or more fixed ends. Similarly, the third capture molecule 11C may have two or more fixed ends. The number of fixed ends of the third capture molecule 11C may be the same as or different from the number of fixed ends of the first capture molecule 11A.
[0102] The enzyme 14 bound to the fourth capture molecule 11D may be the same as or different from the enzyme 14 bound to the second capture molecule 11B. If the enzyme 14 bound to the fourth capture molecule 11D is different from the enzyme 14 bound to the second capture molecule 11B, the substrates 24 contained in each evaluation solution 21 will also be different. The fourth capture molecule 11D may be the same type as or different from the third capture molecule 11C.
[0103] The method for attaching the first target substance 13A to the first capture molecule 11A may be the same as in Example 1 or the same as in Example 2. Similarly, the method for attaching the second target substance 13B to the third capture molecule 11C may be the same as in Example 1 or the same as in Example 2.
[0104] In the example shown in Figure 10, two types of target substances 13 are detected, but three or more types of target substances 13 may also be detected.
[0105] Similar to Example 5, the substrate 12 may have ends to which the first capture molecule 11A, the third capture molecule 11C, etc., are not immobilized.
[0106] The target substance detection apparatus and target substance detection method of the present invention are not limited to the embodiments and examples described above, and various applications and modifications can be made within the scope of the present invention with respect to the configuration of the detection apparatus, manufacturing conditions, etc.
[0107] For example, the enzyme-labeled second capture molecule is composed of the second capture molecule and the enzyme being bound together, but the second capture molecule and the enzyme may be bound directly or indirectly. Similarly, the enzyme-labeled fourth capture molecule is composed of the fourth capture molecule and the enzyme being bound together, but the fourth capture molecule and the enzyme may be bound directly or indirectly.
[0108] Figure 11 is a schematic diagram illustrating another example of a substrate coated with a target substance.
[0109] The target substance-attached substrate 10A shown in Figure 11 includes a substrate 12 on which a first capture molecule 11A is immobilized on at least a portion of its surface, a target substance 13 bound to the first capture molecule 11A, and an enzyme-labeled second capture molecule 15A to which a second capture molecule 11B and an enzyme 14 are bound, with the second capture molecule 11B bound to the target substance 13.
[0110] In the enzyme-labeled secondary capture molecule 15A, the secondary capture molecule 11B and the enzyme 14 are indirectly bound. Specifically, the secondary capture molecule 11b and the enzyme 14 are bound together, and the secondary capture molecule 11b is bound to the second capture molecule 11B, thereby forming the enzyme-labeled secondary capture molecule 17. Furthermore, the enzyme-labeled secondary capture molecule 17 is bound to the second capture molecule 11B, thereby forming the enzyme-labeled secondary capture molecule 15A.
[0111] In the target substance detection device and target substance detection method of the present invention, the structure in which the enzyme is immobilized on the substrate via the target substance may be not only a sandwich structure of (1) a first capture molecule immobilized on the substrate / (2) the target substance / (3) an enzyme-labeled second capture molecule, but also a structure of (1) a first capture molecule immobilized on the substrate / (2) the target substance / (3) a second capture molecule / (4) an enzyme-labeled secondary capture molecule. By adopting the latter structure, it is not necessary to prepare a new enzyme-labeled secondary capture molecule when the target substance changes, and the enzyme-labeled secondary capture molecule can be used in common for various target substances. Furthermore, by making this enzyme-labeled secondary capture molecule a molecule that recognizes multiple sites of the second capture molecule, multiple enzyme-labeled capture molecules can be immobilized on the substrate. As a result, the amount of enzyme immobilized on the substrate increases, making it possible to operate the sensor with high sensitivity. A typical example of such a secondary capture molecule is a polyclonal antibody.
[0112] This specification discloses the following:
[0113] <1> The sensor comprises a substrate with a target substance and a sensor body. The above-mentioned target substance-attached substrate comprises a substrate on which a first capture molecule is immobilized on at least a portion of its surface, a target substance bound to the first capture molecule, and an enzyme-labeled second capture molecule on which a second capture molecule and an enzyme are bound, with the second capture molecule bound to the target substance. The sensor body includes an evaluation solution containing a substrate modified by the enzyme, a container for containing the evaluation solution, and an electrical sensor provided to be in contact with the evaluation solution in order to sense the components in the evaluation solution. The substrate with the target substance is separated independently from the sensor body. A target substance detection device wherein the portion of the substrate with the target substance to which the first capture molecule is immobilized on the surface of the substrate is immersed in the evaluation liquid.
[0114] <2> A portion of the substrate with the target substance protrudes from the evaluation solution. <1> A device for detecting the target substance described above.
[0115] <3> The above substrate is urea, and the above enzyme is urease. <1> or <2> A device for detecting the target substance described above.
[0116] <4> The above electrical sensor is one of the following: an FET sensor, a conductivity sensor, an ion electrode, or an impedance sensor. <1> ~ <3> A detection device for target substances as described in any one of the following.
[0117] <5> The above electrical sensor is an FET sensor using graphene as the semiconductor channel. <1> ~ <4> A detection device for target substances as described in any one of the following.
[0118] <6> The above electrical sensor is an FET sensor. An ion-sensitive film is provided on the surface of the above FET sensor. <1> ~ <5> A detection device for target substances as described in any one of the following.
[0119] <7> The conductivity of the evaluation solution before the substrate with the target substance is immersed in it is 1 mS / cm or less. <1> ~ <6> A detection device for target substances as described in any one of the following.
[0120] <8> The substrate on which the first capture molecule is immobilized is composed of magnetic particles on which the first capture molecule is immobilized and a magnetic substrate that holds the magnetic particles by magnetism. <1> ~ <7> A detection device for target substances as described in any one of the following.
[0121] <9> The above container is divided into multiple wells, each containing the above evaluation solution in a volume of 1 pL or less. <1> and <3> ~ <7> A detection device for target substances as described in any one of the following.
[0122] <10> The end of the above substrate is divided into two or more parts. The first capture molecule is immobilized on at least one end of the above substrate. The first capture molecule is not immobilized on at least one end of the above substrate. Each end of the above substrate is immersed in a separate evaluation solution. <1> ~ <8> A detection device for target substances as described in any one of the following.
[0123] <11> The above target substance includes a first target substance and a second target substance. The end of the above substrate is divided into two or more parts. The first capture molecule is immobilized on at least one end of the above substrate. A third capture molecule, which has a different binding target than the first capture molecule, is immobilized on at least one end of the above substrate. The first target substance is bound to the first capture molecule described above. The second target substance is bound to the third capture molecule described above. The above target substance-attached substrate further comprises an enzyme-labeled fourth capture molecule in which a fourth capture molecule and an enzyme are bound, and the fourth capture molecule is bound to the second target substance. Each end of the above substrate is immersed in a separate evaluation solution. <1> ~ <8> and <10> A detection device for target substances as described in any one of the following.
[0124] <12> A step of preparing a target substance substrate comprising: a substrate on which a first capture molecule is immobilized on at least a portion of its surface; a target substance bound to the first capture molecule; and an enzyme-labeled second capture molecule on which a second capture molecule and an enzyme are bound, with the second capture molecule bound to the target substance; A step of immersing the portion of the target substance-attached substrate where the first capture molecule is immobilized on the surface of the substrate into an evaluation solution containing the substrate modified by the enzyme described above, A method for detecting a target substance, comprising the step of sensing the components in the evaluation liquid using an electrical sensor provided in contact with the evaluation liquid.
[0125] <13> The substrate with the target substance is immersed in the evaluation solution such that a portion of the substrate with the target substance protrudes from the evaluation solution. <12> A method for detecting the target substance described above.
[0126] <14> The target substance-attached substrate is prepared by immersing the substrate, on which the first capture molecule is immobilized on at least a portion of its surface, in a preparation solution containing the enzyme-labeled second capture molecule and the target substance. <12> or <13> A method for detecting the target substance described above.
[0127] <15> The above-mentioned substrate with the target substance is prepared by immersing the substrate, on which the first capture molecule is immobilized on at least a portion of the surface and on which the target substance is bound to the first capture molecule, in a preparation solution containing the above-mentioned enzyme-labeled second capture molecule. <12> or <13> A method for detecting the target substance described above.
[0128] <16> The substrate on which the first capture molecule is immobilized is composed of magnetic particles on which the first capture molecule is immobilized and a magnetic substrate that holds the magnetic particles by magnetism. <12> ~ <15> A method for detecting a target substance as described in any one of the following.
[0129] <17> The above container is divided into multiple wells, each containing the above evaluation solution in a volume of 1 pL or less. <12> , <14> or <15> A method for detecting the target substance described above.
[0130] <18> The end of the above substrate is divided into two or more parts. The first capture molecule is immobilized on at least one end of the above substrate. The first capture molecule is not immobilized on at least one end of the above substrate. Each end of the above substrate is immersed in a separate evaluation solution. <12> ~ <16> A method for detecting a target substance as described in any one of the following.
[0131] <19> The above target substance includes a first target substance and a second target substance. The end of the above substrate is divided into two or more parts. The first capture molecule is immobilized on at least one end of the above substrate. A third capture molecule, which has a different binding target than the first capture molecule, is immobilized on at least one end of the above substrate. The first target substance is bound to the first capture molecule described above. The second target substance is bound to the third capture molecule described above. The above target substance-attached substrate further comprises an enzyme-labeled fourth capture molecule in which a fourth capture molecule and an enzyme are bound, and the fourth capture molecule is bound to the second target substance. Each end of the above substrate is immersed in a separate evaluation solution. <12> ~ <16> and <18> A method for detecting a target substance as described in any one of the following.
[0132] <20> The above electrical sensor is one of the following: an FET sensor, a conductivity sensor, an ion electrode, or an impedance sensor. <12> ~ <19> A method for detecting a target substance as described in any one of the following. [Explanation of Symbols]
[0133] 1. Target substance detection device 10, 10A Substrate with target substance 11A 1st capture molecule 11B Second capture molecule 11C Third capture molecule 11D 4th capture molecule 11b Secondary capture molecule 12 Base material 13 Target substance 13A 1st target substance 13B Second target substance 14 Enzymes 15, 15A Enzyme-labeled second capture molecule 16 Enzyme-labeled fourth capture molecule 17 Enzyme-labeled secondary capture molecules 20 Sensor body 21 Evaluation solution 22 Container 23 Electrical Sensors 24 Substrate 25 Semiconductor layer 26 Source electrodes 27 Drain electrode 28 Insulating substrate 29 Bulkhead 30 Preparation liquid 31 Magnetic particles 32 Magnetic base material 33 wells
Claims
1. The sensor comprises a substrate with a target substance and a sensor body. The target substance-attached substrate comprises a substrate on which a first capture molecule is immobilized on at least a portion of its surface, a target substance bound to the first capture molecule, and an enzyme-labeled second capture molecule on which a second capture molecule and an enzyme are bound, with the second capture molecule bound to the target substance. The sensor body includes an evaluation solution containing a substrate modified by the enzyme, a container for containing the evaluation solution, and an electrical sensor provided to be in contact with the evaluation solution in order to sense the components in the evaluation solution. The substrate with the target substance is separated independently from the sensor body. The portion of the substrate with the target substance to which the first capture molecule is immobilized on the surface of the substrate is immersed in the evaluation liquid. The container is divided into multiple wells, each containing the evaluation solution in a volume of 1 pL or less, and is used as a detection device for target substances.
2. The sensor comprises a substrate with a target substance and a sensor body. The target substance-attached substrate comprises a substrate on which a first capture molecule is immobilized on at least a portion of its surface, a target substance bound to the first capture molecule, and an enzyme-labeled second capture molecule on which a second capture molecule and an enzyme are bound, with the second capture molecule bound to the target substance. The sensor body includes an evaluation solution containing a substrate modified by the enzyme, a container for containing the evaluation solution, and an electrical sensor provided to be in contact with the evaluation solution in order to sense the components in the evaluation solution. The container is divided into a plurality of wells, each containing the evaluation solution. The substrate with the target substance is separated independently from the sensor body. The portion of the substrate with the target substance to which the first capture molecule is immobilized on the surface of the substrate is immersed in the evaluation liquid. The end of the aforementioned substrate is divided into two or more parts. The first capture molecule is immobilized on at least one end of the substrate. The first capture molecule is not immobilized on at least one end of the substrate. A target substance detection device in which the ends of the substrate are each immersed in the evaluation liquid contained in separate wells.
3. The sensor comprises a substrate with a target substance and a sensor body. The target substance-attached substrate comprises a substrate on which a first capture molecule is immobilized on at least a portion of its surface, a target substance bound to the first capture molecule, and an enzyme-labeled second capture molecule on which a second capture molecule and an enzyme are bound, with the second capture molecule bound to the target substance. The sensor body includes an evaluation solution containing a substrate modified by the enzyme, a container for containing the evaluation solution, and an electrical sensor provided to be in contact with the evaluation solution in order to sense the components in the evaluation solution. The container is divided into a plurality of wells, each containing the evaluation solution. The substrate with the target substance is separated independently from the sensor body. The portion of the substrate with the target substance to which the first capture molecule is immobilized on the surface of the substrate is immersed in the evaluation liquid. The aforementioned target substance includes a first target substance and a second target substance. The end of the aforementioned substrate is divided into two or more parts. The first capture molecule is immobilized on at least one end of the substrate. A third capture molecule, which has a different target for binding than the first capture molecule, is immobilized on at least one end of the substrate. The first target substance is bound to the first capture molecule. The second target substance is bound to the third capture molecule. The target substance-attached substrate further comprises an enzyme-labeled fourth capture molecule in which a fourth capture molecule and an enzyme are bound, and the fourth capture molecule is bound to the second target substance. A target substance detection device in which the ends of the substrate are each immersed in the evaluation liquid contained in separate wells.
4. The sensor comprises a substrate with a target substance and a sensor body. The target substance-attached substrate comprises a substrate on which a first capture molecule is immobilized on at least a portion of its surface, a target substance bound to the first capture molecule, and an enzyme-labeled second capture molecule on which a second capture molecule and an enzyme are bound, with the second capture molecule bound to the target substance. The sensor body includes an evaluation solution containing a substrate modified by the enzyme, a container for containing the evaluation solution, and an electrical sensor provided to be in contact with the evaluation solution in order to sense the components in the evaluation solution. The substrate with the target substance is separated independently from the sensor body. The portion of the substrate with the target substance to which the first capture molecule is immobilized on the surface of the substrate is immersed in the evaluation liquid. A target substance detection device wherein the conductivity of the evaluation liquid before the substrate with the target substance is immersed in it is 1 mS / cm or less.
5. The sensor comprises a substrate with a target substance and a sensor body. The target substance-attached substrate comprises a substrate on which a first capture molecule is immobilized on at least a portion of its surface, a target substance bound to the first capture molecule, and an enzyme-labeled second capture molecule on which a second capture molecule and an enzyme are bound, with the second capture molecule bound to the target substance. The sensor body includes an evaluation solution containing a substrate modified by the enzyme, a container for containing the evaluation solution, and an electrical sensor provided to be in contact with the evaluation solution in order to sense the components in the evaluation solution. The substrate with the target substance is separated independently from the sensor body. The portion of the substrate with the target substance to which the first capture molecule is immobilized on the surface of the substrate is immersed in the evaluation liquid. The aforementioned electrical sensor is an FET sensor, A target substance detection device in which an ion-sensitive film is provided on the surface of the FET sensor.
6. A target substance detection device according to claim 2, 3, 4, or 5, wherein a portion of the substrate with the target substance protrudes from the evaluation liquid.
7. The target substance detection device according to claim 1, 2, 3, 4, or 5, wherein the substrate is urea and the enzyme is urease.
8. The target substance detection device according to claim 1, 2, 3, or 4, wherein the electrical sensor is one of an FET sensor, a conductivity sensor, an ion electrode, and an impedance sensor.
9. The target substance detection device according to claim 1, 2, 3, 4, or 5, wherein the electrical sensor is an FET sensor using graphene as a semiconductor channel.
10. The target substance detection device according to claim 2, 3, 4, or 5, wherein the substrate on which the first capture molecule is immobilized comprises magnetic particles on which the first capture molecule is immobilized and a magnetic substrate that holds the magnetic particles magnetically.
11. A step of preparing a target substance-attached substrate comprising: a substrate on which a first capture molecule is immobilized on at least a portion of its surface; a target substance bound to the first capture molecule; and an enzyme-labeled second capture molecule on which a second capture molecule and an enzyme are bound, with the second capture molecule bound to the target substance; A step of immersing the portion of the target substance-coated substrate where the first capture molecule is immobilized on the surface of the substrate into an evaluation solution containing the substrate modified by the enzyme, The process includes a step of sensing the components in the evaluation liquid using an electrical sensor provided in contact with the evaluation liquid, The evaluation liquid is contained in a container. A method for detecting a target substance, wherein the container is divided into a plurality of wells, each containing the evaluation solution in a volume of 1 pL or less.
12. A step of preparing a target substance-attached substrate comprising: a substrate on which a first capture molecule is immobilized on at least a portion of its surface; a target substance bound to the first capture molecule; and an enzyme-labeled second capture molecule on which a second capture molecule and an enzyme are bound, with the second capture molecule bound to the target substance; A step of immersing the portion of the target substance-coated substrate where the first capture molecule is immobilized on the surface of the substrate into an evaluation solution containing the substrate modified by the enzyme, The process includes a step of sensing the components in the evaluation liquid using an electrical sensor provided in contact with the evaluation liquid, The evaluation liquid is contained in a container. The container is divided into a plurality of wells, each containing the evaluation solution. The end of the aforementioned substrate is divided into two or more parts. The first capture molecule is immobilized on at least one end of the substrate. The first capture molecule is not immobilized on at least one end of the substrate. A method for detecting a target substance, wherein the ends of the substrate are immersed in the evaluation solution contained in separate wells.
13. A step of preparing a target substance-attached substrate comprising: a substrate on which a first capture molecule is immobilized on at least a portion of its surface; a target substance bound to the first capture molecule; and an enzyme-labeled second capture molecule on which a second capture molecule and an enzyme are bound, with the second capture molecule bound to the target substance; A step of immersing the portion of the target substance-coated substrate where the first capture molecule is immobilized on the surface of the substrate into an evaluation solution containing the substrate modified by the enzyme, The process includes a step of sensing the components in the evaluation liquid using an electrical sensor provided in contact with the evaluation liquid, The evaluation liquid is contained in a container. The container is divided into a plurality of wells, each containing the evaluation solution. The aforementioned target substance includes a first target substance and a second target substance. The end of the aforementioned substrate is divided into two or more parts. The first capture molecule is immobilized on at least one end of the substrate. A third capture molecule, which has a different target for binding than the first capture molecule, is immobilized on at least one end of the substrate. The first target substance is bound to the first capture molecule. The second target substance is bound to the third capture molecule. The target substance-attached substrate further comprises an enzyme-labeled fourth capture molecule in which a fourth capture molecule and an enzyme are bound, and the fourth capture molecule is bound to the second target substance. A method for detecting a target substance, wherein the ends of the substrate are immersed in the evaluation solution contained in separate wells.
14. The method for detecting a target substance according to claim 12 or 13, wherein the target substance-coated substrate is immersed in the evaluation liquid such that a portion of the target substance-coated substrate protrudes from the evaluation liquid.
15. The method for detecting a target substance according to claim 11, 12, or 13, wherein the target substance-attached substrate is prepared by immersing the substrate, on which the first capture molecule is immobilized on at least a portion of its surface, in a preparation solution containing the enzyme-labeled second capture molecule and the target substance.
16. The method for detecting a target substance according to claim 11, 12, or 13, wherein the target substance-attached substrate is prepared by immersing the substrate, on which the first capture molecule is immobilized on at least a portion of the surface and on which the target substance is bound to the first capture molecule, in a preparation solution containing the enzyme-labeled second capture molecule.
17. The method for detecting a target substance according to claim 12 or 13, wherein the substrate on which the first capture molecule is immobilized comprises magnetic particles on which the first capture molecule is immobilized and a magnetic substrate that holds the magnetic particles by magnetism.
18. The method for detecting a target substance according to claim 11, 12, or 13, wherein the electrical sensor is one of an FET sensor, a conductivity sensor, an ion electrode, and an impedance sensor.