JP2008082988A - Detection method using multistage amplification - Google Patents

Abstract

An object of the present invention is to improve the detection sensitivity of a sensor using the electrical characteristics of a field effect transistor.
A step of reacting a primary binding molecule immobilized on a field effect transistor with a detection target contained in a sample; a step of reacting a secondary binding molecule specifically reacting with the detection target. A method for detecting an object to be detected, comprising the step of measuring an electric signal of a field effect transistor after reacting the secondary binding molecule. The secondary binding molecule is preferably labeled with a substance that amplifies an electrical signal.
[Selection] Figure 23

Description

  The present invention relates to a detection method using multistage amplification. More specifically, the present invention relates to a method for detecting a detection target in a sample by amplifying an electric signal of a field effect transistor as a detection unit in multiple stages.

  A method of detecting a detection target using a broadly defined antigen-antibody reaction is referred to as “immunological detection method”, “immunoassay”, or the like. One “immunological detection method” is an enzyme immunoassay (ELISA).

  The sandwich method, which is one of enzyme immunoassays, generally involves reacting and binding an antibody (capture antibody) bound to a solid phase such as a microtiter well and the detection target; The detection target is reacted with a detection antibody labeled with an enzyme; the detection target is detected by measuring the activity of an enzyme that labels the detection antibody. In general, the sandwich method has higher detection sensitivity and higher specificity than a competitive method using only a capture antibody.

  On the other hand, a field effect transistor (hereinafter also referred to as “FET”) has three terminals of a source electrode, a drain electrode, and a gate electrode, and a current flowing through a channel connected to the source electrode and the drain electrode is applied to the gate electrode. It is a semiconductor element controlled by an electric field generated by a voltage. There is also known a carbon nanotube field effect transistor (hereinafter also referred to as “CNT-FET”) in which a channel is formed of an ultrafine fiber body, for example, a carbon nanotube (hereinafter also referred to as “CNT”).

An example of a CNT-FET is shown in FIGS. 1A and 1B (see, for example, Non-Patent Document 1).
In the CNT-FET shown in FIG. 1A, a source electrode 3 and a drain electrode 4, and a channel connecting these electrodes are disposed on an insulating film 1 formed on a first surface of a substrate 2, A gate electrode 5 electrically connected to the silicon substrate 2 is disposed on the surface. Such an FET may be referred to as a back gate type field effect transistor (hereinafter also referred to as “back gate type FET”) based on the arrangement of the gate electrode.
In the FET shown in FIG. 1B, a source electrode 3, a drain electrode 4, and a gate electrode 5 are disposed on the insulating film 1 formed on the first surface of the substrate 2. Such an FET may be referred to as a side gate type field effect transistor (hereinafter also referred to as “side gate type FET”) based on the arrangement of the gate electrode.

In addition, development of sensors utilizing the electrical characteristics of CNT-FETs is underway (see, for example, Patent Document 1). These sensors make use of the fact that the electrical characteristics of CNTs serving as channels change depending on the state change of the molecular recognition site fixed to the CNT. For example, the reaction between the molecular recognition site and the substance to be detected , A current (hereinafter referred to as “source-drain current”) or a voltage (hereinafter referred to as “source-drain voltage”) between the source electrode and the drain electrode of the CNT-FET through a change in electrical characteristics of CNT induced by reaction. ) Is detected as a change.
International Publication No. 2004/104568 Pamphlet Kazuhiko Matsumoto, “Application of carbon nanotube SET / FET sensor”, IEICE Electronic Materials, Vol.EFM-03, No.35-44, 2003.12.19, p.47-50.

  The present inventor has studied to improve the sensitivity of the sensor using the electrical characteristics of the FET. As a result, like the sandwich method in ELISA, after reacting the detection target to the molecular recognition site (eg, capture antibody) immobilized on the FET, further reacting the binding molecule that reacts specifically with the detection target. As a result, it was found that the change in the electrical characteristics of the FET is amplified. The present invention has been made based on this finding.

  Furthermore, we investigated increasing the detection sensitivity and increasing the degree of structural freedom as a sensor by making the FET a specific structure. In the conventional FET, in order to control the source-drain current, it is necessary to dispose a gate electrode for controlling the electrical characteristics of the channel in the vicinity of the channel.

  That is, in the conventional back gate type FET, the substrate is made to act as a back gate electrode, so that the gate electrode is brought close to the channel through only the insulating film formed on the substrate. Therefore, it has been considered that the gate electrode needs to be in electrical contact with the substrate. That is, the gate electrode is placed in direct electrical contact with the electrically conductive substrate, and the electric field change in the vicinity of the channel due to the potential change of the gate electrode as much as possible, that is, the effect on the source-drain current or the source-drain voltage. Was thought to be necessary.

  Further, in the conventional side gate type FET, since the source-drain current is controlled by the gate electrode, it is considered necessary to arrange the gate electrode close to the channel. That is, the gate electrode disposed on the same surface as the substrate surface on which the source electrode, the drain electrode, and the channel are disposed is brought close to the channel to the nanometer level, and as much as possible to the source-drain current or the source-drain voltage. It was thought necessary to enhance the action.

  In the FET in which the source electrode, the drain electrode, and the channel are formed on the insulating film formed on the support substrate, the present inventor “disposes the gate electrode so that polarization is caused by the movement of free electrons in the support substrate”. We studied the development of FETs based on the new principle (source-drain current control principle).

And while this inventor examined the improvement of the performance of FET, and the application to the biosensor of FET, the gate electrode of FET was arrange | positioned at the back surface of the board | substrate with which the source electrode, the drain electrode, and the channel were arrange | positioned. In some cases, it was found that the source-drain current can be controlled even if an insulating film is formed on the back surface of the substrate.
Furthermore, the present inventor has shown that when the gate electrode of the FET is disposed on the same surface as the substrate surface on which the source electrode, the drain electrode and the channel are disposed, the FET is disposed at a certain distance from the source electrode, the drain electrode and the channel. However, it has been found that the source-drain current can be controlled.
Furthermore, the inventor is separated from the substrate on which the source electrode, the drain electrode and the channel are arranged, but the gate electrode arranged on a separate substrate which is electrically connected controls the source-drain current. I found that I can do it.

  Then, the capture binding molecule fixed to the FET based on these new control principles is reacted with the detection target, and the binding molecule that reacts specifically with the detection target is further reacted to react the FET electrical We have completed a new principle detection method that amplifies changes in characteristics.

That is, the present invention relates to the following detection method.
[1] A step of reacting a primary binding molecule fixed to a field-effect transistor and a detection target contained in a sample; a secondary binding molecule that specifically reacts with the detection target; A method of detecting an object to be detected, comprising: reacting with a substance; and measuring an electric signal of a field effect transistor after reacting the secondary binding molecule.
[2] A step of reacting a primary binding molecule immobilized on a field effect transistor and a detection target contained in a sample; a secondary binding molecule that specifically reacts with the detection target; A step of reacting a secondary binding molecule with a secondary binding molecule, and a step of reacting a higher order binding molecule as required. And a method of detecting an object to be detected, comprising the step of measuring an electric signal of a field effect transistor after reacting the highest-order binding molecule among the binding molecules.
[3] The method further includes the step of blocking the fixed surface of the primary binding molecule in the field effect transistor before reacting the primary binding molecule and the detection target. [1] or [2] The method described in 1.
[4] The method according to any one of [1] to [3], wherein any one of the second to highest binding molecules is labeled with a substance that amplifies an electric signal of a field effect transistor.
[5] The method according to [4], wherein the substance that amplifies the electric signal of the field effect transistor is an electric conductor, an electric insulator, or a semiconductor substance.
[6] The method according to [4], wherein the substance that amplifies the electric signal of the field effect transistor is an electric conductor or a semiconductor substance.
[7] The electric signal of the field effect transistor has an I-Vg characteristic indicating a relationship between a source-drain current and a gate voltage, or an IV characteristic indicating a relationship between the source-drain current and the source-drain voltage. The method according to any one of 1] to [6].

Furthermore, the present invention relates to a detection method using a field effect transistor having a specific structure shown below.
[8] The field-effect transistor flows through the substrate, a source electrode and a drain electrode disposed on the substrate, a channel including an ultrafine fiber body that electrically connects the source electrode and the drain electrode, and the channel Including a gate electrode for controlling the current;
The method according to any one of [1] to [7], wherein the primary binding molecule is fixed to the substrate, the ultrafine fiber body, or the gate electrode.
[9] The field effect transistor flows through the substrate, a source electrode and a drain electrode disposed on the substrate, a channel including an ultrafine fiber body that electrically connects the source electrode and the drain electrode, and the channel. Including a gate electrode for controlling the current;
The substrate has a support substrate made of a semiconductor or metal, a first insulating film formed on the first surface of the support substrate, and a second insulating film formed on the second surface of the support substrate. The source electrode, the drain electrode and the channel are disposed on the first insulating film; the gate electrode is disposed on the second insulating film; and the primary binding molecule is: The method according to any one of [1] to [7], wherein the method is fixed to the second insulating film or the gate electrode.
[10] The field-effect transistor flows through the substrate, a source electrode and a drain electrode disposed on the substrate, a channel including an ultrafine fiber body that electrically connects the source electrode and the drain electrode, and the channel. Including a gate electrode for controlling the current;
The substrate includes a support substrate made of a semiconductor or metal, and a first insulating film formed on a first surface of the support substrate; the source electrode, the drain electrode, the channel, and the gate electrode are Disposed on an insulating film, and a distance between the gate electrode and the ultrafine fiber body is 10 μm or more, and the primary binding molecules are fixed to the first insulating film or the gate electrode, The method according to any one of 1] to [7].
[11] The field-effect transistor flows through the substrate, a source electrode and a drain electrode disposed on the substrate, a channel including an ultrafine fiber body that electrically connects the source electrode and the drain electrode, and the channel A gate electrode for controlling current, and a second substrate electrically connected to the substrate;
The substrate includes a support substrate made of a semiconductor or metal, a first insulating film formed on a first surface of the support substrate, and the source electrode, the drain electrode, and the channel are on the first insulating film. The gate electrode is disposed on the first surface of the second substrate, and the primary binding molecule is fixed to the first surface of the second substrate or the gate electrode. The method according to any one of [1] to [7].
[12] The method according to any one of [1] to [11], wherein the ultrafine fiber body is a carbon nanotube.

  The detection method of the present invention further improves the sensitivity of the sensor using the electrical characteristics of the field effect transistor. Furthermore, expansion of dynamic range and improvement of specificity are expected.

  The detection method of the present invention includes 1) a step of reacting a primary binding molecule fixed to a field effect transistor and a detection target contained in a sample, and 2) a secondary reaction specifically reacting with the detection target. Reacting the binding molecule with the object to be detected, and 3) measuring the electric signal of the field effect transistor after reacting the secondary binding molecule. Furthermore, a step of blocking the bonding surface of the primary binding molecule in the field effect transistor may be included before the step 1).

1. Field Effect Transistor A field effect transistor to which a primary binding molecule is fixed is generally a substrate; a source electrode and a drain electrode disposed on the substrate; and electrically connecting the source electrode and the drain electrode. A channel; including a gate electrode for controlling a current flowing through the channel. Examples of the field effect transistor are also described in Patent Document 1 and Non-Patent Document 1 described above. Furthermore, the field effect transistor of a preferable aspect is demonstrated for every member below.

1-1. Substrate A source electrode, a drain electrode, and a channel are arranged on the substrate of the field effect transistor. Regarding the structure and material of the substrate, it is preferable that polarization (described later) is caused by movement of free electrons in the substrate by applying a voltage to a gate electrode (described later). Usually, the substrate has a support substrate made of a semiconductor or metal; and an insulating film that electrically insulates the support substrate from the source electrode, the drain electrode, and the channel. FIG. 2 shows an example of the substrate. FIG. 2A shows a substrate including a support substrate 400 and a first insulating film 402. FIG. 2B is a substrate including a support substrate 400, a first insulating film 402, and a second insulating film 404.

  The support substrate is preferably a semiconductor or a metal. The semiconductor is not particularly limited, and examples thereof include group 14 elements such as silicon and germanium, III-V compounds such as gallium arsenide and indium phosphide, and II-VI compounds such as zinc telluride. The metal is not particularly limited, and examples thereof include aluminum and nickel. Although the thickness of a support substrate is not specifically limited, It is preferable that it is 0.1-1.0 mm, and 0.3-0.5 mm is especially preferable.

The material of the first insulating film formed on the first surface of the support substrate (the surface on which the source electrode, the drain electrode, and the channel are disposed) is not particularly limited, but silicon oxide, silicon nitride, aluminum oxide, and titanium oxide And inorganic compounds such as acrylic resins and polyimides. A functional group such as a hydroxyl group, an amino group, or a carboxyl group may be introduced on the surface of the first insulating film.
Although the thickness of a 1st insulating film is not specifically limited, 10-1000 nm is preferable and 20-500 nm is especially preferable. If the first insulating film is too thin, a tunnel current may flow. On the other hand, if the first insulating film is too thick, it may be difficult to control the source-drain current using the gate electrode.

  A second insulating film may be formed on the second surface (back surface of the first surface) of the support substrate. The material of the second insulating film is the same as the material of the first insulating film. Similarly to the first insulating film, the thickness of the second insulating film is preferably 10 nm or more, and particularly preferably 20 nm or more, but is not particularly limited. On the other hand, in the case of a back gate type FET (described later) or an isolation gate type FET (described later), the thickness of the second insulating film is not particularly limited, but is preferably 1000 nm or less, like the first insulating film, 500 nm or less is particularly preferable.

  The surface (first surface or second surface) covered with the insulating film of the support substrate is preferably smooth. That is, the interface between the support substrate and the insulating film is preferably smooth. This is because if the surface of the support substrate is smooth, the reliability of the insulating film covering the surface is increased. The surface of the supporting substrate that is covered with the insulating film is not particularly limited, but is preferably polished. The smoothness of the surface of the support substrate can be confirmed by a surface roughness measuring machine or the like.

1-2. Channels The channel is not particularly limited as long as it is semiconducting, but preferably contains an ultrafine fiber body exhibiting semiconducting properties. The ultrafine fiber body is a fiber body having a diameter of several nanometers showing electrical conductivity. Examples of the ultrafine fiber body include CNT, DNA, conductive polymer, silicon fiber, silicon whisker, graphene and the like. Of these, CNT is preferred.

  The number of ultrafine fiber bodies contained in the channel may be one or more. The number of ultrafine fiber bodies can be confirmed by AFM. There may also be a gap between the ultrafine fiber body and the substrate.

  When the ultrafine fiber body is a carbon nanotube, it may be either single-walled CNT or multi-walled CNT, but single-walled CNT is preferable. Moreover, the defect may be introduce | transduced into CNT. “Defect” means a state in which the carbon five-membered ring or six-membered ring constituting the CNT is opened. It is assumed that the CNTs into which defects are introduced have a structure that is barely connected, but the actual structure is not clear. The method for introducing defects into the CNT is not particularly limited, but for example, the CNT may be annealed.

  The ultrafine fiber body may be protected by an insulating protective film in order to prevent damage. By covering the ultrafine fiber body with an insulating protective film, the entire FET can be ultrasonically cleaned or cleaned using a strong acid or a strong base. Furthermore, since the damage to the ultrafine fiber body is prevented by providing the insulating protective film, the life of the FET can be significantly extended.

  The insulating protective film is, for example, a film formed by an insulating adhesive or a passivation film. When the insulating protective film is a silicon oxide film, the primary binding molecules can be easily fixed to the insulating protective film.

1-3. About Source Electrode and Drain Electrode The source electrode and the drain electrode are disposed on the first insulating film of the substrate. The material of the source electrode and the drain electrode is, for example, a metal such as gold, platinum, or titanium. The source electrode and the drain electrode may have a multilayer structure made of two or more kinds of metals. For example, a gold layer may be superimposed on a titanium layer. The source electrode and the drain electrode are formed by evaporating these metals on the first insulating film. When depositing metal, it is preferable to transfer the pattern using lithography.

  Although the space | interval of a source electrode and a drain electrode is not specifically limited, Usually, it is about 2-10 micrometers. This interval may be further reduced in order to facilitate the connection between the electrodes by the ultrafine fiber body.

1-4. About the gate electrode It is preferable that the gate electrode included in the FET causes a polarization due to the movement of free electrons to the substrate on which the source electrode and the drain electrode are arranged by applying a voltage. “Polarization due to movement of free electrons” means that a region biased toward positive charges and a region biased toward negative charges are formed in the substrate as free electrons move in the substrate. In the case of a substrate made of a semiconductor or metal and a substrate made of an insulating film, polarization due to the movement of free electrons occurs in the support substrate having electrical conductivity. Whether or not the substrate is polarized can be confirmed by measuring a potential difference between both surfaces of the substrate.

  The size of the gate electrode is not particularly limited, and may be determined according to the size of the ultrafine fiber element (made of the ultrafine fiber body serving as the source electrode, the drain electrode, and the channel). If the size of the gate electrode is too small for the ultrafine fiber element, it may be difficult for the gate electrode to control the source-drain current. For example, when the distance between the source electrode and the drain electrode is 2 to 10 μm, the size of the gate electrode may be about 0.1 mm × 0.1 mm or more.

  The gate electrode arranged to polarize the substrate is classified into (A) a back gate electrode, (B) a side gate electrode, and (C) an isolation gate electrode.

(A) About Back Gate Electrode The back gate electrode is disposed on the second insulating film of the substrate. Since it is disposed on the back surface of the substrate with respect to the source electrode, the drain electrode, and the channel, it is referred to as a back gate electrode. The back gate electrode may be disposed in direct contact with the second insulating film, or may be disposed physically separated from the second insulating film.

  The back gate electrode may be disposed only on a part of the second insulating film or may be disposed on the entire surface of the second insulating film. If the gate electrode is provided on the entire second surface of the substrate, the primary binding molecules can be fixed on the entire surface of the second insulating film.

In the conventional back gate type FET, in order to control the source-drain current by the back gate electrode, the back gate electrode is arranged in direct contact with a supporting substrate (made of a semiconductor or metal) to obtain an interaction. It was.
On the other hand, the present inventor has found that it is not always necessary to directly contact the back gate electrode and the support substrate. That is, it was found that the source-drain current can be controlled even when an insulating film is provided between the back gate electrode and the support substrate. This is because when a voltage is applied to the gate electrode, polarization occurs due to the presence of free electrons in the support substrate (made of a semiconductor or metal), and the source-drain current is controlled by the polarization. Conceivable. Polarization due to the movement of free electrons includes factors due to capacitive coupling, but other factors are not excluded.

  An example of an FET having a back gate electrode is shown in FIG.

(B) Side gate electrode The side gate electrode is disposed on the first insulating film of the substrate. Since the source electrode, the drain electrode, and the channel are disposed on the same surface, the side electrode is referred to as a side gate electrode. The side gate electrode may be disposed in direct contact with the first insulating film, or may be disposed physically separated from the first insulating film.

  The distance between the side gate electrode disposed on the same surface of the substrate and the ultrafine fiber body is not particularly limited. However, in the FET of the present invention, it can be 10 μm or more, further 100 μm or more, and further 1 mm or more. The upper limit is not particularly limited, but is several cm or less. The “interval between the gate electrode and the ultrafine fiber body” means the shortest distance between each other.

  Conventional side-gate FETs are thought to require direct interaction between the side gate electrode and the source, drain, and channel in order to control the source-drain current with the gate electrode. . Therefore, in the conventional side gate type FET, the distance between the side gate electrode and the channel is made as short as possible (at most about 1 μm).

  On the other hand, the present inventor has found that the side gate electrode does not necessarily need to be close to the source electrode, the drain electrode, and the channel. When a side gate electrode and a source electrode, a drain electrode, and a channel are provided on the same insulating film, when a voltage is applied to the side gate electrode, a supporting substrate (made of a semiconductor or a metal is formed under the insulating film. ), Polarization due to the presence of free electrons in the support substrate occurs, and the source-drain current is controlled by the polarization. Polarization includes factors due to capacitive coupling, but does not exclude other factors.

  In the side gate type FET, the primary binding molecule is fixed to the side gate electrode, and the sample solution may be dropped. In the side gate type FET described here, the distance between the side gate electrode, the source electrode, the drain electrode, and the channel can be increased, so that the contamination of the ultrafine fiber contained in the channel with the sample solution can be prevented.

  An example of an FET having a side gate electrode is shown in FIG.

(C) Separation gate electrode The separation gate electrode is separated from the substrate on which the source electrode, the drain electrode and the channel are arranged, but is arranged on a second substrate which is electrically connected. The second substrate may be a substrate having a support substrate made of a semiconductor or metal and an insulating film formed on at least one surface of the support substrate, or a substrate made of an insulator, preferably the former substrate. is there.

  The second substrate on which the separation gate electrode is disposed is separated from the substrate on which the source electrode, the drain electrode, and the channel are disposed. The distance between the substrate on which the source electrode, the drain electrode and the channel are disposed and the second substrate on which the gate electrode is disposed is not particularly limited, and is 3 mm or more, further 10 mm or more, and further 15 mm or more. Can be done, and more.

  As described above, the second substrate on which the separation gate electrode is disposed is electrically connected to the substrate on which the source electrode, the drain electrode, and the channel are disposed. For example, (a) the substrate and the second substrate are placed on the same conductive substrate, or (b) the substrate and the second substrate are different from each other. This means that each conductive substrate is placed on a conductive substrate and connected by a conductive member. An example of the embodiment (a) is shown in FIG. 5, and an example of the embodiment (b) is shown in FIG.

  The conductive substrate is not particularly limited, and may be a glass substrate on which a gold thin film is deposited or a substrate made of a material such as brass. The conductive member is not particularly limited, and is, for example, a conductive wire such as a copper wire.

  In the isolation gate type FET, since the substrate on which the source electrode, the drain electrode, and the channel are arranged can be separated from the second substrate on which the gate electrode is arranged, the degree of freedom in structure is high. Therefore, a detection device using a separation gate type FET can be a highly practical device.

2. Regarding primary binding molecule 2-1. Types of primary binding molecules The primary binding molecules may be any molecules that specifically react with the target to be detected. Examples of primary binding molecules include antigens or antibodies or aptamers that specifically bind to the antigen; saccharides or lectins that bind to the saccharides; or DNA or DNA complementary to the DNA, etc. Can be used. The primary binding molecule is immobilized on the FET and further reacts with and binds to the detection target. It is preferable that the detection object is provided by being dissolved or dispersed in, for example, a solution.

  The detection target in the detection method of the present invention is not particularly limited as long as it enables the detection method of the present invention. For example, a component detected using an antigen-antibody reaction or a component detected using an enzyme reaction In addition, examples include components detected by other specific reactions, and components detected using an antigen-antibody reaction are preferable.

  Examples of components detected by antigen-antibody reaction include IgG, IgM, IgA, IgE, apoprotein AI, apoprotein AII, apoprotein B, apoprotein E, rheumatoid factor, D-dimer, oxidized LDL, glycated LDL, glyco Albumin, T3, T4, drugs (such as anti-tencan drugs), C-reactive protein (CRP), cytokines, α-fetoprotein (AFP), DUPAN-2, carcinoembryonic antigen (CEA), CA19-9, CA -125, PIVKA-II (Protein induced by vitamin K absence-II), parathyroid hormone (PTH), human chorionic gonadotropin (hCG), thyroid stimulating hormone (TSH), insulin, C-peptide, estrogen, anti-GAD antibody , Pepsinogen, influenza A antigen, influenza B antigen, anti-coronavirus , HBV antigen, anti-HBV antibody, HCV antigen, anti-HCV antibody, HTLV-I antigen, anti-HTLV-I antibody, HIV antibody, tuberculosis antibody, mycoplasma antibody, glycoalbumin, hemoglobin A1c, adiponectin, cystatin C, atrial natriuretic Peptides (ANP), brain natriuretic peptide (BNP), troponin T, troponin I, creatinine kinase-MB (CK-MB), myoglobin, H-FABP (human heart-derived fatty acid binding protein), DON, NIV, T2, etc. Mold toxins, bisphenol A, nonylphenol, dibutyl phthalate, polychlorinated biphenyls (PCBs), dioxins, p, p'-dichlorodiphenyltrichloroethane, endocrine disruptors such as tributyltin, fungi such as E. coli, eggs, milk , Wheat, buckwheat, Food allergens and Konahyoudani and allergens mites such as Toyahyoudani flower production, etc., an antiallergic agent antibodies, and the like.

  Examples of biological components detected using an enzymatic reaction include glucose, 1,5-anhydroglucitol, hemoglobin A1c, glycoalbumin, fucose, urea, uric acid, ammonia, creatinine, total cholesterol, free cholesterol, high density Cholesterol in lipoprotein (HDL-C), cholesterol in low density lipoprotein (LDL-C), cholesterol in very low density lipoprotein (VLDL-C), cholesterol in remnant-like lipoprotein (RLP-C) , Triglyceride, phospholipid, total protein, albumin, globulin, bilirubin, bile acid, sialic acid, lactic acid, pyruvic acid, free fatty acid, ceruloplasmin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), Creatine phosphokinase (CPK), phosphokinase (PK), amylase, lipase, cholinesterase, γ-glutamyl transpeptidase, leucine aminopeptidase, L-lactate dehydrogenase (LDH), aldolase, alkaline phosphatase, acid phosphatase, N-acetylglucosaminidase, Examples include guanase and monoamine oxidase.

  Examples of other components detected by specific binding include methods using nucleic acids, lectins and the like. For example, DNA or RNA encoding cancer genes such as ras, cancer suppressor genes such as p53, peptide nucleic acids, aptamers, Examples include glycoproteins.

  Examples of the sample containing the detection target include biological samples such as whole blood, plasma, serum, spinal fluid, saliva, amniotic fluid, urine, sweat, pancreatic juice, tears, stool, and food and soil-derived samples. Can be mentioned. These samples can also be subjected to the method of the present invention by processing. Examples of the processing method include dilution, extraction and concentration with an aqueous medium or an organic solvent.

2-2. About the site | part of the field effect transistor to which a primary bond molecule is fixed The primary bond molecule should just be fixed to FET. The fixing site is not particularly limited, and includes a substrate, a gate electrode, and an ultrafine fiber body (including a film that protects the ultrafine fiber body) included in a channel. Hereinafter, an example in which the primary binding molecule is fixed to the FET will be described with reference to the drawings.

  7 to 11 show examples in which the primary binding molecule is fixed to the back gate type FET.

  7 and 8 show an example in which a primary binding molecule is immobilized on a channel. In FIG. 7, the primary binding molecule is directly immobilized on the channel. On the other hand, in FIG. 8, since the primary binding molecules are fixed to the channel via the insulating protective film, the sample solution does not come into contact with the channel, and noise is reduced.

  FIG. 9 shows an example in which primary binding molecules are fixed to the second insulating film of the substrate. Since the second insulating film can be cleaned without damaging the ultrafine fiber body, it can be reused. In addition, primary binding molecules may be fixed to the entire second insulating film, and therefore a relatively large number of primary binding molecules can be fixed.

  In FIG. 9A, the primary binding molecules are fixed to the entire surface of the second insulating film, which is useful when the back gate electrode is not fixed to the second insulating film. On the other hand, FIGS. 9B and 9C are useful when the primary binding molecules are fixed to a part of the second insulating film and the back gate electrode is fixed to the second insulating film. In FIG. 9D, a plurality of back gate electrodes are arranged on the second insulating film, and a plurality of types of primary binding molecules are fixed to the second insulating film.

FIG. 10 shows an example in which a concave portion is formed on the second surface of the substrate, and the primary binding molecules are fixed to the second insulating film located at the bottom of the concave portion. Although the material of the side wall of a recessed part is not specifically limited, For example, it is a silicon oxide. In this example, a certain amount of sample solution can be provided by adjusting the volume of the recess. In addition, the added sample solution is not easily dissipated and can be stably held at the site where the primary binding molecules are fixed.
10A and 10B are diagrams illustrating an example in which the back gate electrode functions as a lid of a recess. FIG. 10C is a diagram showing an example in which the back gate electrode is arranged on the side wall of the recess. FIG. 10D is a diagram illustrating an example in which the back gate electrode is disposed on the side wall of the recess. FIG. 10E is a diagram showing an example in which the back gate electrode is arranged on the second insulating film outside the recess.

FIG. 11 shows an example in which the primary binding molecule is fixed to the gate electrode. In this example, since the second surface of the substrate can be cleaned without damaging the ultrafine fiber body, it can be easily reused.
FIG. 11A is a diagram showing an example in which primary binding molecules are fixed to a back gate electrode when one back gate electrode is arranged. FIG. 11B is a diagram illustrating an example in which a plurality of types of primary binding molecules are fixed to different back gate electrodes when a plurality of back gate electrodes are arranged.

  12 to 16 show examples in which primary binding molecules are fixed to side-gate FETs.

FIG. 12 shows an example in which the primary binding molecule is fixed to the ultrafine fiber body. In this example, the primary binding molecule is directly fixed to the ultrafine fiber body which is a channel. FIG. 13 shows an example in which the primary binding molecules are fixed to an insulating protective film that protects the ultrafine fiber body. In this example, the primary biopolymer can be placed physically close to the ultrafine fiber body without the sample solution being in direct contact with the ultrafine fiber body and the electrode, and noise is reduced by the protective film. Therefore, a highly sensitive sensor can be provided.
FIG. 13A shows an example in which the primary binding molecule is fixed to an insulating protective film that protects the ultrafine fiber element. FIG. 13B is a diagram showing an example in which the primary binding molecules are fixed to an insulating protective film that protects the ultrafine fiber element and the gate electrode.

  FIG. 14 shows an example in which the primary binding molecules are fixed to the first insulating film when the side gate electrode is arranged so as to be in contact with the first insulating film. The sample solution may or may not contact the side gate electrode (FIG. 14A) (FIG. 14B).

FIG. 15 shows an example in which a recess is formed on the second surface of the substrate and the primary binding molecules are fixed to the second insulating film located at the bottom of the recess. Although the material of the side wall of a recessed part is not specifically limited, For example, it is a silicon oxide. In this example, the sample solution can be accurately positioned at a site where the primary binding molecule is fixed (that is, in the recess).
FIG. 16 shows an example in which the primary binding molecule is fixed to the gate electrode.

  17 to 22 show examples in which the primary binding molecule is fixed to the separation gate type FET.

FIG. 17 is a diagram illustrating an example in which the primary binding molecules are fixed to the insulating film when the separation gate electrode is arranged without being in contact with the insulating film.
FIG. 18 is a diagram showing an example in which the primary binding molecules are fixed to the insulating film when the separation gate electrode is arranged so as to be in contact with the insulating film. The sample solution may or may not be in contact with the separation gate electrode (FIG. 18A) (FIG. 18B). FIG. 18C is a diagram illustrating an example in which a plurality of types of primary binding molecules are respectively fixed to an insulating film when a plurality of separation gate electrodes are arranged.

  FIG. 19 is a diagram showing an example in which the primary binding molecule is fixed to the gate electrode. FIG. 19A is a diagram showing an example in which primary binding molecules are fixed to a separation gate electrode when one separation gate electrode is arranged. FIG. 19B is a diagram showing an example in which a plurality of types of primary binding molecules are fixed to different separation gate electrodes when a plurality of separation gate electrodes are arranged.

FIG. 20 is a diagram illustrating an example in which when there are a plurality of gate element portions (including a gate electrode and a second substrate), a plurality of types of primary binding molecules are fixed to different separation gate electrodes.
FIG. 21 shows a case where a plurality of types of primary fiber elements and gate elements are arranged so that a conductive substrate is sandwiched between them and a plurality of separation gate electrodes are arranged on the gate elements. It is a figure which shows the example which each fixed the binding molecule to the insulating film. In this example, the gate element portion can be easily detached from the ultrafine fiber element portion. Therefore, it is possible to replace a plurality of gate element portions with respect to one ultrafine fiber element portion.
FIG. 22 shows a case in which a plurality of types of primary fibers are obtained when the ultrafine fiber element portion and the gate element portion are electrically connected by a conductive member and a plurality of separation gate electrodes are arranged on the gate element portion. It is a figure which shows the example which each fixed the binding molecule to the insulating film.

  As described above, it is possible to fix a plurality of types of primary binding molecules to one FET. However, when the detection target is an influenza virus, a plurality of types of primary binding molecules are converted to A A combination of an antibody against type B virus and an antibody against type B virus.

2-3. Method for immobilizing primary binding molecule to FET The method for immobilizing the primary binding molecule to FET is not particularly limited, and for example, there is a method of immobilizing via a bivalent crosslinking reagent. The bivalent crosslinking reagent has two functional groups, one functional group is used for bonding to the FET, and another functional group is used for bonding to the primary binding molecule. The bivalent crosslinking reagent has, for example, two functional groups and a hydrophilic polymer chain (such as polyethylene glycol chain) or a hydrophobic chain (such as alkyl chain) that connects the functional groups. Examples of the two functional groups include a combination of a functional group that reacts with an amino group and a functional group that reacts with a thiol group.

For example, when the primary binding molecule is fixed to the insulating film via a bivalent crosslinking reagent, the following procedure may be used.
After reacting the primary binding molecule and the bivalent crosslinking reagent, the unreacted bivalent crosslinking reagent is removed by dialysis or the like to obtain a primary binding molecule-bivalent crosslinking reagent complex. An insulating film of the substrate treated with the silanized coupling agent and the primary binding molecule-bivalent crosslinking reagent complex are reacted and fixed. Alternatively, the substrate insulating film treated with the silanized coupling agent is reacted with the divalent crosslinking reagent, and the primary binding molecules are further reacted to be fixed.

  The method for immobilizing the primary binding molecule is not limited to a method using a bivalent crosslinking reagent, and for example, a method of immobilizing a histag fusion molecule via an NTA-Ni complex or the like can also be used.

3. Regarding secondary binding molecules and higher order binding molecules 3-1. Regarding the secondary binding molecule The detection method of the present invention is characterized in that after the primary binding molecule and the detection target are reacted, the secondary binding molecule is further reacted. The secondary binding molecule may be any molecule that specifically reacts with the detection target, and the type thereof is the same as the primary binding molecule, such as an antigen or an antibody or aptamer that specifically binds to the antigen; Examples include lectins that bind to saccharides; or DNA or DNA complementary to the DNA, and one of them can be used. The secondary binding molecule is preferably a different type of molecule from the primary binding molecule, but may be the same type. FIG. 23 shows a schematic diagram of the state in which the secondary binding molecules have reacted.

  In the detection method of the present invention, it is preferable to provide the secondary binding molecule dissolved or dispersed in the solution to the detection target bound to the primary binding molecule. The solvent of the solution is not particularly limited, and may be water or a buffer solution such as PBS (Phosphate buffered saline).

  By reacting the secondary binding molecule, the change in charge becomes larger than the state in which only the detection target object reacts with the primary binding molecule, and the electric signal of the FET can be amplified.

3-2. Third-order or higher binding molecule The detection method of the present invention may further comprise a step of reacting a second-order binding molecule and then a third-order or higher binding molecule. It is preferable that the tertiary binding molecule specifically reacts with the secondary binding molecule, the fourth binding molecule with the tertiary binding molecule, and the (X + 1) th binding molecule with the Xth binding molecule.

3-3. As described above, the detection method of the present invention uses a secondary binding molecule and, if necessary, a tertiary or higher binding molecule, any of which may be labeled. However, it is preferable that the highest order binding molecule is labeled. FIG. 24 shows a schematic diagram of a state in which a labeled secondary binding molecule is reacted.

  The labeling substance is preferably a substance that amplifies the electric signal of the FET when a binding molecule labeled with the substance reacts. Examples of the substance that amplifies the electric signal of the FET include an electric conductor substance such as a metal, a semiconductor substance such as a carbon nanotube, or an insulator substance, preferably an electric conductor substance and a semiconductor substance.

4). Regarding measurement of electric signal The detection method of the present invention includes a step of measuring an electric signal of an FET after reacting a secondary binding molecule or, if necessary, a tertiary binding molecule. Examples of electrical signals include “the relationship between source-drain current and gate voltage (also referred to as“ I-Vg characteristics ”)” when the source-drain voltage is constant, or “source” when the gate voltage is constant. -The relationship between the drain current and the source-drain voltage (also referred to as “IV characteristics”) ”.

  The electrical signal of the FET can be measured using a normal semiconductor parameter analyzer. What is necessary is just to observe the change of the electrical property in a dynamic range. If the calibration curve is obtained by examining the relationship between the change amount and the concentration of the detection object in advance, the concentration of the detection object can also be measured from the observation result.

5. About blocking of the binding surface of the primary binding molecule in the field effect transistor The detection method of the present invention may include a step of blocking the binding surface of the primary binding molecule in the field effect transistor. The blocking is preferably performed before the primary binding molecule reacts with the detection target. Blocking can be performed using a blocking agent. Examples of the blocking agent include proteins such as bovine serum albumin (BSA) and casein, and synthetic polymers such as polyethylene glycol. By blocking, binding of a substance other than the detection target in the sample to the binding surface of the primary binding molecule in the field effect transistor can be suppressed.

6). About washing | cleaning operation The detection method of this invention may include the step which wash | cleans a reaction material before the step which measures an electrical signal. Before the step of measuring the electric signal, for example, after the reaction between the primary binding molecule and the detection target, after the reaction between the detection target and the secondary binding molecule, or a higher-order binding molecule. It means after reacting. The washing operation may be performed using a washing solution, and the washing solution is preferably water, a buffer solution such as PBS or Good Buffer, and a surfactant, a synthetic polymer, a preservative, and the like are appropriately added. The detection accuracy can be improved by the washing.

(1) Fabrication of sensor The field effect transistor shown in FIG. 9A was prepared. The substrate is composed of a support substrate made of silicon and having a thickness of 550 μm; and a first and second insulating films made of silicon oxide and having a thickness of 300 nm. The area of the substrate is 1 cm ² (1 cm × 1 cm). The channel is a single-walled carbon nanotube which is an ultrafine fiber body and is usually connected by several single-walled carbon nanotubes, which is confirmed by AFM. The gate electrode is brought into contact with the front surface of the second insulating film. The distance between the source electrode and the drain electrode is 5 μm.

A commercially available anti-influenza virus type A nucleoprotein antibody (Fitzgerald) was added to 100 mmol / L phosphate buffer (pH 7.5) to a final concentration of 1 mg / mL, and the final concentration was 4.15 mmol. Sulfo-EMCS (manufactured by Dojindo Laboratories) was added so as to be / L. The obtained solution was mixed and reacted at 25 ° C. for 30 minutes.
After the reaction, the buffer of the antibody solution was replaced with a 100 mmol / L phosphate buffer (pH 6.0) containing 5 mmol / L EDTA (manufactured by Kanto Chemical Co., Inc.) by ultrafiltration.

To the second insulating film (silicon oxide film: 1 cm ² ) of the substrate, 2 mol / L NaOH (50 μL) was added and treated at 45 ° C. for 2 minutes. Thereafter, 3 μL of Sila Ace S810 (manufactured by Chisso Corporation), which is a silane coupling agent, was added and treated at 45 ° C. for 15 minutes and then at 200 ° C. for 30 minutes. Thereafter, 50 μL of 1 mmol / L sodium tetrahydroborate aqueous solution (manufactured by Wako Pure Chemical Industries, Ltd.) was added and treated for 15 minutes.

  20 μL of the above antibody solution was dropped on the second insulating film of the treated substrate, and reacted at 4 ° C. for 4 to 16 hours. The antibody solution after the reaction was removed, and 100 μL of PBS (pH 7.4) to which 1% BSA (manufactured by Sigma) was added was dropped and reacted at 25 ° C. for 10 minutes to produce the desired sensor.

(2) Preparation of influenza antigen As an influenza A virus serving as an antigen, commercially available purified virus (Fitzgerald) was diluted with 0.5% Nonidet-P40 (nonionic surfactant, manufactured by Fluka), PBS pH 7.4) containing 0.5% BSA and 0.1% NaN ₃ was diluted to a virus concentration of 10 μg / mL. The 10 μg / mL virus solution was approximately 10 ⁷ pfu / mL.

(3) Preparation of secondary binding molecule (secondary antibody) A commercially available antibody (manufactured by Fitzgerald) was used as the anti-influenza A virus type A nucleoprotein antibody serving as the secondary antibody. Secondary antibody solutions with four concentrations of 0.1 μg / 10 μL, 1 μg / 10 μL, and 10 μg / 10 μL were prepared with PBS (pH 7.4) containing 0.5% BSA and 0.1% NaN ₃ .

(4) Detection The influenza A antigen was detected by the following procedure using the produced sensor.
As shown in the following procedures, each solution (10 μL) was added to the substrate surface, and then reacted at 25 ° C. for 10 minutes. Then, it was washed 3 times with 50 μL of PBS (pH 7.4). The solution was removed with N ₂ gas. An I-Vg characteristic (relationship between source-drain current and gate voltage) at a gate voltage of −20 V to 20 V was measured with a semiconductor parameter analyzer (apparatus) to obtain an I-Vg curve.

Procedure 1: PBS containing 10 μL of 0.5% Nonidet-P40 and 0.5% BSA was added to the antibody binding surface of the substrate of the field effect transistor of the fabricated sensor. Thereby, the antibody binding surface was blocked.
Procedure 2: 10 μL of virus solution (10 ⁷ pfu / mL) was added. Thereby, the primary antibody as the primary binding molecule and the virus as the antigen were reacted and bound.
Procedure 3: 10 μL of secondary antibody solution (0.1 μg / 10 μL) was added.
Procedure 4: 10 μL of secondary antibody solution (1 μg / 10 μL) was added.
Procedure 5: 10 μL of secondary antibody solution (10 μg / 10 μL) was added.

  The I-Vg curves obtained after each procedure are shown in FIGS. The vertical axis represents the source-drain current I, and the horizontal axis represents the gate voltage Vg. FIG. 26 is a graph in which the vertical axis shown in FIG. 25 is enlarged.

  Furthermore, FIG. 27 shows the source-drain current when the gate voltage is −12 V in the I-Vg curve after each procedure. As shown, when the virus (antigen) was reacted (procedure 2), the source-drain current did not change much, but when the secondary antibody was reacted (procedures 3-5), the source-drain current was It turns out that it has changed greatly. It can also be seen that the amount of change increases as the concentration of the secondary antibody is increased.

On the other hand, FIG. 28 shows the gate voltage at the source-drain current −2.5 × 10 ⁻⁸ A of the I-Vg curve after each procedure. As shown, when the secondary antibody is reacted (procedures 3 to 5) with respect to the amount of change in the gate voltage when the virus (antigen) is reacted (procedure 2), it can be seen that the change further occurs. It can also be seen that the amount of change increases as the concentration of the secondary antibody is increased.

  From the above results, the secondary binding molecule that reacts specifically with the detection target was further reacted with the primary binding molecule fixed to the field effect transistor, compared with the case where the detection target was merely reacted. In the case, it was suggested that the electric signal change of the field effect transistor becomes larger (amplified). Therefore, it is considered that a larger amplification effect can be obtained if the secondary binding molecule is labeled with an appropriate substance.

  Since the signal of the FET biosensor is amplified by using the detection method of the present invention, highly sensitive detection is possible. Moreover, since a secondary binding molecule and a higher-order binding molecule are used if necessary, an improvement in specificity is expected. Further expansion of the dynamic range is expected. The detection method of the present invention can enable various clinical diagnoses by detecting a detection target in a sample.

FIG. 1A is a schematic diagram of a conventional back gate FET. FIG. 1B is a schematic diagram of a conventional side-gate FET. Reference numeral 1 denotes an insulating film, 2 denotes a substrate, 3 denotes a source electrode, 4 denotes a drain electrode, and 5 denotes a gate electrode. It is a figure which shows the example of the board | substrate of FET. Reference numeral 400 denotes a support substrate, 402 denotes a first insulating film, and 404 denotes a second insulating film. It is a figure which shows an example of a back gate type FET. 100 is a back gate type FET, 102 is a supporting substrate, 104 is a first insulating film, 106 is a second insulating film, 108 is a source electrode, 110 is a drain electrode, 112 is an ultrafine fiber body, and 114 is a gate electrode. Show. It is a figure which shows an example of side gate type FET. 150 is a side gate type FET, 102 is a supporting substrate, 104 is a first insulating film, 106 is a second insulating film, 108 is a source electrode, 110 is a drain electrode, 112 is an ultrafine fiber body, and 114 is a gate electrode. Show. It is a figure which shows an example of isolation gate type FET. 200 is an isolation gate type FET, 102 is a first support substrate, 104 is a first insulating film, 106 is a second insulating film, 108 is a source electrode, 110 is a drain electrode, 112 is an ultrafine fiber body, 202 is The second support substrate, 204 is a third insulating film, 206 is a fourth insulating film, 208 is a gate electrode, 210 is a conductive substrate, 212 is an ultrafine fiber element portion, and 214 is a gate element portion. It is a figure which shows an example of isolation gate type FET. 300 is an isolation gate type FET, 102 is a first support substrate, 104 is a first insulating film, 106 is a second insulating film, 108 is a source electrode, 110 is a drain electrode, 112 is an ultrafine fiber body, 202 is The second support substrate, 204 is the third insulating film, 206 is the fourth insulating film, 208 is the gate electrode, 212 is the ultrafine fiber element portion, 214 is the gate element portion, and 302 is the first conductive substrate. , 304 is a second conductive substrate, and 306 is a conductive member. It is a figure which shows the example which fixed the primary binding molecule to the ultrafine fiber body in back gate type FET. 100 is a back gate type FET, 102 is a supporting substrate, 104 is a first insulating film, 106 is a second insulating film, 108 is a source electrode, 110 is a drain electrode, 112 is an ultrafine fiber body, 114 is a gate electrode, Reference numeral 472 denotes a primary binding molecule, and 482 denotes a sample solution. It is a figure which shows the example which fixed the primary bond molecule | numerator to the insulating protective film in back gate type FET. 100 is a back gate type FET, 102 is a supporting substrate, 104 is a first insulating film, 106 is a second insulating film, 108 is a source electrode, 110 is a drain electrode, 112 is an ultrafine fiber body, 114 is a gate electrode, 472 is a primary binding molecule, 482 is a sample solution, and 640 is an insulating protective film. It is a figure which shows the example which fixed the primary bond molecule | numerator to the 2nd insulating film in back gate type FET. 510, 520 and 520a are back gate type FETs, 102 is a supporting substrate, 104 is a first insulating film, 106 is a second insulating film, 108 is a source electrode, 110 is a drain electrode, 112 is an ultrafine fiber body, 512 Reference numerals 522, 522a and 522b denote gate electrodes, 472, 472a and 472b denote primary binding molecules, and 490, 490a and 490b denote sample solutions. It is a figure which shows the other example which fixed the primary bond molecule | numerator to the 2nd insulating film in back gate type FET. 102 is a supporting substrate, 104 is a first insulating film, 106 is a second insulating film, 108 is a source electrode, 110 is a drain electrode, 112 is an ultrafine fiber body, 114 is a gate electrode, 116 is a recess side wall, 472 is The primary binding molecule, 482, represents the sample solution. In a back gate type FET, it is a figure showing an example which fixed a primary bond molecule to a gate electrode. 530 and 530a are back gate type FETs, 102 is a support substrate, 104 is a first insulating film, 106 is a second insulating film, 108 is a source electrode, 110 is a drain electrode, 112 is an ultrafine fiber body, 532 and 532a And 532b are gate electrodes, 472, 472a and 472b are primary binding molecules, and 490, 490a and 490b are sample solutions. It is a figure which shows the example which fixed the primary bond molecule | numerator to the ultrafine fiber body in side gate type FET. Reference numeral 102 denotes a supporting substrate, 104 denotes a first insulating film, 108 denotes a source electrode, 110 denotes a drain electrode, 112 denotes an ultrafine fiber body, 114 denotes a gate electrode, 472 denotes a primary binding molecule, and 490 denotes a sample solution. It is a figure which shows the example which fixed the primary bond molecule | numerator to the insulating protective film in side gate type FET. 102 is a supporting substrate, 104 is a first insulating film, 108 is a source electrode, 110 is a drain electrode, 112 is an ultrafine fiber body, 114 is a gate electrode, 472 is a primary binding molecule, 490 is a sample solution, 640 is a sample solution An insulating protective film is shown. It is a figure which shows the example which fixed the primary bond molecule | numerator to the 1st insulating film in side gate type FET. Reference numeral 102 denotes a supporting substrate, 104 denotes a first insulating film, 108 denotes a source electrode, 110 denotes a drain electrode, 112 denotes an ultrafine fiber body, 114 denotes a gate electrode, 472 denotes a primary binding molecule, and 490 denotes a sample solution. FIG. 6 is a diagram showing another example in which a primary binding molecule is fixed to a second insulating film in a side gate type FET. 102 is a supporting substrate, 104 is a first insulating film, 106 is a second insulating film, 108 is a source electrode, 110 is a drain electrode, 112 is an ultrafine fiber body, 114 is a gate electrode, 116 is a recess side wall, 472 is The primary binding molecule, 482, represents the sample solution. It is a figure which shows the example which fixed the primary bond molecule | numerator to the gate electrode in side gate type FET. Reference numeral 102 denotes a supporting substrate, 104 denotes a first insulating film, 108 denotes a source electrode, 110 denotes a drain electrode, 112 denotes an ultrafine fiber body, 114 denotes a gate electrode, 472 denotes a primary binding molecule, and 490 denotes a sample solution. It is a figure which shows the example which fixed the primary bond molecule | numerator to the insulating film of a gate element part in isolation | separation gate type FET. 600 is an isolation gate type FET, 102 is a first support substrate, 104 is a first insulating film, 106 is a second insulating film, 108 is a source electrode, 110 is a drain electrode, 112 is an ultrafine fiber body, 202 is Second support substrate, 204 is a third insulating film, 206 is a fourth insulating film, 472 is a primary binding molecule, 490 is a sample solution, 602 is a gate electrode, 210 is a conductive substrate, and 212 is ultrafine. A fiber element portion 214 is a gate element portion. FIG. 5 is a diagram showing another example in which a primary binding molecule is fixed to an insulating film of a gate element part in an isolated gate type FET. 610 and 610a are gate element portions, 202 is a second support substrate, 204 is a third insulating film, 206 is a fourth insulating film, 472, 472a and 472b are primary binding molecules, 490, 490a and 490b are Sample solutions 612, 612a and 612b represent gate electrodes. It is a figure which shows the example which fixed the primary bond molecule | numerator to the gate electrode of the gate element part in isolation | separation gate type FET. 620 and 620a are gate element portions, 202 is a second support substrate, 204 is a third insulating film, 206 is a fourth insulating film, 472, 472a and 472b are primary binding molecules, 490, 490a and 490b are Sample solutions 622, 622a and 622b represent gate electrodes. FIG. 5 is a diagram showing an example in which a plurality of types of primary binding molecules are fixed to each gate electrode when there are a plurality of gate element portions in an isolated gate type FET. 630 is an isolation gate type FET, 102 is a first support substrate, 104 is a first insulating film, 106 is a second insulating film, 108 is a source electrode, 110 is a drain electrode, 112 is an ultrafine fiber body, 202 is Second supporting substrate, 204 is a third insulating film, 206 is a fourth insulating film, 472a and 472b are primary binding molecules, 490a and 490b are sample solutions, 622 is a gate electrode, 210 is a conductive substrate, Reference numeral 212 denotes an ultrafine fiber element part, and 214a and 214b denote gate element parts. FIG. 5 is a diagram showing an example in which a plurality of types of primary binding molecules are fixed to an insulating film of a gate element portion in an isolated gate type FET. 800 is an isolation gate type FET, 102 is a first support substrate, 104 is a first insulating film, 106 is a second insulating film, 108 is a source electrode, 110 is a drain electrode, 112 is an ultrafine fiber body, 202 is Second support substrate, 204 is a third insulating film, 206 is a fourth insulating film, 472a and 472b are primary binding molecules, 490a and 490b are sample solutions, 612a and 612b are gate electrodes, and 210 is conductive A substrate, 212 is an ultrafine fiber element portion, and 214 is a gate element portion. FIG. 6 is a diagram showing another example in which a plurality of types of primary binding molecules are respectively fixed to an insulating film of a gate element portion in an isolated gate type FET. 900 is an isolation gate type FET, 102 is a first support substrate, 104 is a first insulating film, 106 is a second insulating film, 108 is a source electrode, 110 is a drain electrode, 112 is an ultrafine fiber body, 202 is Second support substrate, 204 is a third insulating film, 206 is a fourth insulating film, 472a and 472b are primary binding molecules, 490a and 490b are sample solutions, 612a and 612b are gate electrodes, and 302 is a first electrode , 304 is a second conductive substrate, 306 is a conductive member, 212 is an ultrafine fiber element portion, and 214 is a gate element portion. It is a figure which shows typically a mode that the detection target object 11 reacted with the primary binding molecule 10 fixed to the board | substrate 20 of FET, and the secondary binding molecule 12 couple | bonded with the detection target object 11. FIG. For example, the primary binding molecule 10 is fixed to the back surface 20-2 of the surface 20-1 on the substrate where the channel is formed. It is a figure which shows the state which the secondary binding molecule 13 labeled with the labeling substance 13-1 couple | bonded instead of the secondary binding molecule 12 shown by FIG. It is the graph which showed the I-Vg characteristic obtained by the detection of the Example by the relationship between the source-drain current I (vertical axis) and the gate voltage Vg (horizontal axis). It is the graph which expanded the graph shown in FIG. 25 by the vertical axis | shaft. It is a graph which shows the source-drain electric current I when the gate voltage Vg is -12V of the I-Vg characteristic obtained in the Example. It is a graph which shows the gate voltage Vg when the source-drain current I of the I-Vg characteristic obtained in the Example is -2.5 * 10 < ^-8 >.

Claims (12)

Reacting a primary binding molecule immobilized on a field effect transistor with a detection target contained in a sample;
Reacting a secondary binding molecule specifically reacting with the detection target with the detection target, and measuring an electric signal of a field effect transistor after reacting the secondary binding molecule. A method for detecting an object to be detected. Reacting a primary binding molecule immobilized on a field effect transistor with a detection target contained in a sample;
Reacting a secondary binding molecule that specifically reacts with the detection object with the detection object;
Reacting a tertiary binding molecule that specifically binds to a secondary binding molecule with the secondary binding molecule, and, if necessary, reacting a higher order binding molecule; and A method for detecting an object to be detected, comprising a step of measuring an electric signal of a field effect transistor after reacting a highest-order binding molecule.   The method according to claim 1, further comprising blocking a binding surface of the primary binding molecule in the field effect transistor before reacting the primary binding molecule with the detection target.   The method according to any one of claims 1 to 3, wherein any of the second to highest binding molecules is labeled with a substance that amplifies an electric signal of a field effect transistor.   5. The method of claim 4, wherein the material that amplifies the electrical signal of the field effect transistor is an electrical conductor, electrical insulator, or semiconductor material.   The method of claim 4, wherein the material that amplifies the electric signal of the field effect transistor is an electrical conductor or a semiconductor material.   The electric signal of the field effect transistor has an IV characteristic indicating a relationship between a source-drain current and a gate voltage, or an IV characteristic indicating a relationship between the source-drain current and the source-drain voltage. 7. The method according to any one of 6. The field effect transistor controls a substrate, a source electrode and a drain electrode disposed on the substrate, a channel including an ultrafine fiber body that electrically connects the source electrode and the drain electrode, and a current flowing through the channel Including a gate electrode,
The method according to claim 1, wherein the primary binding molecule is fixed to the substrate, the ultrafine fiber body, or the gate electrode. The field effect transistor controls a substrate, a source electrode and a drain electrode disposed on the substrate, a channel including an ultrafine fiber body that electrically connects the source electrode and the drain electrode, and a current flowing through the channel Including a gate electrode,
The substrate has a support substrate made of a semiconductor or metal, a first insulating film formed on the first surface of the support substrate, and a second insulating film formed on the second surface of the support substrate. And
The source electrode, the drain electrode and the channel are disposed on the first insulating film;
The gate electrode is disposed on the second insulating film, and the primary binding molecules are fixed to the second insulating film or the gate electrode. The method according to one item. The field effect transistor controls a substrate, a source electrode and a drain electrode disposed on the substrate, a channel including an ultrafine fiber body that electrically connects the source electrode and the drain electrode, and a current flowing through the channel Including a gate electrode,
The substrate has a support substrate made of a semiconductor or metal, a first insulating film formed on the first surface of the support substrate,
The source electrode, the drain electrode, the channel, and the gate electrode are disposed on the first insulating film, an interval between the gate electrode and the ultrafine fiber body is 10 μm or more, and the primary binding molecule is The method according to claim 1, wherein the method is fixed to the first insulating film or the gate electrode. The field effect transistor controls a substrate, a source electrode and a drain electrode disposed on the substrate, a channel including an ultrafine fiber body that electrically connects the source electrode and the drain electrode, and a current flowing through the channel And a second substrate electrically connected to the substrate,
The substrate has a support substrate made of a semiconductor or metal, a first insulating film formed on the first surface of the support substrate,
The source electrode, the drain electrode and the channel are disposed on the first insulating film;
The gate electrode is disposed on a first surface of the second substrate, and the primary binding molecule is fixed to the first surface or gate electrode of the second substrate; The method according to any one of claims 1 to 7.   The method according to claim 1, wherein the ultrafine fiber body is a carbon nanotube.

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