Method for Detecting Small Uncharged Molecules

Abstract

To provide a method capable of detecting small uncharged molecules simply and highly sensitively using an FET.SOLUTION: A method for detecting small uncharged molecules comprises (A) a step of interacting a small uncharged molecules with probe molecules that interacts with the small uncharged molecules immobilized on a semiconductor sensing device; (B) after the step (A), a step of further interacting protein whose small uncharged molecules are modified with the probe molecules, and (C) after the step (B), a step of detecting a potential change in a gate channel region due to the interaction.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for detecting small uncharged molecules.

Background Art

[0002] A field-effect transistor (FET) type biosensor (hereinafter abbreviated as FET biosensor) is a very promising tool for detecting molecules in a biological sample. When using an FET biosensor, since the change in the charge density on the gate surface due to the adsorption of the target molecule is directly detected as an electrical signal, label-free detection is possible, and low-cost and rapid detection of biomolecules is possible. Therefore, research on the detection of molecules using FET biosensors has been widely conducted.

[0003] An FET biosensor is a device that measures the change in the potential of the gate channel region caused by the charge of a detection target molecule (hereinafter also referred to as a target molecule) captured by a probe molecule within the device length, which is the charge detection range. Therefore, it has been difficult to directly detect small uncharged molecules that do not have a charge. In order to detect small uncharged molecules, the application of a sandwich assay using two types of antibodies has been attempted so far (Non-Patent Documents 1 and 2). However, since small molecules have a small molecular size and are enclosed within the antigen recognition domain, multiple antibodies cannot bind simultaneously, and detection by an FET biosensor using a sandwich assay has been difficult.

[0004] 5-Fluorouracil (5-FU), an anticancer agent widely used in the chemotherapy of cancer treatment, has a dosage determined by the patient's body surface area conversion. However, since its metabolic rate depends on liver function, there are individual differences, and only about 20% of patients are administered within the optimal concentration range. Furthermore, current methods (for example, ELISA and HPLC) also have problems such as long measurement time and high cost. Therefore, rapid blood concentration monitoring is required.

Prior Art Documents

Non-Patent Documents

[0005] [Non-Patent Document 1] Anal. Chem., 2017, Vol. 89, pp. 11325-11331 [Non-Patent Document 2] ACS Omega, 2019, Vol. 4, pp. 14765-14771 [Summary of the Invention] [Problems to be Solved by the Invention]

[0006] The present invention has been made in view of the above circumstances, and an object thereof is to provide a method capable of simply and highly sensitively detecting small uncharged molecules using an FET biosensor. [Means for Solving the Problems]

[0007] As a result of intensive studies to achieve the above object, the present inventors utilize the competition with a probe molecule that interacts with the small uncharged molecule immobilized on the gate insulating film between the small uncharged molecule and the protein modified with the small uncharged molecule, and found that it is possible to detect small uncharged molecules using an FET biosensor, and thus completed the present invention.

[0008] Therefore, the present invention provides the following method for detecting small uncharged molecules. 1. (A) A step of allowing a small uncharged molecule to interact with a probe molecule that interacts with the small uncharged molecule immobilized on a semiconductor sensing device, (B) After step (A), a step of further allowing a protein modified with the small uncharged molecule to interact with the probe molecule, and (C) After step (B), a step of detecting a potential change in the gate channel region due to the interaction A method for detecting small uncharged molecules comprising the steps of. 2. A method for detecting a small uncharged molecule, wherein a semiconductor sensing device with the probe molecule immobilized thereon comprises forming a first organic monolayer film composed of an organic monolayer having a reactive functional group on the first insulating layer of a FET in which a first insulating layer containing silicon oxide or an inorganic oxide is formed as a reaction gate insulating film on a semiconductor, and binding the probe molecule to the first organic monolayer film directly through the reactive functional group or using a crosslinking molecule, and having a probe molecule / organic monolayer film / insulating layer / semiconductor structure as a detection unit. 3. A method for detecting a small uncharged molecule according to 1 or 2, wherein the probe molecule is a Fab. 4. A method for detecting a small uncharged molecule according to any one of 1 to 3, which is a method for detecting a small uncharged molecule contained in a biological sample. 5. A method for detecting a small uncharged molecule according to 4, wherein the biological sample containing the small uncharged molecule is serum. 6. A method for detecting a small uncharged molecule according to 4 or 5, which includes a step of pretreating the biological sample containing the small uncharged molecule with a surfactant before step (A). 7. A method for detecting a small uncharged molecule according to any one of 1 to 5, wherein the small uncharged molecule is 5-fluorouracil (5-FU).

Advantages of the Invention

[0009] According to the present invention, it becomes possible to detect small uncharged molecules with high sensitivity using an FET biosensor.

Brief Description of the Drawings

[0010]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Mode for Carrying Out the Invention

[0011] The method for detecting a small uncharged molecule of the present invention includes: (A) a step of allowing a small uncharged molecule to interact with a probe molecule that interacts with the small uncharged molecule immobilized on a semiconductor sensing device; (B) after step (A), further allowing a protein modified with the small uncharged molecule to interact with the probe molecule; and (C) after step (B), detecting a potential change in the gate channel region due to the interaction. In the present invention, the small uncharged molecule means a molecule having a molecular weight of generally 1,000 or less and having no charge.

[0012] The semiconductor sensing device used in the present invention is such that a first organic monolayer film composed of an organic monolayer having a reactive functional group is formed on the first insulating layer of an FET in which a first insulating layer containing silicon oxide or inorganic oxide is formed as a reaction gate insulating portion on a semiconductor, and a probe molecule is bonded to the first organic monolayer film directly through the reactive functional group or using a crosslinking molecule. It preferably comprises a probe molecule / organic monolayer film / insulating layer / semiconductor structure as a detection unit.

[0013] Among the above-mentioned detection units, the insulating layer / semiconductor structure portion is obtained by removing the gate electrode from a normal FET, and its configuration can utilize those conventionally known. The insulating layer is preferably silicon oxide. This FET structure can be either n-type or p-type. Examples of such an FET include those shown in Fig. 1(A). In Fig. 1, 1 represents a silicon substrate, 1a represents a gate channel region, 2 represents an insulating layer containing silicon oxide or inorganic oxide (such as glass, alumina, etc.), 4 represents a gate electrode, 5 represents a source electrode, 6 represents a drain electrode, and 7 represents a doped region.

[0014] Then, as shown in Fig. 1(B), a first organic monolayer film 3 is formed on the insulating layer 2. Here, in the present invention, as a basic principle, it is configured to detect the potential change in the gate channel region accompanying the binding reaction between the probe molecule and the target molecule on the surface of the insulating layer as an electrical signal. The thickness of the insulating layer is preferably 30 to 300 nm, particularly preferably 50 to 150 nm.

[0015] The first organic monolayer film is composed of an organic monolayer having a reactive functional group. The organic monolayer having a reactive functional group is preferably a monolayer film of an alkoxysilane represented by the following formula (1).

Chemical formula

[0016] In formula (1), R is an amino group, an aminooxy group, a carboxy group, or a thiol group.

[0017] In formula (1), R 1 is a linear alkanediyl group having 3 to 22 carbon atoms. The linear alkanediyl group preferably has 3 to 18 carbon atoms, more preferably 3 to 8 carbon atoms. A shorter carbon chain is preferable because it weakens the hydrophobicity of the organic monolayer film and can suppress non-specific adsorption due to the hydrophobic interaction of the target molecule.

[0018] R 1 Specific examples of the linear alkanediyl group represented by include propane-1,3-diyl group, butane-1,4-diyl group, pentane-1,5-diyl group, hexane-1,6-diyl group, heptane-1,7-diyl group, octane-1,8-diyl group, nonane-1,9-diyl group, decane-1,10-diyl group, undecane-1,11-diyl group, dodecane-1,12-diyl group, tridecane-1,13-diyl group, tetradecane-1,14-diyl group, pentadecane-1,15-diyl group, hexadecane-1,16-diyl group, heptadecane-1,17-diyl group, octadecane-1,18-diyl group, nonadecane-1,19-diyl group, eicosane-1,20-diyl group, heneicosane-1,21-diyl group, docosane-1,22-diyl group. Among these, those having 3 to 18 carbon atoms are preferable, and those having 3 to 8 carbon atoms are more preferable.

[0019] In formula (1), R 2 ~R 4 are each independently a linear or branched alkyl group having 1 to 5 carbon atoms or a linear or branched alkoxyalkyl group having 2 to 5 carbon atoms. Examples of the alkyl group include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert-butyl group, etc. Among these, a methyl group or an ethyl group is preferable. Examples of the alkoxyalkyl group include methoxymethyl group, ethoxymethyl group, 2-methoxyethyl group, 2-ethoxyethyl group, etc. Among these, an alkoxyalkyl group having 2 to 3 carbon atoms is preferable. R 2 ~R 4Particularly preferred are a methyl group, an ethyl group, a 2-methoxyethyl group, etc.

[0020] As the alkoxysilane in which R is an amino group, a carboxy group or a thiol group, commercially available products can be used. Further, the alkoxysilane in which R is an aminooxy group can be synthesized according to the following scheme. [Chemical formula] (In the formula, R 1 ~R 4 is the same as described above. R 5 is a linear alkanediyl group in which the number of carbon atoms is reduced by 2 from R 1 .)

[0021] The alkoxysilane in which R is an aminooxy group can be prepared by treating a trialkoxysilane and an O-alkenylhydroxyamine with a platinum-based catalyst. For example, in a nitrogen atmosphere, a platinum-based catalyst such as chloroplatinic(IV) acid is added to a mixture of a trialkoxysilane and an O-alkenylhydroxyamine, and the mixture is reacted at 10 to 200 °C for 1 to 1,200 hours, more preferably at 60 to 120 °C for 12 to 48 hours to prepare it. For the film-forming operation, it is preferable to use a product obtained by removing excess trialkoxysilane by an operation such as distillation.

[0022] The first organic monolayer film is formed on the insulating layer by a gas-phase chemical reaction or a liquid-phase reaction of the alkoxysilane, and an optimized film in which single molecules are most densely packed is formed by, for example, the self-assembly function of organic molecules. When forming a monolayer film by a gas-phase chemical reaction, for example, a substrate and an alkoxysilane are enclosed in a container and reacted in an argon inert atmosphere preferably at 80 to 200 °C for 1 to 24 hours, more preferably at 100 to 130 °C for 2 to 5 hours to form a film. When forming a monolayer film by a liquid-phase reaction, for example, a substrate is immersed in an organic solvent containing an alkoxysilane in an argon inert atmosphere and left standing preferably at 20 to 80 °C for 1 minute to 24 hours, more preferably at 55 to 65 °C for 5 to 20 minutes to form a film.

[0023] Examples of the organic solvent include toluene, methanol, ethanol, etc., and toluene, methanol, etc. are particularly preferable.

[0024] On the semiconductor of the FET, a second insulating layer containing silicon oxide or inorganic oxide can be further formed as a reference gate insulating portion. On this second insulating layer, a self-assembled monolayer composed of organic molecules that do not react with either the probe molecule or the small uncharged molecule that is the target molecule can be formed as a second organic monolayer, and this monolayer / insulating layer / semiconductor structure can be used as a reference portion. Note that if the reaction gate insulating portion and the reference gate insulating portion are separated so as not to affect each other in the potential change measurement, the first insulating layer of the reaction gate insulating portion and the second insulating layer of the reference gate insulating portion can also be provided in the same layer.

[0025] FIG. 2 shows an example of the unit configuration of an on-chip device in which an organic self-assembled monolayer / insulating layer / semiconductor structure is applied to the detection unit 9 and the reference unit 8. In FIG. 2, 1 is a silicon substrate, 2 is an insulating layer, and 10 is a template portion. The unit configuration of this device is not limited to the illustrated configuration, and the detection unit and the reference unit do not necessarily need to be arranged in a one-to-one relationship, and the number and combination of the detection unit and the reference unit can be appropriately changed and arranged as needed. In addition, the detection unit and the reference unit can each be formed with a size of several to several tens of μm.

[0026] As the second organic self-assembled monolayer, a self-assembled monolayer of alkoxysilane having a linear alkyl group with 8 to 22 carbon atoms which may be fluorinated is preferable. Note that when a self-assembled monolayer of alkoxysilane is used as the organic self-assembled monolayer, the second insulating layer is preferably formed of silicon oxide.

[0027] The second organic self-assembled monolayer is preferably a self-assembled monolayer in order to form a uniform film on the insulating layer. Specifically, it is preferably a self-assembled monolayer of trialkoxysilane represented by the following formula (2). [Chemical formula]

[0028] In formula (2), R 6 is a linear alkyl group having 8 to 22 carbon atoms, preferably 10 to 18 carbon atoms, and part or all of the hydrogen atoms may be substituted with fluorine atoms. The linear alkyl group preferably has 10 to 18 carbon atoms. Specific examples of the linear alkyl group include n-octyl group, n-nonyl group, n-decyl group, n-undecyl group, n-dodecyl group, n-tridecyl group, n-tetradecyl group, n-pentadecyl group, n-hexadecyl group, n-heptadecyl group, n-octadecyl group, n-nonadecyl group, n-eicosyl group, n-henicosyl group, n-docosyl group, etc.

[0029] In formula (2), R 7 ~R 9 are each independently a linear or branched alkyl group having 1 to 5 carbon atoms or a linear or branched alkoxyalkyl group having 2 to 5 carbon atoms. Examples of the alkyl group include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert-butyl group, etc. Among these, a methyl group or an ethyl group is preferred. Examples of the alkoxyalkyl group include methoxymethyl group, ethoxymethyl group, 2-methoxyethyl group, 2-ethoxyethyl group, etc. Among these, an alkoxyalkyl group having 2 to 3 carbon atoms is preferred.

[0030] Specific examples of the trialkoxysilane represented by formula (2) include CH3(CH2)7Si(OCH3)3, CH3(CH2)7Si(OC2H5)3, CH3(CH2)8Si(OCH3)3, CH3(CH2)8Si(OC2H5)3, CH3(CH2)9Si(OCH3)3, CH3(CH2)9Si(OC2H5)3, CH3(CH2) 10 Si(OCH3)3, CH3(CH2) 10 Si(OC2H5)3, CH3(CH2) 11 Si(OCH3)3, CH3(CH2) 11 Si(OC2H5)3, CH3(CH2)12 Si(OCH3)3, CH3(CH2) 12 Si(OC2H5)3, CH3(CH2) 13 Si(OCH3)3, CH3(CH2) 13 Si(OC2H5)3, CH3(CH2) 14 Si(OCH3)3, CH3(CH2) 14 Si(OC2H5)3, CH3(CH2) 15 Si(OCH3)3, CH3(CH2) 15 Si(OC2H5)3, CH3(CH2) 16 Si(OCH3)3, CH3(CH2) 16 Si(OC2H5)3, CH3(CH2) 17 Si(OCH3)3, CH3(CH2) 17 Si(OC2H5)3, CH3(CH2) 18 Si(OCH3)3, CH3(CH2) 18 Si(OC2H5)3, CH3(CH2) 19 Si(OCH3)3, CH3(CH2) 19 Si(OC2H5)3, CH3(CH2) 20 Si(OCH3)3, CH3(CH2) 20 Si(OC2H5)3, CH3(CH2) 21 Si(OCH3)3, CH3(CH2) 21 Si(OC2H5)3, CF3(CF2)5(CH2)2Si(OCH3)3, CF3(CF2)5(CH2)2Si(OC2H5)3, CF3(CF2)6(CH2)2Si(OCH3)3, CF3(CF2)6(CH2)2Si(OC2H5)3, CF3(CF2)7(CH2)2Si(OCH3)3, CF3(CF2)7(CH2)2Si(OC2H5)3, CF3(CF2)8(CH2)2Si(OCH3)3, CF3(CF2)8(CH2)2Si(OC2H5)3, CF3(CF2)9(CH2)2Si(OCH3)3, CF3(CF2)9(CH2)2Si(OC2H5)3, CF3(CF2) 10 (CH2)2Si(OCH3)3, CF3(CF2) 10 (CH2)2Si(OC2H5)3, CF3(CF2) 11 (CH2)2Si(OCH3)3, CF3(CF2)11 (CH2)2Si(OC2H5)3, CF3(CF2) 12 (CH2)2Si(OCH3)3, CF3(CF2) 12 (CH2)2Si(OC2H5)3, CF3(CF2) 13 (CH2)2Si(OCH3)3, CF3(CF2) 13 (CH2)2Si(OC2H5)3, CF3(CF2) 14 (CH2)2Si(OCH3)3, CF3(CF2) 14 (CH2)2Si(OC2H5)3, CF3(CF2) 15 (CH2)2Si(OCH3)3, CF3(CF2) 15 (CH2)2Si(OC2H5)3, CF3(CF2) 16 (CH2)2Si(OCH3)3, CF3(CF2) 16 (CH2)2Si(OC2H5)3, CF3(CF2) 17 (CH2)2Si(OCH3)3, CF3(CF2) 17 (CH2)2Si(OC2H5)3, CF3(CF2) 18 (CH2)2Si(OCH3)3, CF3(CF2) 18 (CH2)2Si(OC2H5)3, CF3(CF2) 19 (CH2)2Si(OCH3)3, CF3(CF2) 19 (CH2)2Si(OC2H5)3 and the like can be mentioned.

[0031] Note that the first and second organic monolayers can be formed at desired positions by patterning. In particular, for forming an integrated device on-chip, patterning of the organic monolayer is effective. For example, on the surface of the insulating layer of the detection part, a first monolayer composed of organic molecules having reactive functional groups for immobilizing probe molecules is formed, while in the reference part and further in the non-gate part (template part), in order to avoid non-specific adsorption of target molecules, a second organic monolayer composed of organic molecules that do not react with either probe molecules or target molecules is formed by patterning in a position-selective manner.

[0032] As a reference part, it is also possible to use a monomolecular film similar to the first organic monomolecular film as the second organic monomolecular film, on which a compound that does not interact with the target molecule is immobilized. That is, a compound / organic monomolecular film / insulating layer / semiconductor structure that does not interact with the target molecule can also be used as the reference part. In this case, the reference part can be formed according to the same method as the organic monomolecular film formation method in the detection part described above and the compound immobilization method described below.

[0033] In the semiconductor sensing device, probe molecules are immobilized on the first organic monomolecular film of the detection part. For example, as shown in FIG. 1(C), probe molecules 11 are bound to the first organic monomolecular film 3.

[0034] The probe molecules are immobilized on the organic monomolecular film directly or via a cross-linking molecule. Examples of the cross-linking molecule include glutaraldehyde. In this case, the method for modifying the organic monomolecular film with glutaraldehyde is not particularly limited. For example, it may be reacted in an aqueous solution of glutaraldehyde with a concentration of 0.01 to 25% by mass at 10 to 50 ° C for 1 minute to 24 hours.

[0035] Next, the probe molecules are immobilized by reacting reactive functional groups such as amino groups in the probe molecules with glutaraldehyde. Specifically, for example, it may be reacted in a solution containing probe molecules (the solvent is ultrapure water, phosphate buffered saline, etc.) at 10 to 50 ° C for 1 minute to 24 hours. Preferably, it may be reacted at 10 to 35 ° C for 1 minute to 60 minutes. The concentration of the probe molecules is preferably 1 pg / mL to 1 mg / mL, and more preferably 100 ng / mL to 100 μg / mL.

[0036] The probe molecule may be appropriately selected as long as it binds to the small uncharged molecule to be detected. Examples of the probe molecule include antibodies. In the present invention, the antibody includes immunoglobulins such as IgG, IgM, IgA, IgD, and IgE, and those containing antibody fragments (Fab, F(ab')2). The antibody may be a polyclonal antibody or a monoclonal antibody. In the present invention, it is particularly preferable to use Fab as the probe molecule.

[0037] Figure 3 shows a conceptual diagram of a method for detecting a small uncharged molecule using the semiconductor sensing device of the present invention. In this detection method, after allowing the immobilized probe molecule directly immobilized on the organic monolayer film to interact with the small uncharged molecule, the protein modified with the small uncharged molecule is allowed to interact with the probe molecule, and the potential change in the gate channel region 1a due to the interaction is detected. In FIG. 3, 12 is a small uncharged molecule, 13 is a protein modified with a small uncharged molecule, and the other components are denoted by the same reference numerals as in FIG. 1, and the description thereof is omitted.

[0038] Since the small uncharged molecule does not have a charge, the amount of charge within the device length does not change due to the interaction with the probe molecule. However, after allowing the small uncharged molecule to interact with the probe molecule, by further allowing the protein modified with the small uncharged molecule to interact with the probe molecule, the adsorption amount of the protein modified with the small uncharged molecule onto the sensor surface changes depending on the concentration of the small uncharged molecule to be measured. That is, the potential of the gate channel region changes depending on the concentration of the small uncharged molecule to be measured. In this case, a potential shift can be detected as a signal under a constant current, and a current shift can be detected as a signal under a constant voltage. Note that the shift of the threshold voltage is opposite between the case of using an n-type FET and the case of using a p-type FET.

[0039] In step (A), to allow the probe molecules immobilized on the device to interact with small uncharged molecules, a solution containing the small uncharged molecules may be diluted as necessary and placed on the gate electrode. At this time, as the solution, a general solution used for molecule detection can be used, but a solution that particularly satisfies physiological conditions is preferred. For example, physiological saline, phosphate-buffered saline, Tris-buffered saline, MES-buffered saline, MOPS-buffered saline, PIPES-buffered saline, HEPES-buffered saline, etc. can be preferably used. Note that the pH of the solution is preferably 5 to 10, and more preferably 6 to 8.

[0040] Also, ions such as Ca 2+ , Mg 2+ , chelating agents such as ethylenediaminetetraacetic acid (EDTA) and glycol ether diamine tetraacetic acid (EGTA), and surfactants such as Tween (registered trademark) 20, Triton (registered trademark) X-100, and Nonidet (registered trademark) P-40 may be added to the solution. When adding the ions, the concentration is preferably 0.1 to 10 mM, and more preferably 0.5 to 5 mM. When adding the chelating agent, the concentration is preferably 0.1 to 10 mM, and more preferably 0.5 to 5 mM. When adding the surfactant, the concentration is preferably 0.001 to 10% by volume, and more preferably 0.05 to 5% by volume.

[0041] In step (A), the temperature when allowing the probe molecules immobilized on the device to interact with small uncharged molecules is preferably 0 to 40°C, more preferably 10 to 30°C, and even more preferably room temperature (20 to 25°C). The reaction time is preferably 30 seconds to 2 hours, more preferably 1 minute to 1 hour, and even more preferably 5 to 30 minutes.

[0042] The concentration of detectable small uncharged molecules varies depending on their type, but is usually about 0 to 1,000 ng / mL, preferably about 0 to 500 ng / mL, and more preferably 0 to 200 ng / mL.

[0043] A cleaning step may be provided after step (A) and before step (B). The cleaning solution used in the cleaning step is preferably a solution that satisfies the physiological conditions used when allowing the probe molecules immobilized on the above-described device to interact with the protein. Further, the above-described ions, chelating agents, surfactants, etc. may be added to the cleaning solution. In this case, it is preferable to add them so as to achieve the above-described concentrations.

[0044] Next, in step (B), as the protein modified with the small uncharged molecule added after allowing the probe molecule immobilized on the organic monolayer to interact with the small uncharged molecule, those in which the small uncharged molecule is modified on bovine serum albumin (BSA), cationized bovine serum albumin (cationized BSA), keyhole limpet hemocyanin (KLH), Blue Carrier protein, ovalbumin (OVA), etc. are preferable.

[0045] As a method for modifying a small uncharged molecule to a protein, a known method can be adopted. For example, a method of binding a small uncharged molecule to a protein via a crosslinking agent such as 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide, sodium 4-(N-maleimidomethyl)cyclohexane-1-carboxylate 3-sulfo-N-succinimidyl, glutaraldehyde, etc. can be mentioned. Further, if necessary, a functional group that reacts with the crosslinking agent may be introduced into the small uncharged molecule, and the small uncharged molecule may be bound to the protein using the crosslinking agent. For example, when binding 5-FU to BSA, the method described in Macromol. Biosci. 2014, Vol.14, pp. 428-439 can be referred to. As the protein modified with the small uncharged molecule, commercially available products can also be used.

[0046] When adding a protein modified with a small uncharged molecule, a solution containing the protein modified with the small uncharged molecule may be diluted as necessary and placed on the gate electrode. At this time, the solution is not particularly limited, but those satisfying physiological conditions are preferable. For example, physiological saline, phosphate buffered saline, Tris buffered saline, MES buffered saline, MOPS buffered saline, PIPES buffered saline, HEPES buffered saline, etc. can be preferably used. Note that the pH of the solution is preferably 5 to 10, more preferably 6 to 8.

[0047] The concentration of the protein modified with the small uncharged molecule in the solution is preferably 1 to 100 μg / mL, more preferably 20 to 50 μg / mL.

[0048] Also, ions such as Ca 2+ , Mg 2+ , chelating agents such as ethylenediaminetetraacetic acid (EDTA), glycol ether diamine tetraacetic acid (EGTA), and surfactants such as Tween (registered trademark) 20, Triton (registered trademark) X-100, Nonidet (registered trademark) P-40, etc. may be added. When adding the ions, the concentration is preferably 0.1 to 10 mM, more preferably 0.5 to 5 mM. When adding the chelating agent, the concentration is preferably 0.1 to 10 mM, more preferably 0.5 to 5 mM. When adding the surfactant, the concentration is preferably 0.001 to 10% by volume, more preferably 0.05 to 5% by volume.

[0049] In step (B), the temperature when the probe molecule immobilized on the device and the protein modified with the small uncharged molecule interact is preferably 0 to 40 °C, more preferably 10 to 30 °C, and even more preferably room temperature (20 to 25 °C). The reaction time is preferably 30 seconds to 2 hours, more preferably 1 minute to 1 hour, and even more preferably 5 to 30 minutes.

[0050] A washing step may be provided after step (B) and before step (C). The washing solution used in the washing step is preferably a solution that satisfies the physiological conditions used when the probe molecule immobilized on the above-described device interacts with the protein. Further, the above-described ions, chelating agents, surfactants, etc. may be added to the washing solution. In this case, it is preferable to add them so as to reach the above-described concentration.

[0051] In step (C), the potential change in the gate channel region derived from the protein modified with the small uncharged molecule is detected using the semiconductor sensing device. Specifically, for example, an electrolytic solution is filled between the detection unit and the gate electrode, and a gate voltage is applied to detect the potential change in the gate channel region. Examples of the electrolytic solution include a phosphate buffer solution and a solution that satisfies the physiological conditions used when the probe molecule immobilized on the above-described device interacts with the protein. When there are few small uncharged molecules that interacted with the probe molecule in step (A) (that is, the concentration of small uncharged molecules in the measurement sample is low), since there are many proteins modified with small uncharged molecules that interact with the probe molecule, a large potential change is observed. On the other hand, when there are many small uncharged molecules that interacted with the probe molecule in step (A) (that is, the concentration of small uncharged molecules in the measurement sample is high), the opposite is true, and the potential change becomes small. Thus, it becomes possible to quantitatively detect small uncharged molecules in the measurement sample.

[0052] According to the method of the present invention, it is possible to detect small uncharged molecules with high sensitivity. Further, it is possible to detect small uncharged molecules under physiological conditions. The small uncharged molecules are not particularly limited as long as they have the property of interacting with the probe molecule. For example, anticancer agents such as 5-fluorouracil (5-FU), mitomycin C, cyclophosphamide, busulfan, cisplatin, carboplatin, irinotecan, docetaxel, paclitaxel, vinorelbine, tegafur, methotrexate, anastrozole, gefitinib, imatinib, ubenimex, etc., estradiol, progesterone, cortisol, thyroid hormone, adrenaline, noradrenaline, dopamine, etc. can be mentioned.

[0053] The method of the present invention can be particularly preferably used for detecting small uncharged molecules in a biological sample. Examples of the biological sample include serum, blood, urine, sweat, saliva, interstitial fluid, tissue extract, cell extract, etc. Among these, it is particularly useful for detecting small uncharged molecules in serum, urine, sweat, and saliva. When detecting small uncharged molecules in these biological samples, the biological sample may be added directly onto the gate electrode of the device in step (A), or may be diluted if necessary and then added onto the gate electrode of the small uncharged molecule. As the solution for dilution, physiological saline, phosphate buffered saline, Tris buffered saline, MES buffered saline, MOPS buffered saline, PIPES buffered saline, HEPES buffered saline, etc. can be preferably used. The pH of the solution is preferably 5 to 10, more preferably 6 to 8.

[0054] Further, Ca 2+ , Mg 2+Ions such as ethylenediaminetetraacetic acid (EDTA) and glycol ether diaminetetraacetic acid (EGTA), chelating agents such as Tween (registered trademark) 20, Triton (registered trademark) X-100, and Nonidet (registered trademark) P-40, and other surfactants may be added. When the ions are added, the concentration is preferably 0.1 to 10 mM, and more preferably 0.5 to 5 mM. When the chelating agents are added, the concentration is preferably 0.1 to 10 mM, and more preferably 0.5 to 5 mM. When a surfactant is added, the concentration is preferably 0.001 to 10 vol.%, and more preferably 0.05 to 5 vol.%. In particular, it is preferable to add a surfactant from the viewpoint of suppressing the influence of contaminating factors in a biological sample. EXAMPLES

[0055] The present invention will be specifically described below with reference to Production Examples, Examples and Comparative Examples, but the present invention is not limited to the following Examples.

[0056] [Production Example 1] We constructed a semiconductor sensing device with an organic monolayer / insulating layer / semiconductor structure as the detection section. Silicon oxide was used as the insulating layer. Aminopropyltriethoxysilane was used to form a monolayer as the first organic monolayer of the detection section. The device fabrication method is described below.

[0057] The photoresist film was removed by ultrasonic treatment using acetone from a 10 μm long, 1,000 μm wide n-type FET manufactured by Toppan Printing Co., Ltd. To create active sites by introducing hydroxyl groups onto the gate surface, the gate surface was exposed to 200 W O2 plasma for 1 minute using a Plasma Reactor PR301 (manufactured by Yamato Scientific Co., Ltd.). The device was immersed in toluene containing 1% by mass of aminopropyltriethoxysilane (Sigma-Aldrich) and left to stand for 7 minutes at 60°C under an argon atmosphere to form a monolayer on the gate electrode. The FET on which the monolayer was formed was ultrasonically cleaned using a methanol / toluene mixed solvent (mass ratio 1:1) and rinsed with ethanol. After cleaning the substrate, it was left to stand for 60 minutes at 160°C under an argon atmosphere.

[0058] [Reference Example 1] The response upon addition of 5-FU-modified BSA (BSA / 5-FU, manufactured by Cosmo Bio Co., Ltd.) was evaluated as follows using a device in which an antibody fragment (Fab) derived from an anti-5-fluorouracil (5-FU) antibody was immobilized on the gate electrode of the detection unit modified with an amino-based self-assembled monolayer. First, glutaraldehyde was reacted as a cross-linking molecule for cross-linking the amino group of the organic self-assembled monolayer and Fab. The reaction was carried out by immersing the detection unit of the device on which a self-assembled monolayer with an amino group at the end was formed in 0.01 mL of a 2.5 mass% aqueous glutaraldehyde solution at room temperature for 30 minutes. Thereafter, the device was placed in a holder, and the gate electrode portion cross-linked with glutaraldehyde was immersed in phosphate-buffered saline containing 50 μg / mL of Fab (137 mM NaCl, 2.682 mM KCl, 8.1 mM Na2HPO4·12H2O, 1.469 mM KH2PO4, pH 7.4, hereinafter referred to as PBS) for 1 hour. After washing the substrate, capping treatment with PBS containing 10 mM ethanolamine was performed for 1 hour. Thereafter, the detection unit of the device was immersed in 0.5 mL of phosphate buffer (0.12 mM NaH2PO4·2H2O, 0.51 mM Na2HPO4·12H2O, pH 7.4, hereinafter referred to as PB) for 3 minutes, and then at room temperature, the current-voltage curve of the Fab-immobilized device was measured using an Ag / AgCl reference electrode with a digital source meter (manufactured by Keithley Instruments, Inc., 2612). The measurement conditions were a gate voltage of -3 V to 0.5 V and a drain voltage of 0.1 V. Subsequently, 20 μL of PBS containing 25 μg / mL of 5-FU-modified BSA (BSA / 5-FU) was added onto the gate surface of the Fab-immobilized device, allowed to stand for 30 minutes, and then rinsed with 3 mL of PBS and 3 mL of PB. Thereafter, 0.5 mL of PB was added onto the gate surface, allowed to stand for 3 minutes, and then the current-voltage curve of the BSA / 5-FU adsorption device was measured, and the gate voltage shift ΔV g was evaluated.

[0059] [Comparative Reference Example 1] As a control experiment, the response of the Fab-immobilized device upon addition of a protein that does not show specificity, human serum albumin (HSA), was evaluated as follows. First, glutaraldehyde was reacted as a cross-linking molecule for cross-linking the amino group of the organic monolayer and Fab. The reaction was carried out by immersing the detection part of the device on which a monolayer with an amino group-terminated end was formed in 0.01 mL of an aqueous solution of 2.5% by mass glutaraldehyde at room temperature for 30 minutes. Then, the device was placed in a holder, and the gate electrode part cross-linked with glutaraldehyde was immersed in PBS containing 50 μg / mL of Fab for 1 hour. After washing the substrate, capping treatment with PBS containing 10 mM ethanolamine was carried out for 1 hour. Thereafter, the detection part of the device was immersed in 0.5 mL of PB for 3 minutes, and then at room temperature, the current-voltage curve of the Fab-immobilized device was measured using an Ag / AgCl reference electrode with a digital source meter (manufactured by Keithley Instruments, Inc., 2612). The measurement conditions were a gate voltage of -3 V to 0.5 V and a drain voltage of 0.1 V. Subsequently, 20 μL of PBS containing 1,000 μg / mL of HSA was added onto the gate surface of the Fab-immobilized device, allowed to stand for 30 minutes, and then rinsed with 3 mL of PBS and 3 mL of PB. Thereafter, 0.5 mL of PB was added onto the gate surface, allowed to stand for 3 minutes, and then the current-voltage curve of the HSA-adsorbed device was measured, and the gate voltage shift ΔV g was evaluated.

[0060] The evaluation results of the shift amount of the semiconductor characteristics before and after the addition of BSA / 5-FU or HSA to the Fab-immobilized device are shown in Fig. 4. When BSA / 5-FU was added to the Fab-immobilized device, the gate voltage shift ΔV g in the current-voltage curve, which is a semiconductor characteristic, was +39 mV. On the other hand, when HSA was added to the Fab-immobilized device, almost no change in the current-voltage curve was observed. From this, it was confirmed that the Fab-immobilized device has specificity for BSA / 5-FU.

[0061] [Example 1] As for the detection of 5-FU using the Fab-immobilized device, the response measurement by sequential addition of 5-FU and BSA / 5-FU was evaluated as follows. First, glutaraldehyde was reacted as a cross-linking molecule for cross-linking the amino group of the organic monolayer and Fab. The reaction was carried out by immersing the detection part of the device on which a monolayer with an amino group at the end was formed in 0.01 mL of a 2.5 mass% aqueous glutaraldehyde solution at room temperature for 30 minutes. Then, the device was placed on a holder, and the gate electrode part cross-linked with glutaraldehyde was immersed in PBS containing 50 μg / mL of Fab for 1 hour. After substrate cleaning, capping treatment with PBS containing 10 mM ethanolamine was performed for 1 hour. Thereafter, after immersing the detection part of the device in 0.5 mL of PB for 3 minutes, the current-voltage curve of the Fab-immobilized device was measured at room temperature using an Ag / AgCl reference electrode and a digital source meter (manufactured by Case Relay Co., Ltd., 2612). The measurement conditions were a gate voltage of -3 V to 0.5 V and a drain voltage of 0.1 V. Subsequently, 20 μL of PBS containing 0 to 1,000 ng / mL of 5-FU was added to the gate surface, allowed to stand for 30 minutes, and then rinsed with 5 mL of PBS. Thereafter, 20 μL of PBS containing 25 μg / mL of BSA / 5-FU was added onto the gate surface of the Fab-immobilized device, allowed to stand for 30 minutes, and then rinsed with 3 mL of PBS and 3 mL of PB. Then, 0.5 mL of PB was added onto the gate surface and allowed to stand for 3 minutes, and the current-voltage curve of the BSA / 5-FU adsorption device was measured, and the gate voltage shift ΔV g was evaluated.

[0062] The evaluation results of the shift amount of semiconductor characteristics by sequential addition of 5-FU and BSA / 5-FU to the Fab-immobilized device are shown in Fig. 5. After adding 5-FU at each concentration of 0 to 1,000 ng / mL to the Fab-immobilized device, the gate voltage shift ΔV gWhen obtained, it was confirmed that the shift amount decreased as the concentration increased. This means that the adsorption amount of BSA / 5-FU changed due to the decrease in the unreacted Fab molecular weight on the FET gate surface as the 5-FU concentration increased. Therefore, the quantitative detectability of 5-FU by sequential addition of 5-FU and BSA / 5-FU to the Fab-immobilized device was shown.

[0063] [Example 2] As a pretreatment of the serum sample when detecting 5-FU in serum by the Fab-immobilized device, dilution with phosphate buffered saline containing Tween 20 was performed as follows, and the influence of interfering factors in the serum was evaluated. First, glutaraldehyde was reacted as a cross-linking molecule for cross-linking the amino group of the organic monolayer and Fab. The reaction was carried out by immersing the detection part of the device on which a monolayer with an amino group at the end was formed in 0.01 mL of a 2.5 mass% aqueous glutaraldehyde solution at room temperature for 30 minutes. Then, the device was placed in a holder, and the gate electrode part cross-linked with glutaraldehyde was immersed in PBS containing 50 μg / mL of Fab for 1 hour. After washing the substrate, a capping treatment with PBS containing 10 mM ethanolamine was performed for 1 hour. Thereafter, the detection part of the device was immersed in 0.5 mL of PB for 3 minutes, and then at room temperature, the current-voltage curve of the Fab-immobilized device was measured with a digital source meter (manufactured by Keithley Instruments, Inc., 2612) using an Ag / AgCl reference electrode. The measurement conditions were a gate voltage of -3 V to 0.5 V and a drain voltage of 0.1 V. Subsequently, human serum containing 5-FU at 500 ng / mL or 0 ng / mL was diluted 5-fold with PBS (PBS-T) containing 0.05% (v / v) Tween 20, and then 20 μL was added to the FET gate surface and allowed to stand for 30 minutes. Thereafter, rinsing was performed using 3 mL of PBS and 3 mL of PB. Thereafter, only when diluted human serum containing 5-FU at 500 ng / mL was added, 20 μL of PBS containing 25 μg / mL of BSA / 5-FU was added onto the gate surface of the Fab-immobilized device, allowed to stand for 30 minutes, and then rinsing was performed using 3 mL of PBS and 3 mL of PB. Then, 0.5 mL of PB was added onto the gate surface and allowed to stand for 3 minutes, and then the current-voltage curve of the device was measured, and the gate voltage shift ΔV g was evaluated.

[0064] [Example 3] As a pretreatment of the serum sample for detecting 5-FU in serum using the Fab-immobilized device, dilution with PBS was performed as follows, and evaluation of the influence of serum interfering factors was carried out. First, glutaraldehyde was reacted as a cross-linking molecule for cross-linking the amino group of the organic monolayer and Fab. The reaction was carried out by immersing the detection part of the device on which a monolayer with an amino group at the end was formed in 0.01 mL of an aqueous solution of 2.5% (m / m) glutaraldehyde at room temperature for 30 minutes. Thereafter, the device was placed in a holder, and the gate electrode part cross-linked with glutaraldehyde was immersed in PBS containing 50 μg / mL of Fab for 1 hour. After washing the substrate, capping treatment with PBS containing 10 mM ethanolamine was performed for 1 hour. Thereafter, the detection part of the device was immersed in 0.5 mL of PB for 3 minutes, and then at room temperature, the current-voltage curve of the Fab-immobilized device was measured using an Ag / AgCl reference electrode with a digital source meter (manufactured by Case Relay Co., Ltd., 2612). The measurement conditions were a gate voltage of -3 V to 0.5 V and a drain voltage of 0.1 V. Subsequently, human serum containing 5-FU at 500 ng / mL or 0 ng / mL was diluted 5-fold with PBS, then 20 μL was added to the FET gate surface and left standing for 30 minutes. Thereafter, rinsing was performed using 3 mL of PBS and 3 mL of PB. Then, only when diluted human serum containing 5-FU at 500 ng / mL was added, 20 μL of PBS containing 25 μg / mL of BSA / 5-FU was added onto the gate surface of the Fab-immobilized device, left standing for 30 minutes, and then rinsing was performed using 3 mL of PBS and 3 mL of PB. Then, 0.5 mL of PB was added onto the gate surface and left standing for 3 minutes, and then the current-voltage curve of the BSA / 5-FU adsorption device was measured, and the gate voltage shift ΔV g was evaluated.

[0065] As a pretreatment of the serum sample during the measurement of the 5-FU concentration in blood using the Fab-immobilized device, the evaluation results of the shift amount of the semiconductor characteristics before and after the sequential addition of serum containing 5-FU at 500 ng / mL and BSA / 5-FU, which were diluted with PBS-T or PBS, or before and after the addition of serum containing 5-FU at 0 ng / mL that had been subjected to each dilution treatment, are shown in Fig. 6. When the serum diluted with PBS-T was dropped, the change range of the gate voltage shift ΔV g became smaller, while when the serum diluted with PBS was dropped, ΔV g showed a large change range. Therefore, it was shown that the influence of contaminants in the serum on the sensor response can be suppressed by the dilution operation with PBS-T.

[0066] [Example 4] As the measurement of the 5-FU concentration in serum using this device, the response measurement by the sequential addition of 5-FU and BSA / 5-FU was evaluated as follows. First, glutaraldehyde was reacted as a cross-linking molecule for cross-linking the amino group of the organic monolayer and Fab. The reaction was carried out by immersing the detection part of the device on which a monolayer with an amino group at the end was formed in 0.01 mL of a 2.5% by mass aqueous solution of glutaraldehyde at room temperature for 30 minutes. Then, the device was placed on a holder, and the gate electrode part cross-linked with glutaraldehyde was immersed in PBS containing 50 μg / mL of Fab for 1 hour. After washing the substrate, a capping treatment was performed with PBS containing 10 mM ethanolamine for 1 hour. Then, the detection part of the device was immersed in 0.5 mL of PB for 3 minutes, and then at room temperature, the current-voltage curve of the Fab-immobilized device was measured using an Ag / AgCl reference electrode with a digital source meter (manufactured by Keithley Instruments, Inc., 2612). The measurement conditions were a gate voltage of -3 V to 0.5 V and a drain voltage of 0.1 V. Subsequently, human serum containing 0 to 1,000 ng / mL of 5-FU was diluted 5-fold with PBS-T, then 20 μL was added to the FET gate surface and left standing for 30 minutes. Then, rinsing was performed using 5 mL of PBS. Then, 20 μL of PBS containing 25 μg / mL of BSA / 5-FU was added onto the gate surface of the Fab-immobilized device, left standing for 30 minutes, and then rinsing was performed using 3 mL of PBS and 3 mL of PB. Then, 0.5 mL of PB was added onto the gate surface and left standing for 3 minutes, and then the current-voltage curve of the BSA / 5-FU adsorbed device was measured, and the gate voltage shift ΔV g was evaluated.

[0067] The evaluation results of the shift amount of semiconductor characteristics by sequential addition of PBS-T diluted 5-FU-containing serum and BSA / 5-FU to the Fab-immobilized device are shown in Fig. 7. To the Fab-immobilized device, 5-FU-containing serum adjusted to a concentration in the range of 0 to 200 ng / mL by PBS-T dilution was added, and then BSA / 5-FU was added sequentially. As a result, it was confirmed that the gate voltage shift ΔV g decreased with an increase in the 5-FU concentration. From this, the quantitative detectability of the 5-FU concentration in serum was demonstrated by the method of the present invention.

Description of Symbols

[0068] 1 Silicon substrate 1a Gate channel region 2 Insulating layer 3 First organic monomolecular film 4 Gate electrode 5 Source electrode 6 Drain electrode 7 Doped region 8 Reference part 9 Detection part 10 Template part 11 Probe molecule 12 Small non-charged molecule 13 Protein modified with small non-charged molecule

Claims

1. (A) A step of causing a small uncharged molecule to interact with a probe molecule that interacts with the small uncharged molecule immobilized on a semiconductor sensing device; (B) After step (A), a step of further causing a protein modified with the small uncharged molecule to interact with the probe molecule; and (C) After step (B), a step of detecting a potential change in a gate channel region due to the interaction A method for detecting a small uncharged molecule, comprising the above steps.

2. The semiconductor sensing device on which the probe molecule is immobilized has a first insulating layer containing silicon oxide or an inorganic oxide as a reaction gate insulating film formed on a semiconductor. On the first insulating layer, a first organic monomolecular film composed of an organic monomolecular film having a reactive functional group is formed, and a probe molecule is bonded to the first organic monomolecular film directly through the reactive functional group or using a crosslinking molecule. The detection method for a small uncharged molecule according to claim 1, comprising a probe molecule / organic monomolecular film / insulating layer / semiconductor structure as a detection unit.

3. The detection method for a small uncharged molecule according to claim 1 or 2, wherein the probe molecule is Fab.

4. The detection method for a small uncharged molecule according to any one of claims 1 to 3, which is a method for detecting a small uncharged molecule contained in a biological sample.

5. The detection method for a small uncharged molecule according to claim 4, wherein the biological sample containing the small uncharged molecule is serum.

6. The detection method for a small uncharged molecule according to claim 4 or 5, comprising a step of pretreating the biological sample containing the small uncharged molecule with a surfactant before step (A).

7. The detection method for a small uncharged molecule according to any one of claims 1 to 5, wherein the small uncharged molecule is 5-fluorouracil.

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