JP7276774B2 - Aptamer-immobilized semiconductor sensing device and method for detecting uncharged molecules - Google Patents

Description

The present invention relates to an aptamer-immobilized semiconductor sensing device and a method for detecting uncharged molecules.

Field effect transistors (FETs) are very promising tools for the detection of biomolecules. When the FET is used, the change in charge density on the gate surface accompanying the adsorption of biomolecules is directly detected as an electrical signal, so label-free detection is possible, and biomolecules can be rapidly detected at low cost. Therefore, research on detection of biomolecules using FETs has been extensively conducted.

Since FET biosensors are easy to operate, small, and inexpensive, they are expected to be applied in various fields such as medicine, food, and environment. A FET biosensor is a device that measures changes in surface potential on a gate electrode due to the charge of a biomolecule trapped in a receptor within the Debye length, which is the charge detection range. Therefore, it has been difficult to detect uncharged molecules that have no charge.

Therefore, it is effective to use a nucleic acid molecule (aptamer) as a probe molecule that changes its structure as it captures a molecule to be measured (hereinafter also referred to as a target molecule) to change the amount of charge in the vicinity of the sensor interface. was (Non-Patent Document 1). This is a method of indirectly detecting the target molecule by measuring the change in the amount of charge within the Debye length caused by the binding of the target molecule, utilizing the structural change of the aptamer as it captures the target molecule. This makes it possible to detect uncharged molecules. However, when the target molecule is dropped, the adjacent aptamers may cause steric hindrance with each other, resulting in insufficient change in the amount of charge, resulting in a decrease in sensitivity.

N. Nakatsuka et al., Science, 362, pp. 319-324 (2018)

It is an object of the present invention to provide a semiconductor sensing device and a method for detecting uncharged molecules that make it possible to detect uncharged molecules simply and with high sensitivity using an FET. do.

As a result of intensive research to achieve the above object, the present inventors immobilized the aptamer in a state of forming a higher-order structure in an FET biosensor using an aptamer as a probe molecule, and then fixed the higher-order structure. The present inventors have found that the target molecule can be detected with high sensitivity by using a device obtained by solving the problem, and have completed the present invention.

Accordingly, the present invention provides the following aptamer-immobilized semiconductor sensing device and uncharged molecule detection method.
1. An aptamer-immobilized semiconductor sensing device comprising a field-effect transistor having a detection portion on which an aptamer is immobilized as a probe molecule,
An aptamer-immobilized semiconductor sensing device, wherein the aptamer is immobilized on a detecting portion while forming a higher-order structure, and then the higher-order structure is dissolved.
2. 1. The aptamer-immobilized semiconductor sensing device of 1, wherein the aptamer is a DNA aptamer.
3. 1 or 2, wherein the higher-order structure is a G-quartet structure.
4. The detection part comprises a first insulating layer containing silicon oxide or inorganic oxide as a reaction gate insulating part formed on a semiconductor, and an organic single molecule having a reactive functional group on the first insulating layer. A first organic monomolecular film composed of a film is formed, and an aptamer in a state of forming a higher-order structure as a probe molecule is directly applied to the first organic monomolecular film via the reactive functional group or a cross-linking molecule. 4. The aptamer-immobilized semiconductor sensing device according to any one of 1 to 3, which comprises an aptamer/organic monomolecular film/insulating layer/semiconductor structure formed by binding using a stoichiometer and then eliminating the higher-order structure.
5. Further formed on the semiconductor is a second insulating layer comprising a silicon oxide or an inorganic oxide as a reference gate insulator, on which the second insulating layer reacts with both the aptamer and uncharged molecules. 4. An aptamer-immobilized semiconductor sensing device comprising an organic monomolecular film/insulating layer/semiconductor structure as a reference portion, which is formed by forming a second organic monomolecular film composed of organic molecules that do not bind to each other.
6. A step of interacting the aptamer immobilized on the aptamer-immobilized semiconductor sensing device of any one of 1 to 5 with an uncharged molecule;
and detecting a surface potential change on the gate electrode due to said interaction.
7. 6. The method for detecting an uncharged molecule according to 6, wherein the uncharged molecule is a compound having a steroid skeleton.

By using the device of the present invention, it becomes possible to detect uncharged molecules with higher sensitivity.

BRIEF DESCRIPTION OF THE DRAWINGS It is sectional drawing which shows the semiconductor sensing device of this invention, (A) is a field effect transistor, (B) is the state which formed the organic monomolecular film on the insulating layer of the gate electrode of a field effect transistor, (C) is an organic This shows a state in which probe molecules are immobilized on a monomolecular film. An example of the unit configuration of an on-chip device is shown, (A) is a partial plan view, and (B) is an enlarged cross-sectional view thereof. 1 is a conceptual diagram of uncharged molecule detection using the semiconductor sensing device of the present invention. FIG. It is the fluorescence observation image by the scanner type image-analysis apparatus measured by the reference example. 2 is a current-voltage curve measured in Example 2. FIG. It is a current-voltage curve measured in Comparative Example 2-1. It is a current-voltage curve measured in Comparative Example 2-2. 10 is a graph showing gate voltage shift values measured in Example 3 and Comparative Example 3; 10 is a graph showing gate voltage shift values measured in Examples 5-1 to 5-3 and Comparative Example 4; 10 is a graph showing gate voltage shift values measured in Examples 7-1 and 7-2 and Comparative Example 6. FIG.

[Aptamer-immobilized semiconductor sensing device]
The aptamer-immobilized semiconductor sensing device of the present invention comprises an FET having a detection part on which an aptamer is immobilized as a probe molecule, wherein the aptamer is bound to the detection part in a state of forming a higher-order structure, After that, it is obtained by eliminating the higher-order structure.

FETs that can be applied to the device of the present invention are not particularly limited, and conventionally known structures such as ion sensitive FETs and extended gate FETs can be used. Semiconductors used in FETs are not particularly limited, and oxide semiconductors such as zinc oxide and indium oxide, molybdenum disulfide, graphene, and two-dimensional material MXene can be used in addition to silicon.

In the present invention, the aptamer to be immobilized on the aptamer-immobilized semiconductor sensing device may be appropriately selected according to the target to be detected. That is, an aptamer that binds to the target uncharged molecule may be selected.

Nucleic acid aptamers are preferred as the aptamers used in the present invention. A nucleic acid aptamer is a molecule containing nucleotide residues, and may be a molecule consisting only of nucleotide residues or a molecule containing nucleotide residues. The nucleotides include ribonucleotides, deoxyribonucleotides and derivatives thereof. The aptamer may be one containing deoxyribonucleotides and/or derivatives thereof (DNA aptamers), one containing ribonucleotides and/or derivatives thereof (RNA aptamers), or one containing both of these (DNA/RNA aptamers). ) can be used. A DNA aptamer is preferable as the aptamer. Moreover, the aptamer may be a single-stranded aptamer or a double-stranded aptamer.

The nucleotides may contain both natural bases and non-natural bases (artificial bases) as bases. The natural bases include adenine, cytosine, guanine, thymine, uracil and modified bases thereof. Examples of the modification include methylation, fluorination, amination, thiolation and the like. Examples of the non-natural base include 2'-fluoropyrimidine, 2'-O-methylpyrimidine, etc. Specifically, 2'-fluorouracil, 2'-aminouracil, 2'-O-methyluracil , 2′-thiouracil and the like.

Said nucleotides may be modified nucleotides. Examples of the modified nucleotides include 2'-methylated uracil nucleotide, 2'-methylated cytosine nucleotide, 2'-fluorinated uracil nucleotide, 2'-fluorinated-cytosine nucleotide, 2'-aminated-uracil nucleotide, 2'. -aminated-cytosine nucleotide, 2'-thiolated-uracil nucleotide, 2'-thiolated-cytosine nucleotide and the like. The aptamer may contain non-nucleotides such as PNA (peptide nucleic acid) and LNA (Locked Nucleic Acid).

The number of bases of the aptamer is not particularly limited, but is usually about 25-200, preferably about 35-120, more preferably about 40-80.

The aptamer is immobilized on the detection part while forming a higher-order structure. Here, higher-order structure means secondary structure, tertiary structure and quaternary structure. Examples of the higher-order structure include G-quartet structure, hairpin loop structure, stem loop structure, pseudoknot structure, bulge loop structure, i-motif structure and the like. As the aptamer, an aptamer that can form any of the higher-order structures described above and that can interact with the desired target molecule is used.

A higher-order structure can be formed by a known method according to the desired higher-order structure. For example, if the aptamer can form a G-quartet structure as a higher-order structure, the G-quartet structure can be formed by adding the aptamer to a solution containing potassium ions. The G-quartet structure is a planar structure formed by four guanine (G) bases. In the present invention, it is preferable to immobilize an aptamer having a G-quartet structure as its higher-order structure.

At this time, the potassium ion concentration in the solution is preferably 30 to 500 mM, more preferably 50 to 300 mM, even more preferably 80 to 200 mM. Examples of the solution include sodium in saline, phosphate-buffered saline, Tris-buffered saline, MES-buffered saline, MOPS-buffered saline, PIPES-buffered saline, HEPES-buffered saline, and the like. Potassium ions substituted for potassium ions can be preferably used. The pH of the solution is preferably 5-10, more preferably 6-8.

In addition, ions such as Ca ²⁺ and Mg ²⁺ , chelating agents such as ethylenediaminetetraacetic acid (EDTA) and glycol etherdiaminetetraacetic acid (EGTA), Tween (registered trademark) 20 and Triton (registered trademark) X are added to the solution. Surfactants such as -100, Nonidet® P-40, and the like may be added. When the ions are added, the concentration is preferably 0.1 to 10 mM, more preferably 0.5 to 5 mM. When the chelating agent is added, its concentration is preferably 0.1-10 mM, more preferably 0.5-5 mM. When a surfactant is added, its concentration is preferably 0.001-10% by volume, more preferably 0.05-5% by volume.

The amount of aptamer added to the solution is preferably such that the concentration in the solution is 0.01 to 10 μM, more preferably 0.01 to 0.1 μM.

As a method for immobilizing the aptamer, conventionally known methods can be applied. Examples thereof include a method of immobilization via a thiol group, a method of forming a graphene sheet in the detection portion and immobilizing it thereon via a pyrene derivative such as 1-pyrenebutanoic acid succinimidyl ester. These methods may be appropriately selected depending on the FET used.

When immobilizing an aptamer via an organic monolayer, the aptamer is immobilized on the organic monolayer directly or via a cross-linking molecule. At this time, glutaraldehyde or the like can be used as the cross-linking molecule. In this case, the method for modifying the organic monomolecular film with glutaraldehyde is not particularly limited. ℃ for 1 minute to 24 hours.

Next, the aptamer is immobilized by reacting the reactive functional groups in the aptamer with glutaraldehyde. Specifically, the reaction may be carried out in a solution containing the aptamer, preferably at 10 to 50°C for 1 minute to 24 hours, more preferably at 10 to 35°C for 1 minute to 60 minutes. The concentration of the aptamer is preferably 0.01-10 μM, more preferably 0.01-0.1 μM.

Finally, the conformation of the aptamer is resolved. As a method for eliminating the higher-order structure, there is a method of immersing the aptamer in a solution having a lower potassium ion concentration than the solution used for immobilizing the aptamer or in a solution containing no potassium ions. Preferred examples of such a solution include physiological saline, phosphate-buffered saline, Tris-buffered saline, MES-buffered saline, MOPS-buffered saline, PIPES-buffered saline, HEPES-buffered saline, and the like. .

In the present invention, the detection part of the FET is formed by forming a first insulating layer containing silicon oxide or inorganic oxide as a reaction gate insulating part on a semiconductor, and forming a reactive function on the first insulating layer. A first organic monomolecular film made of an organic monomolecular film having a group is formed, and an aptamer in a state of forming a higher-order structure is applied to the first organic monomolecular film as a probe molecule via the reactive functional group. It is preferable to have an aptamer/organic monomolecular film/insulating layer/semiconductor structure formed by bonding directly or using a cross-linking molecule, and then eliminating the higher-order structure.

In the detection part, the insulating layer/semiconductor structure part can use an FET in which an insulating layer containing silicon oxide or inorganic oxide is formed as a reaction gate insulating part on a semiconductor. A known one can be used. Preferably, the insulating layer is silicon oxide. FETs may be n-type or p-type. This FET includes, for example, the one shown in FIG. In FIG. 1, 1 is a silicon substrate, 2 is an insulating layer containing silicon oxide or inorganic oxide (glass, alumina, etc.), 4 is a gate electrode, 5 is a source electrode, 6 is a drain electrode, and 7 is a doped region. indicates

Then, as shown in FIG. 1B, a first organic monomolecular film 3 is formed on the insulating layer 2 . Here, in the present invention, as a basic principle, a configuration is adopted in which a change in surface potential accompanying a binding reaction between probe molecules and target molecules on the surface of the insulating layer is detected as an electric signal. The thickness of the insulating layer is preferably 30-300 nm, more preferably 50-150 nm.

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

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

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. The shorter the carbon chain, the weaker the hydrophobicity possessed by the organic monomolecular film, and the less nonspecific adsorption caused by the hydrophobic interaction of the target molecule.

Specific examples of linear alkanediyl groups represented by R ¹ include propane-1,3-diyl, butane-1,4-diyl, pentane-1,5-diyl, 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, and docosane-1,22-diyl group. . Among these, those having 3 to 18 carbon atoms are preferred, and those having 3 to 8 carbon atoms are more preferred.

In formula (1), R ² to 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. be. Examples of the alkyl group include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group and tert-butyl group. Among these, a methyl group or an ethyl group is preferred. Examples of the alkoxyalkyl group include methoxymethyl group, ethoxymethyl group, 2-methoxyethyl group and 2-ethoxyethyl group. Among these, an alkoxyalkyl group having 2 to 3 carbon atoms is preferred. Methyl group, ethyl group, 2-methoxyethyl group and the like are particularly preferable as R ² to R ⁴ .

（式中、Ｒ¹～Ｒ⁴は、前記と同じ。Ｒ⁵は、Ｒ¹から炭素数が２減少した直鎖状アルカンジイル基である。） A commercially available product can be used as the alkoxysilane in which R is an amino group, a carboxyl group, or a thiol group. Moreover, the alkoxysilane in which the R is an aminooxy group can be synthesized according to the following scheme.

(In the formula, R ¹ to R ⁴ are the same as above. ^{R 5} is a linear alkanediyl group with 2 carbon atoms less than R ¹ .)

Alkoxysilanes in which R is an aminooxy group can be prepared by treating a trialkoxyhydrosilane and an O-alkenylhydroxyamine with a platinum-based catalyst. For example, in a nitrogen atmosphere, a mixture of trialkoxyhydrosilane and O-alkenylhydroxyamine is added with a platinum-based catalyst such as hexachloroplatinic (IV) acid, and the mixture is heated at 10 to 200° C. for 1 to 1,200 hours, more preferably 60 hours. It can be prepared by reacting at ~120°C for 12-48 hours. For the film-forming operation, it is preferable to use a product from which excess trialkoxyhydrosilane has been removed by an operation such as distillation.

The first organic monomolecular film is formed by vapor-phase chemical reaction or liquid-phase reaction of the alkoxysilane on the insulating layer, and the optimization thereof, for example, the self-assembly function of the organic molecules, allows close-packing of the monomolecules. A thin film is formed. When forming a monomolecular film by a vapor phase chemical reaction, for example, the substrate and alkoxysilane are enclosed in a container, and the temperature is preferably 80 to 200° C. for 1 to 24 hours, more preferably 100 to 130° C. in a dry room. A film can be formed by reacting for 2 to 5 hours at . When forming a monomolecular film by a liquid phase reaction, for example, the substrate is immersed in an organic solvent containing alkoxysilane, preferably at 20 to 80°C for 1 minute to 24 hours, more preferably at 55 to 65°C. A film can be formed by standing still for 5 to 20 minutes.

Examples of the organic solvent include toluene, methanol, ethanol and the like, with toluene and methanol being particularly preferred.

A second insulating layer comprising silicon oxide or inorganic oxide can be further formed on the semiconductor of the FET as a reference gate insulator. On the second insulating layer, a monomolecular film composed of organic molecules that do not react with either the aptamer that is the probe molecule or the uncharged molecule that is the target molecule is formed as the second organic monomolecular film. , this monolayer/insulating layer/semiconductor structure can be used as a reference. If the reaction gate insulating portion and the reference gate insulating portion are separated from each other to such an extent that they do not affect each other in potential change measurement, the first insulating layer of the reaction gate insulating portion and the second insulating layer of the reference gate insulating portion layers can also be provided in the same layer.

FIG. 2 shows a unit configuration example of an on-chip device in which an organic monomolecular film/insulating layer/semiconductor structure is applied to the detection section 9 and the reference section 8. As shown in FIG. 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. can be placed as Also, the detection section and the reference section can each be formed with a size of several to several tens of μm.

The second organic monomolecular film is preferably an alkoxysilane monomolecular film having a linear alkyl group having 8 to 22 carbon atoms which may be fluorinated. When an alkoxysilane monomolecular film is used as the organic monomolecular film, the second insulating layer is preferably made of silicon oxide.

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

In formula (2), R ⁶ is a linear alkyl group having 8 to 22 carbon atoms, preferably 10 to 18 carbon atoms, and some 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 and n-pentadecyl. group, n-hexadecyl group, n-heptadecyl group, n-octadecyl group, n-nonadecyl group, n-eicosyl group, n-heneicosyl group, n-docosyl group and the like.

In formula (2), R ⁷ to 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. be. Examples of the alkyl group include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group and tert-butyl group. Among these, a methyl group or an ethyl group is preferred. Examples of the alkoxyalkyl group include methoxymethyl group, ethoxymethyl group, 2-methoxyethyl group and 2-ethoxyethyl group. Among these, an alkoxyalkyl group having 2 to 3 carbon atoms is preferred.

Specific examples of _{trialkoxysilanes} 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 ) 10Si} ( _OCH3 ) ₃ , _CH3 ( _CH2 ) _10Si ( _OC2H5 ) ₃ , _CH3 ( _CH2 ) _11Si ( _OCH3 ) ₃ , _CH3 ( _CH2 ) _11Si ( _OC2H5 ) ₃ , _CH3 ( _{CH2 ) 12Si} ( _OCH3 ) ₃ , _CH3 ( _CH2 ) _12Si ( _OC2H5 ) ₃ , _{CH3 ( CH2} ) _13Si ( _OCH3 ) ₃ , _CH3 ( _{CH2 ) 13Si} ( _OC2H5 ) ₃ , _CH3 ( _CH2 ) _14Si ( _OCH3 ) ₃ , _CH3 ( _CH2 ) _14Si ( _{OC2H5 ) 3} , _CH3 ( _{CH2 ) 15Si} ( _OCH3 ) ₃ , _CH3 ( _CH2 ) _15Si ( _OC2H5 ) ₃ , _CH3 ( _CH2 ) _16Si ( _OCH3 ) ₃ , _CH3 ( _CH2 ) _16Si ( _OC2H5 ) ₃ , _CH3 ( _{CH2 ) 17Si} ( _{OCH3 ) 3} , _CH3 ( _CH2 ) _17Si ( _OC2H5 ) ₃ , _CH3 ( _CH2 ) _18Si ( _OCH3 ) ₃ , _CH3 ( _CH2 ) _18Si ( _OC2H5 ) ₃ , _CH3 ( _CH2 ) _19Si ( _OCH3 ) _{3 , CH3} ( _CH2 ) _19Si ( _{OC2H5 ) 3} , _CH3 ( _{CH2 ) 20Si} ( _OCH3 ) ₃ , _CH3 ( _CH2 ) _20Si ( _OC2H5 ) ₃ , _CH3 ( _CH2 ) _21Si ( _OCH3 ) ₃ , _CH3 ( _CH2 ) _21Si ( _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 (CF ₂ ) 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 )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} (CF ₂ ) 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 )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} (CF _{2 ) 19} ( _CH2 ) _2Si ( _OC2H5 ) ₃ and the like.

The first and second organic monomolecular films can be formed at desired positions by patterning. In particular, patterning of organic monomolecular films is effective for forming on-chip integrated devices. For example, on the surface of the insulating layer of the detection section, a first monomolecular film composed of organic molecules having a reactive functional group for immobilizing probe molecules is formed on the surface of the reference section, and further the non-gate section ( In the template part), in order to avoid non-specific adsorption of the target molecule, a second organic monomolecular film composed of organic molecules that do not react with either the probe molecule or the target molecule is position-selectively patterned. Form.

As the reference portion, it is also possible to use a second organic monomolecular film in which a compound that does not interact with the target molecule is immobilized on the same monomolecular film as the first organic monomolecular film. That is, a compound/organic monomolecular film/insulating layer/semiconductor structure that does not interact with the target molecule can be used as the reference portion. In this case, the reference portion can be formed according to the same method as the method for forming the organic monomolecular film in the detection portion described above and the method for immobilizing the compound described later.

In the semiconductor sensing device, an aptamer, which is a probe molecule, is immobilized on the first organic monomolecular film of the detection section. For example, as shown in FIG. 1(C), an aptamer 11 is bound to the first organic monomolecular film 3 .

[Method for detecting uncharged molecules]
The method for detecting an uncharged molecule of the present invention comprises a step of allowing the aptamer immobilized on the aptamer-immobilized semiconductor sensing device to interact with the uncharged molecule, and detecting a change in surface potential on the gate electrode due to the interaction. and the step of

FIG. 3 shows a conceptual diagram of a method for detecting uncharged molecules based on aptamer-uncharged molecule interaction using the aptamer-immobilized semiconductor sensing device of the present invention. In this detection method, the aptamer directly immobilized on the organic monomolecular film is allowed to interact with an uncharged molecule, and the surface potential change of the insulating layer caused by this interaction is detected as an electrical signal. In addition, in FIG. 3, 12 is an uncharged molecule. Further, other configurations are denoted by the same reference numerals as in FIG. 1, and descriptions thereof are omitted.

Since the aptamer has a phosphate group, when the aptamer immobilized on the device interacts with an uncharged molecule, the structure of the aptamer changes as the target molecule is captured, resulting in a charge within the Debye length. The amount changes and the surface potential on the gate electrode shifts. In this case, potential shift can be detected under constant current, and current shift can be detected under constant voltage. It should be noted that the shift of the threshold voltage shows the same behavior when using an n-type FET and when using a p-type FET.

In order to allow the aptamer immobilized on the device to interact with the uncharged molecule, a solution containing the uncharged molecule may be diluted as necessary and placed on the gate electrode. At this time, as the solution, a general solution used for detection of uncharged molecules can be used, but a solution that satisfies physiological conditions is particularly preferable. For example, physiological saline, phosphate-buffered physiological saline, Tris-buffered physiological saline, MES-buffered physiological saline, MOPS-buffered physiological saline, PIPES-buffered physiological saline, HEPES-buffered physiological saline and the like can be preferably used. The pH of the solution is preferably 5-10, more preferably 6-8.

In addition, ions such as Ca ²⁺ and Mg ²⁺ , chelating agents such as ethylenediaminetetraacetic acid (EDTA) and glycol etherdiaminetetraacetic acid (EGTA), Tween (registered trademark) 20 and Triton (registered trademark) X are added to the solution. Surfactants such as -100, Nonidet® P-40, and the like may be added. When the ions are added, the concentration is preferably 0.1 to 10 mM, more preferably 0.5 to 5 mM. When the chelating agent is added, its concentration is preferably 0.1-10 mM, more preferably 0.5-5 mM. When a surfactant is added, its concentration is preferably 0.001-10% by volume, more preferably 0.05-5% by volume.

The temperature at which the aptamer immobilized on the device and the 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, even more preferably 5 to 30 minutes.

The uncharged molecule to be detected in the present invention is not particularly limited as long as it has the property of interacting with the aptamer. Examples thereof include compounds having a steroid skeleton, peptides, amino acid derivatives, aromatic compounds, sugars and the like.

Examples of compounds having a steroid skeleton include cortisol, cholesterol, progesterone, 11-deonesicorticosterone, corticosterone, aldosterone, cortisone, dehydroepiandrosterone, dehydroepiandrosterone sulfate, dihydrotestosterone, androsterone, epiandro Examples include sterone, testosterone, 17β-estradiol, estrone, and estriol.

Examples of the peptide include calcitonin, thyrotropin-releasing hormone, vasopressin, adrenocorticotrophic hormone, gonadotropin-releasing hormone, growth hormone, oxytocin, glucagon, secretin, and the like.

Examples of the amino acid derivative include melatonin, thyroid hormone, catecholamine, and the like.

The aromatic compounds include toluene, ethylbenzene, cumene, benzyl alcohol, anisole, benzaldehyde, acetophenone, nitrobenzene, thiophenol, benzonitrile, styrene, xylene, biphenyl, benzophenone, triphenylmethane, naphthalene, anthracene, tetracene, pentacene, phenanthrene, chrysene, triphenylene, tetraphene, pyrene, picene, pentaphene, perylene, helicene, coronene and the like.

The sugars include glucose, allose, talose, gulose, altrose, mannose, galactose, idose, sedoheptulose, colyose, psicose, fructose, sorbose, tagatose, ribose, lyxose, xylose, arabinose, apiose, ribulose, xylulose, erythrose, Monosaccharides such as threose, erythrulose and glyceraldehyde, and oligosaccharides and polysaccharides composed of these monosaccharides are included.

The concentration of the detectable uncharged molecule varies depending on its type, but is usually about 10 nM to 10 mM, preferably about 100 nM to 1 mM.

The device of the present invention is obtained by immobilizing an aptamer having a higher-order structure and then removing the higher-order structure. This allows highly sensitive detection of uncharged molecules. It also allows the detection of uncharged molecules under physiological conditions. Although the reason for this is not clear, when aptamers that do not form higher-order structures are immobilized, steric hindrance between adjacent aptamers inhibits structural changes, which reduces the amount of change in interfacial charge and reduces sensitivity. However, by immobilizing the aptamer in a state of forming a higher-order structure, it is immobilized at an appropriate density, and the steric hindrance between adjacent aptamers against structural changes during measurement of the target molecule is alleviated. An increase in response is expected.

EXAMPLES The present invention will be specifically described below with reference to reference examples, examples, and comparative examples, but the present invention is not limited to the following examples. Aptamers used in Examples were provided by NEC Solution Innovators.

[1] Construction of semiconductor sensing device -1
[Example 1] Construction of aptamer-immobilized semiconductor sensing device 1 (1) Formation of organic monomolecular film An n-type FET with a length of 10 µm and a width of 1,000 µm manufactured by Toppan Printing Co., Ltd. is subjected to ultrasonic treatment using acetone. This removed the photoresist. A plasma reactor PR301 (manufactured by Yamato Scientific Co., Ltd.) was used to expose the gate surface to 200 W O ₂ plasma for 1 minute in order to introduce a hydroxyl group to the gate surface and create an active site.
The device was immersed in toluene containing 1% by mass of aminopropyltriethoxysilane (APS, manufactured by Sigma-Aldrich) and allowed to stand at 60° C. for 7 minutes in an argon atmosphere to form a monomolecular film on the gate. filmed. The FET with the monomolecular film formed thereon was ultrasonically cleaned using a mixed solvent of methanol/toluene (mass ratio 1:1) and rinsed with ethanol to fabricate an FET with the APS monomolecular film formed on the gate surface.

(2) Fixation of aptamer Glutaraldehyde was reacted as a cross-linking molecule for cross-linking the amino group of the monomolecular film and the aptamer. The reaction was carried out in phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, _{8.1 mM} Na2HPO4.12H2O, _1.5 mM _KH2PO4 , pH _7.0 ) containing 2.5% by mass of glutaraldehyde. 4, hereinafter also referred to as 1×PBS.) 10 μL was dropped onto the detection part of the device on which the monomolecular film was formed, and left to stand at room temperature for 30 minutes.

Next, a buffer solution (140 mM KCl, 8.1 mM K ₂ HPO ₄ , 1.5 mM KH ₂ PO ₄ , pH 7.4, hereinafter also referred to as 1×PBSK in which all sodium ions in 1×PBS are replaced with potassium ions). ), the aptamer was added to 100 nM.

The gate electrode section crosslinked with glutaraldehyde was immersed in 1×PBSK containing the aptamer for 1 hour to immobilize the aptamer on the organic monolayer. The aptamer-immobilized device was washed once with 1×PBS containing 0.05% by mass Triton X-100 and three times with 1×PBS, ultrasonically washed, and three times with 0.04×PBSK. Washed and dried. After drying, 20 μL of 1×PBS containing 10 mM Tris was dropped onto the aptamer-immobilized organic monolayer and allowed to stand at room temperature for 60 minutes to construct an aptamer-immobilized device 1 .

[Comparative Example 1] Construction of aptamer-immobilized semiconductor sensing device 2 In the same manner as in Example 1, except that 1xPBS containing 100nM aptamer was used instead of 1xPBSK containing 100nM aptamer. An immobilization device 2 was constructed.

[Reference example]
An aptamer-immobilized SiO ₂ substrate prepared in the same manner as in Comparative Example 1 was treated with 1×PBS containing 200 nM of N-methylmesoporphyrin IX (hereinafter referred to as NMM), which is a fluorescent reagent that binds to the guanine quadruplex structure. Alternatively, it was immersed in 1×PBSK for 1 hour. Then, using a scanner-type image analyzer (Typhoon 9410, manufactured by GE Healthcare Japan), the fluorescence intensity of each substrate depending on the amount of NMM binding was observed.
The results are shown in FIG. The higher fluorescence intensity after immersion in PBSK suggested that the aptamer formed a guanine quadruplex structure with potassium ions. That is, in Example 1, it was suggested that the aptamer was immobilized while forming a guanine quadruplex structure.

[2] Detection of uncharged molecules-1
[Example 2]
After washing the device 1, it was placed on a holder, and the detection part of the device was immersed in 0.04×PBSK 0.5 mL for 3 minutes. After immersion, the current-voltage curve of the aptamer-immobilized device was measured at room temperature with a digital source meter (Keithley, 2612) using an Ag/AgCl reference electrode. The measurement conditions were a gate voltage (V _g ) of -3.0 to 0.5V and a drain voltage (V _d ) of 0.1V. Subsequently, 20 μL of 1×PBS containing 1 mM cortisol was added onto the gate surface of the aptamer-immobilized device, allowed to stand for 30 minutes, and then rinsed three times with 1 mL of 1×PBS and 1 mL of 0.04×PBSK. After that, 0.5 mL of 0.04×PBSK was added to the gate surface and allowed to stand for 3 minutes, and then the current-voltage curve of the cortisol adsorption device was measured to evaluate the gate voltage shift ΔV _g before and after adding cortisol. . The results are shown in FIG.

[Comparative Example 2-1]
The current-voltage curve of the cortisol adsorption device was measured in the same manner as in Example 2, except that Device 2 was used instead of Device 1, and the gate voltage shift ΔV _g before and after the addition of cortisol was evaluated. The results are shown in FIG.

[Comparative Example 2-2]
The current-voltage curve of the cortisol adsorption device was measured in the same manner as in Example 2, except that 1×PBS containing 20 μM α-amylase was used instead of 1×PBS containing 1 mM cortisol. Gate voltage shift ΔV _g was evaluated. The results are shown in FIG.

5 to 7, dotted lines represent device characteristics before cortisol addition, and solid lines represent device characteristics after cortisol addition. When cortisol was added to the aptamer-immobilized device 1, the current-voltage curve shifted significantly in the positive direction.

[3] Detection of uncharged molecules-2
[Example 3, Comparative Example 3]
Devices 1 and 2 were used to evaluate the gate voltage shift ΔV _g before and after addition of 1×PBS containing 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 100 μM and 1 mM cortisol. The results are shown in FIG.

The results shown in FIGS. 5 to 8 indicate that using a device in which aptamers are immobilized in a solution containing high concentrations of potassium ions is advantageous in terms of detection sensitivity.

[4] Construction of semiconductor sensing device -2
[Example 4-1] Construction of aptamer-immobilized semiconductor sensing device 3 Instead of adding the aptamer to 1 × PBSK so that the aptamer became 100 nM, the aptamer was added to a solution in which equal amounts of 1 × PBS and 1 × PBSK were mixed. An aptamer-immobilized device 3 was constructed in the same manner as in Example 1, except that the was added so as to be 100 nM.

[Example 4-2] Construction of aptamer-immobilized semiconductor sensing device 4 Instead of adding 100 nM of aptamer to 1 x PBSK, 1 x PBS and 1 x PBSK were mixed at a ratio of 1:9. An aptamer-immobilized device 4 was constructed in the same manner as in Example 1, except that 100 nM of aptamer was added to the resulting solution.

[5] Detection of uncharged molecules-3
[Example 5-1]
Using Device 1, the gate voltage shift ΔV _g before and after adding cortisol was evaluated in the same manner as in Example 2. The results are shown in FIG.

[Example 5-2]
The gate voltage shift ΔV _g before and after adding cortisol was evaluated in the same manner as in Example 2, except that Device 3 was used instead of Device 1. The results are shown in FIG.

[Example 5-3]
The gate voltage shift ΔV _g before and after adding cortisol was evaluated in the same manner as in Example 2, except that Device 4 was used instead of Device 1. The results are shown in FIG.

[Comparative Example 4]
The gate voltage shift ΔV _g before and after the addition of cortisol was evaluated in the same manner as in Example 2, except that Device 2 was used instead of Device 1. The results are shown in FIG.

[6] Construction of semiconductor sensing device -3
[Example 6] Construction of aptamer-immobilized semiconductor sensing device 5 Instead of adding 100 nM of aptamer to 1 x PBSK, 0.1 x PBSK was added to 100 nM of aptamer. , an aptamer-immobilized device 5 was constructed in the same manner as in Example 1.

[Comparative Example 5] Construction of aptamer-immobilized semiconductor sensing device 6 An aptamer-immobilized device 6 was constructed in the same manner as in Example 1.

[7] Detection of uncharged molecules-4
[Example 7-1]
Using Device 1, the gate voltage shift ΔV _g before and after adding cortisol was evaluated in the same manner as in Example 2. The results are shown in FIG.

[Example 7-2]
The gate voltage shift ΔV _g before and after cortisol addition was evaluated in the same manner as in Example 2, except that Device 5 was used instead of Device 1. The results are shown in FIG.

[Comparative Example 6]
The gate voltage shift ΔV _g before and after adding cortisol was evaluated in the same manner as in Example 2, except that Device 6 was used instead of Device 1. The results are shown in FIG.

1 silicon substrate 2 insulating layer 3 first organic monomolecular film 4 gate electrode 5 source electrode 6 drain electrode 7 doped region 8 reference portion 9 detection portion 10 template portion 11 aptamer (probe molecule)
12 uncharged molecules

Claims (7)

A method for producing an aptamer-immobilized semiconductor sensing device comprising a field-effect transistor having a detection portion on which a nucleic acid aptamer is immobilized as a probe molecule,
A method for producing an aptamer-immobilized semiconductor sensing device, wherein the nucleic acid aptamer is immobilized in a state of forming a G-quartet structure on a detection part, and then the G-quartet structure is eliminated . 2. The method for producing an aptamer-immobilized semiconductor sensing device according to claim 1, wherein the nucleic acid aptamer is a DNA aptamer. 3. The method for producing an aptamer-immobilized semiconductor sensing device according to claim 2, wherein the nucleic acid aptamer is added to a solution containing potassium ions to form a G-quartet structure. The detection part comprises a first insulating layer containing silicon oxide or inorganic oxide as a reaction gate insulating part formed on a semiconductor, and an organic single molecule having a reactive functional group on the first insulating layer. A first organic monomolecular film composed of a film is formed, and a nucleic acid aptamer in a state of forming a G-quartet structure as a probe molecule is directly or crosslinked to the first organic monomolecular film via the reactive functional group. 4. The method according to any one of claims 1 to 3, comprising an aptamer/organic monolayer/insulating layer/semiconductor structure formed by bonding using molecules and then eliminating the G-quartet structure. A method for producing an aptamer-immobilized semiconductor sensing device. Further formed on the semiconductor is a second insulating layer comprising silicon oxide or inorganic oxide as a reference gate insulator, on which the second insulating layer reacts with both the aptamer and uncharged molecules. 5. The method for producing an aptamer-immobilized semiconductor sensing device according to claim 4, wherein the organic monomolecular film/insulating layer/semiconductor structure formed by forming a second organic monomolecular film composed of organic molecules that do not bind to each other is provided as a reference part. a step of interacting the aptamer immobilized on the aptamer-immobilized semiconductor sensing device obtained by the method for producing an aptamer-immobilized semiconductor sensing device according to any one of claims 1 to 5 with an uncharged molecule;
and detecting a surface potential change on the gate electrode due to said interaction. 7. The method of detecting an uncharged molecule according to claim 6, wherein the uncharged molecule is a compound having a steroid skeleton.

Images