JP2021076529A - Aptamer immobilization semiconductor sensing device and method for detecting uncharged molecules - Google Patents

Abstract

To provide a semiconductor sensing device and a method for detecting uncharged molecules that allow detection of uncharged molecules easily and sensitively by using a field-effect transistor.SOLUTION: The present invention relates to an aptamer immobilization semiconductor sensing device having a field-effect transistor including a detection unit with an aptamer immobilized as a probe molecule. The aptamer is immobilized in the detection unit while a high-order structure is formed, and the high-order structure is thereafter removed.SELECTED DRAWING: None

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 due to the adsorption of biomolecules is directly detected as an electric signal, so that label-free detection is possible, and biomolecules can be detected quickly at low cost. Therefore, studies on the detection of biomolecules using FETs have been widely conducted.

Since FET biosensors are easy to operate, small in size, and inexpensive, they are expected to be applied in various fields such as medicine, food, and the environment. The FET biosensor is a device that measures the change in surface potential on the gate electrode due to the charge of the biomolecule captured by the receptor within the Debye length, which is the charge detection range. Therefore, it has been difficult to detect uncharged molecules that do not have an electric charge.

Therefore, it is effective to change the amount of charge near the sensor interface by using a nucleic acid molecule (aptamer) whose structure changes as the probe molecule captures the molecule to be measured (hereinafter, also referred to as a target molecule). (Non-Patent Document 1). This is a method of indirectly detecting a target molecule by measuring the change in the amount of charge within the Debye length caused by the binding of the target molecule by utilizing the structural change of the aptamer due to the capture of the target molecule. This makes it possible to detect uncharged molecules. However, when the target molecule is dropped, adjacent aptamers cause steric hindrance to each other, so that the amount of charge does not change sufficiently, and the sensitivity may decrease.

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

The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a semiconductor sensing device and a method for detecting uncharged molecules, which enable simple and highly sensitive detection of uncharged molecules using FETs. To do.

As a result of diligent research to achieve the above object, the present inventors fixed the aptamer in a state in which a higher-order structure was formed in the FET biosensor using the aptamer as a probe molecule, and then obtained the higher-order structure. We have found that it is possible to detect the target molecule with high sensitivity by using the device obtained by solving the problem, and have completed the present invention.

Therefore, the present invention provides the following aptamer-immobilized semiconductor sensing device and a method for detecting uncharged molecules.
1. 1. An aptamer-immobilized semiconductor sensing device including a field-effect transistor having an aptamer-immobilized detector as a probe molecule.
An aptamer-immobilized semiconductor sensing device in which the aptamer is immobilized on a detection unit in a state where a higher-order structure is formed, and then the higher-order structure is eliminated.
2. The aptamer-immobilized semiconductor sensing device of 1 in which the aptamer is a DNA aptamer.
3. 3. The aptamer-immobilized semiconductor sensing device of 1 or 2 in which the higher-order structure is a G-quartet structure.
4. In the detection unit, a first insulating layer containing a silicon oxide or an inorganic oxide is formed on the semiconductor as a reaction gate insulating portion, and an organic single molecule having a reactive functional group is formed on the first insulating layer. A first organic monomolecular film composed of a film is formed, and an aptamer in a state in which a higher-order structure is formed as a probe molecule is directly or crosslinked via the reactive functional group on the first organic monomolecular film. 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, which is bonded by using and then eliminated the higher-order structure.
5. A second insulating layer containing a silicon oxide or an inorganic oxide is further formed on the semiconductor as a reference gate insulating portion, and reacts with either the aptamer or the uncharged molecule on the second insulating layer. 4. An aptamer-immobilized semiconductor sensing device having an organic monomolecular film / insulating layer / semiconductor structure as a reference portion, which forms a second organic monomolecular film composed of organic molecules that do not.
The step of interacting the aptamer immobilized on the aptamer-immobilized semiconductor sensing device according to any one of 6.1 to 5 with an uncharged molecule, and
A method for detecting an uncharged molecule, which comprises a step of detecting a change in surface potential on a gate electrode due to the interaction.
7. 6. A method for detecting an uncharged molecule in 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.

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

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

The FET applicable to the device of the present invention is not particularly limited, and conventionally known configurations such as an ion sensitive FET, an extended gate FET, and the like can be used. The semiconductor used for the FET is not particularly limited, and in addition to silicon, oxide semiconductors such as zinc oxide and indium oxide, molybdenum disulfide, graphene, MXene, which is a two-dimensional material, and the like can be used.

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

As the aptamer used in the present invention, a nucleic acid aptamer is preferable. The nucleic acid aptamer is a molecule containing a nucleotide residue, and may be a molecule consisting of only a nucleotide residue or a molecule containing a nucleotide residue. Examples of the nucleotide include ribonucleotides, deoxyribonucleotides and derivatives thereof. The aptamer may be one containing a deoxyribonucleotide and / or a derivative thereof (DNA aptamer), one containing a ribonucleotide and / or a derivative thereof (RNA aptamer), or one containing both of them (DNA / RNA aptamer). ) May be. As the aptamer, a DNA aptamer is preferable. Further, the aptamer may be a single-stranded aptamer or a double-stranded aptamer.

The nucleotide may contain either a natural base or an unnatural base (artificial base) as a base. Examples of the natural base include adenine, cytosine, guanine, thymine, uracil and modified bases thereof. Examples of the modification include methylation, fluorolysis, amination, thiolation and the like. Examples of the unnatural base include 2'-fluoropyrimidine, 2'-O-methylpyrimidine, and the like, and specifically, 2'-fluorouracil, 2'-aminouracil, and 2'-O-methyluracil. , 2'-thiouracil and the like.

The nucleotide may be a modified nucleotide. The modified nucleotides include 2'-methylated urasyl nucleotides, 2'-methylated cytosine nucleotides, 2'-fluoroylated urasyl nucleotides, 2'-fluoroylated-cytosine nucleotides, 2'-aminoated-urasyl nucleotides, 2'. Examples thereof include -aminated-cytosine nucleotides, 2'-thiolated-uracil nucleotides, 2'-thiolated-cytosine nucleotides 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 to 200, preferably about 35 to 120, and more preferably about 40 to 80.

The aptamer is immobilized on the detection unit in a state where a higher-order structure is formed. Here, the higher-order structure means a secondary structure, a tertiary structure, and a quaternary structure. Examples of the higher-order structure include a G-quartet structure, a hairpin loop structure, a stem loop structure, a pseudoknot structure, a bulge loop structure, an i-motif structure and the like. As the aptamer, one that can form any of the above-mentioned higher-order structures and that can interact with the target molecule of interest is used.

The higher-order structure can be formed by a known method according to the higher-order structure of interest. For example, when 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 a 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, and even more preferably 80 to 200 mM. Examples of the solution include sodium such as physiological saline, phosphate buffered saline, Tris buffered saline, MES buffered saline, MOPS buffered saline, PIPES buffered saline, and HEPES buffered saline. Those in which the ions are replaced with potassium ions can be preferably used. The pH of the solution is preferably 5 to 10, more preferably 6 to 8.

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

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

As a method for immobilizing an aptamer, a conventionally known method can be applied. For example, a method for immobilizing a detection unit via an organic monolayer having a reactive functional group, a method using a gold electrode as a detection unit, and the like. Examples thereof include a method of immobilizing via a thiol group, a method of forming a graphene sheet in the detection part, and immobilizing the graphene sheet via a pyrene derivative such as 1-pyrenebutanoic acid succinimidyl ester. These methods may be appropriately selected depending on the FET used.

When the aptamer is immobilized via an organic monolayer, the aptamer is immobilized on the organic monolayer either directly or via a crosslinked molecule. At this time, examples of the crosslinked molecule include glutaraldehyde and the like. In this case, the method for modifying the organic monomolecular film with glutaraldehyde is not particularly limited, but for example, 10 to 50 in phosphate buffered saline (PBS) containing 0.01 to 25% by mass of glutaraldehyde. The reaction may be carried out at ° C. for 1 minute to 24 hours.

Next, the aptamer is immobilized by reacting the reactive functional group 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 to 10 μM, more preferably 0.01 to 0.1 μM.

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

In the present invention, in the FET detection unit, a first insulating layer containing a silicon oxide or an inorganic oxide is formed on the semiconductor as a reaction gate insulating portion, and the reactive functional group is formed on the first insulating layer. A first organic monomolecular film composed of an organic monomolecular film having a group is formed, and an aptamer in a state in which a higher-order structure is formed as a probe molecule is formed on the first organic monomolecular film via the reactive functional group. It is preferable to have an aptamer / organic monomolecular film / insulating layer / semiconductor structure which is directly or by using a crosslinked molecule and then the higher-order structure is eliminated.

Among the detection parts, the insulating layer / semiconductor structure part can utilize an FET in which an insulating layer containing a silicon oxide or an inorganic oxide is formed as a reaction gate insulating part on the semiconductor, and the configuration thereof is conventional. Known ones can be used. The insulating layer is preferably a silicon oxide. The FET may be n-type or p-type. Examples of this FET include those shown in FIG. 1 (A). In FIG. 1, 1 is a silicon substrate, 2 is an insulating layer containing a silicon oxide or an inorganic oxide (glass, alumina, etc.), 4 is a gate electrode, 5 is a source electrode, 6 is a drain electrode, and 7 is a dope region. Is shown.

Then, as shown in FIG. 1 (B), the first organic monolayer 3 is formed on the insulating layer 2. Here, in the present invention, as a basic principle, the surface potential change accompanying the bonding reaction between the probe molecule and the target molecule on the surface of the insulating layer is detected as an electric signal. The thickness of the insulating layer is preferably 30 to 300 nm, particularly preferably 50 to 150 nm.

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

In the 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, and more preferably 3 to 8 carbon atoms. A shorter carbon chain is preferable because the hydrophobicity of the organic monolayer is weakened and non-specific adsorption due to the hydrophobic interaction of the target molecule can be suppressed.

Specific examples of the linear alkanediyl group represented by R ¹ include propane-1,3-diyl group, butane-1,4-diyl group, pentadecane-1,5-diyl group, and hexane-1,6. -Diyl group, heptane-1,7-diyl group, octane-1,8-diyl group, nonan-1,9-diyl group, decan-1,10-diyl group, undecane-1,11-diyl group, docosane -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- Examples include diyl group, octadecane-1,18-diyl group, nonadecane-1,19-diyl group, eikosan-1,20-diyl group, heneicosane-1,21-diyl group, docosane-1,22-diyl group. .. Of these, those having 3 to 18 carbon atoms are preferable, and those having 3 to 8 carbon atoms are more preferable.

In the formula (1), R ^{2 to} R ⁴ are independently linear or branched alkyl groups having 1 to 5 carbon atoms or linear or branched alkoxyalkyl groups having 2 to 5 carbon atoms. is there. Examples of the alkyl group include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group and the like. Of these, a methyl group or an ethyl group is preferable. Examples of the alkoxyalkyl group include a methoxymethyl group, an ethoxymethyl group, a 2-methoxyethyl group, a 2-ethoxyethyl group and the like. Of these, an alkoxyalkyl group having 2 to 3 carbon atoms is preferable. As R ^{2 to} R ⁴ , a methyl group, an ethyl group, a 2-methoxyethyl group and the like are particularly preferable.

（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。） As the alkoxysilane in which R is an amino group, a carboxy group or a thiol group, a commercially available product can be used. Further, the alkoxysilane in which 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 described above. R ⁵ is a linear alkanediyl group having 2 fewer carbon atoms than ^{R 1.)}

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

In the first organic monomolecular film, the alkoxysilane is formed on the insulating layer by a vapor phase chemical reaction or a liquid phase reaction, and the monomolecules are densely packed by the optimization thereof, for example, the self-integration function of the organic molecules. A film is formed. When a monomolecular film is formed by a vapor phase chemical reaction, for example, a substrate and an alkoxysilane are sealed 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 with the above for 2 to 5 hours. 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 allowing it to stand for 5 to 20 minutes.

Examples of the organic solvent include toluene, methanol, ethanol and the like, and toluene, methanol and the like are particularly preferable.

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

FIG. 2 shows an example of a unit configuration of an on-chip device in which an organic monolayer / insulating layer / semiconductor structure is applied to a detection unit 9 and a 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 configuration shown in the figure, and the detection unit and the reference unit do not necessarily have to be arranged in a one-to-one relationship, and the number and combination of the detection unit and the reference unit are appropriately changed as necessary. Can be placed. Further, the detection unit and the reference unit can each be formed in a size of several to several tens of μm.

As the second organic monolayer, a monolayer of alkoxysilane having a linear alkyl group having 8 to 22 carbon atoms, which may be fluorinated, is preferable. When a monolayer of alkoxysilane is used as the organic monolayer, the second insulating layer is preferably formed of silicon oxide.

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

In the formula (2), R ⁶ is a linear alkyl group having 8 to 22 carbon atoms, preferably 10 to 18 carbon atoms, and a part or all of hydrogen atoms may be substituted with fluorine atoms. The linear alkyl group preferably has 10 to 18 carbon atoms. Specifically, the linear alkyl group includes an n-octyl group, an n-nonyl group, an n-decyl group, an n-undecyl group, an n-dodecyl group, an n-tridecylic group, an n-tetradecyl group, and an n-pentadecylic group. Examples thereof include a group, an n-hexadecyl group, an n-heptadecyl group, an n-octadecyl group, an n-nonadecil group, an n-eicosyl group, an n-heneicosyl group, an n-docosyl group and the like.

In formula (2), R ^{7 to} R ⁹ are independently linear or branched alkyl groups having 1 to 5 carbon atoms or linear or branched alkoxyalkyl groups having 2 to 5 carbon atoms. is there. Examples of the alkyl group include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group and the like. Of these, a methyl group or an ethyl group is preferable. Examples of the alkoxyalkyl group include a methoxymethyl group, an ethoxymethyl group, a 2-methoxyethyl group, a 2-ethoxyethyl group and the like. Of these, an alkoxyalkyl group having 2 to 3 carbon atoms is preferable.

Specifically, as the trialkoxysilane represented by the formula (2), CH ₃ (CH ₂ ) ₇ Si (OCH ₃ ) ₃ , CH ₃ (CH ₂ ) ₇ Si (OC ₂ H ₅ ) ₃ , CH ₃ ( CH ₂ ) ₈ Si (OCH ₃ ) ₃ , CH ₃ (CH ₂ ) ₈ Si (OC ₂ H ₅ ) ₃ , CH ₃ (CH ₂ ) ₉ Si (OCH ₃ ) ₃ , CH ₃ (CH ₂ ) ₉ Si ( OC ₂ H ₅ ) ₃ , CH ₃ (CH ₂ ) ₁₀ Si (OCH ₃ ) ₃ , CH ₃ (CH ₂ ) ₁₀ Si (OC ₂ H ₅ ) ₃ , CH ₃ (CH ₂ ) ₁₁ Si (OCH ₃ ) ₃ , CH ₃ (CH ₂ ) ₁₁ Si (OC ₂ H ₅ ) ₃ , CH ₃ (CH ₂ ) ₁₂ Si (OCH ₃ ) ₃ , CH ₃ (CH ₂ ) ₁₂ Si (OC ₂ H ₅ ) ₃ , CH ₃ ( CH ₂ ) ₁₃ Si (OCH ₃ ) ₃ , CH ₃ (CH ₂ ) ₁₃ Si (OC ₂ H ₅ ) ₃ , CH ₃ (CH ₂ ) ₁₄ Si (OCH ₃ ) ₃ , CH ₃ (CH ₂ ) ₁₄ Si ( OC ₂ H ₅ ) ₃ , CH ₃ (CH ₂ ) ₁₅ Si (OCH ₃ ) ₃ , CH ₃ (CH ₂ ) ₁₅ Si (OC ₂ H ₅ ) ₃ , CH ₃ (CH ₂ ) ₁₆ Si (OCH ₃ ) ₃ , CH ₃ (CH ₂ ) ₁₆ Si (OC ₂ H ₅ ) ₃ , CH ₃ (CH ₂ ) ₁₇ Si (OCH ₃ ) ₃ , CH ₃ (CH ₂ ) ₁₇ Si (OC ₂ H ₅ ) ₃ , CH ₃ ( CH ₂ ) ₁₈ Si (OCH ₃ ) ₃ , CH ₃ (CH ₂ ) ₁₈ Si (OC ₂ H ₅ ) ₃ , CH ₃ (CH ₂ ) ₁₉ Si (OCH ₃ ) ₃ , CH ₃ (CH ₂ ) ₁₉ Si ( OC ₂ H ₅ ) ₃ , CH ₃ (CH ₂ ) ₂₀ Si (OCH ₃ ) ₃ , CH ₃ (CH ₂ ) ₂₀ Si (OC ₂ H ₅ ) ₃ , CH ₃ (CH ₂ ) ₂₁ Si (OCH ₃ ) ₃ , CH ₃ (CH ₂ ) ₂₁ Si (OC ₂ H ₅ ) ₃ , CF ₃ (CF ₂ ) ₅ (CH ₂ ) ₂ Si (OCH ₃₎ ) ₃ , CF ₃ (CF ₂ ) ₅ (CH ₂ ) ₂ Si (OC ₂ H ₅ ) ₃ , CF ₃ (CF ₂ ) ₆ (CH ₂ ) ₂ Si (OCH ₃ ) ₃ , CF ₃ (CF ₂ ) ₆ (CH ₂ ) ₂ Si (OC ₂ H ₅ ) ₃ , CF ₃ (CF ₂ ) ₇ (CH ₂ ) ₂ Si (OCH ₃ ) ₃ , CF ₃ (CF ₂ ) ₇ (CH ₂ ) ₂ Si (OC ₂ H) ₅ ) ₃ , CF ₃ (CF ₂ ) ₈ (CH ₂ ) ₂ Si (OCH ₃ ) ₃ , CF ₃ (CF ₂ ) ₈ (CH ₂ ) ₂ Si (OC ₂ H ₅ ) ₃ , CF ₃ (CF ₂ ) ₉ (CH ₂ ) ₂ Si (OCH ₃ ) ₃ , CF ₃ (CF ₂ ) ₉ (CH ₂ ) ₂ Si (OC ₂ H ₅ ) ₃ , CF ₃ (CF ₂ ) ₁₀ (CH ₂ ) ₂ Si (OCH ₃₎ ) ₃ , CF ₃ (CF ₂ ) ₁₀ (CH ₂ ) ₂ Si (OC ₂ H ₅ ) ₃ , CF ₃ (CF ₂ ) ₁₁ (CH ₂ ) ₂ Si (OCH ₃ ) ₃ , CF ₃ (CF ₂ ) ₁₁ (CH ₂ ) ₂ Si (OC ₂ H ₅ ) ₃ , CF ₃ (CF ₂ ) ₁₂ (CH ₂ ) ₂ Si (OCH ₃ ) ₃ , CF ₃ (CF ₂ ) ₁₂ (CH ₂ ) ₂ Si (OC ₂ H) ₅ ) ₃ , CF ₃ (CF ₂ ) ₁₃ (CH ₂ ) ₂ Si (OCH ₃ ) ₃ , CF ₃ (CF ₂ ) ₁₃ (CH ₂ ) ₂ Si (OC ₂ H ₅ ) ₃ , CF ₃ (CF ₂ ) ₁₄ (CH ₂ ) ₂ Si (OCH ₃ ) ₃ , CF ₃ (CF ₂ ) ₁₄ (CH ₂ ) ₂ Si (OC ₂ H ₅ ) ₃ , CF ₃ (CF ₂ ) ₁₅ (CH ₂ ) ₂ Si (OCH ₃₎ ) ₃ , CF ₃ (CF ₂ ) ₁₅ (CH ₂ ) ₂ Si (OC ₂ H ₅ ) ₃ , CF ₃ (CF ₂ ) ₁₆ (CH ₂ ) ₂ Si (OCH ₃ ) ₃ , CF ₃ (CF ₂ ) ₁₆ (CH ₂ ) ₂ Si (OC ₂ H ₅ ) ₃ , CF ₃ (CF ₂ ) ₁₇ (CH ₂ ) ₂ Si (OCH ₃ ) ₃ , CF ₃ (C F ₂ ) ₁₇ (CH ₂ ) ₂ Si (OC ₂ H ₅ ) ₃ , CF ₃ (CF ₂ ) ₁₈ (CH ₂ ) ₂ Si (OCH ₃ ) ₃ , CF ₃ (CF ₂ ) ₁₈ (CH ₂ ) ₂ Si (OC ₂ H ₅ ) ₃ , CF ₃ (CF ₂ ) ₁₉ (CH ₂ ) ₂ Si (OCH ₃ ) ₃ , CF ₃ (CF ₂ ) ₁₉ (CH ₂ ) ₂ Si (OC ₂ H ₅ ) ₃ etc. Be done.

The first and second organic monolayers can be formed at desired positions by patterning. In particular, patterning of an organic monolayer is effective for forming an on-chip integrated device. For example, on the surface of the insulating layer of the detection part, a first monomolecular film composed of an organic molecule having a reactive functional group for immobilization of a probe molecule is formed, while a reference part and a non-gate part ( 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 regioselectively formed by patterning. Form.

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

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

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

FIG. 3 shows a conceptual diagram of a method for detecting an uncharged molecule based on an aptamer-uncharged molecule interaction using the aptamer-immobilized semiconductor sensing device of the present invention. In this detection method, an uncharged molecule is allowed to interact with an aptamer directly immobilized on an organic monolayer, and a change in the surface potential of the insulating layer caused by this interaction is detected as an electric signal. In FIG. 3, 12 is an uncharged molecule. Further, other configurations are designated by the same reference numerals as those in FIG. 1, and the description thereof will be 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 with the capture of the target molecule, so that the charge within the Debye length is charged. The amount changes and the surface potential on the gate electrode shifts. In this case, the potential shift can be detected as a signal when the current is constant, and the current shift can be detected as a signal when the voltage is constant. The shift of the threshold voltage shows the same behavior regardless of whether the n-type FET or the p-type FET is used.

In order for the aptamer immobilized on the device to interact with the uncharged molecule, the 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 detecting uncharged molecules can be used, but a solution satisfying physiological conditions is particularly preferable. For example, physiological saline, phosphate buffered saline, Tris buffered saline, MES buffered saline, MOPS buffered saline, PIPES buffered saline, HEPES buffered saline and the like can be preferably used. The pH of the solution is preferably 5 to 10, more preferably 6 to 8.

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

The temperature at which the aptamer immobilized on the device and the uncharged molecule are allowed to 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 from 30 seconds to 2 hours, more preferably from 1 minute to 1 hour, and even more preferably from 5 to 30 minutes.

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

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

Examples of the peptide include calcitonin, thyrotropin-releasing hormone, vasopressin, adrenocorticotropic 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.

Examples of the aromatic compound include toluene, ethylbenzene, cumene, benzyl alcohol, anisole, benzaldehyde, acetophenone, nitrobenzene, thiophenol, benzonitrile, styrene, xylene, biphenyl, benzophenone, triphenylmethane, naphthalene, anthracene, tetracene and pentacene. Examples thereof include phenanthrene, chrysene, triphenylene, tetraphene, pyrene, picene, pentaphene, perylene, helisene and coronene.

Examples of the sugar include glucose, allose, tallow, gulose, altrose, mannose, galactose, idose, sedoheptulose, corios, psicose, fructose, sorbose, tagatos, ribose, lyxose, xylose, arabinose, apiose, ribulose, xylrose, erythrose, Examples thereof include monosaccharides such as treose, allose, and glyceraldehyde, and oligosaccharides or polysaccharides composed of these monosaccharides.

The concentration of the detectable uncharged molecule varies depending on the 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 eliminating the higher-order structure. This makes it possible to detect uncharged molecules with high sensitivity. In addition, uncharged molecules can be detected under physiological conditions. The reason is not clear, but when aptamers that do not form higher-order structures are immobilized, steric hindrance between adjacent aptamers inhibits structural changes, reducing the amount of change in interfacial charges and reducing sensitivity. However, by immobilizing the aptamer in the state where the higher-order structure is formed, it is immobilized at an appropriate density, and the steric hindrance between adjacent aptamers due to the structural change at the time of measurement of the target molecule is alleviated, and as a result, the sensor Responses are expected to increase.

Hereinafter, the present invention will be specifically described with reference to Reference Examples, Examples and Comparative Examples, but the present invention is not limited to the following Examples. The aptamer used in the examples was provided by NEC Solution Innovators, Ltd.

[1] Construction of semiconductor sensing device-1
[Example 1] Construction of aptamer-immobilized semiconductor sensing device 1 (1) Formation of organic monolayer Film Sonication is performed using acetone from a 10 μm long, 1,000 μm wide n-type FET manufactured by Toppan Printing Co., Ltd. This removed the photoresist. In order to introduce a hydroxy group into the gate surface to prepare an active site, a plasma reactor PR301 (manufactured by Yamato Kagaku Co., Ltd.) was used, and the mixture _{was exposed to 200 W of O 2} plasma for 1 minute.
A monomolecular film is formed on the gate by immersing the device in toluene containing 1% by mass of aminopropyltriethoxysilane (APS, manufactured by Sigma-Aldrich) and allowing it to stand at 60 ° C. for 7 minutes in an argon atmosphere. Membrane. The FET having a monomolecular film formed was ultrasonically washed with a mixed solvent of methanol / toluene (mass ratio 1: 1) and rinsed with ethanol to prepare an FET having an APS monolayer 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 monolayer and the aptamer. The reaction is phosphate-buffered saline containing 2.5% glutaraldehyde (137mM NaCl, 2.7mM KCl, 8.1mM Na 2 HPO 4 · 12H 2 O, 1.5mM KH 2 PO 4, pH7. 4. Hereinafter referred to as 1 × PBS.) 10 μL was added dropwise to the detection part of the device on which the monomolecular film was formed, and the mixture was allowed to stand at room temperature for 30 minutes.

Next, a buffer solution in which all sodium ions of 1 × PBS are replaced with potassium ions (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. ), The aptamer was added so as to be 100 nM.

The gate electrode portion crosslinked with glutaraldehyde was immersed in 1 × PBSK containing the aptamer for 1 hour, and the aptamer was immobilized on an organic monolayer. The aptamer-immobilized device was washed once with 1 x PBS containing 0.05 mass% Triton X-100 and 3 times with 1 x PBS, ultrasonically washed, and then 3 times with 0.04 x PBSK. It was washed and dried. After drying, 20 μL of 1 × PBS containing 10 mM Tris was dropped onto an organic monolayer on which an aptamer was immobilized, 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 An aptamer is used in the same manner as in Example 1 except that 1 × PBS containing 100 nM aptamer is used instead of 1 × PBSK containing 100 nM aptamer. The immobilization device 2 was constructed.

[Reference example]
1 × PBS containing 200 nM of N-methylmesoporphyrin IX (hereinafter referred to as NMM), which is a fluorescent reagent that binds an _{aptamer-immobilized SiO 2} substrate to a guanine quadruplex structure, prepared by the same method as in Comparative Example 1. Alternatively, it was immersed in 1 × PBSK for 1 hour. Then, a scanner-type image analyzer (Typhoon 9410, manufactured by GE Healthcare Japan Co., Ltd.) was used to observe the fluorescence intensity of each substrate depending on the amount of NMM binding.
The results are shown in FIG. The fluorescence intensity was higher when immersed in PBSK, suggesting that the aptamer formed a guanine quadruplex structure by potassium ions. That is, in Example 1, it was suggested that the aptamer was immobilized in a state of forming a guanine quadruple chain structure.

[2] Detection of uncharged molecules-1
[Example 2]
After cleaning the device 1, the device 1 was placed in a holder, and the detection part of the device was immersed in 0.04 × PBSK 0.5 mL for 3 minutes. After immersion, at room temperature, the current-voltage curve of the aptamer-immobilized device was measured with a digital source meter (Caseley, 2612) using an Ag / AgCl reference electrode. The measurement conditions were a gate voltage (V _g ) of -3.0 to 0.5 V and a drain voltage (V _d ) of 0.1 V. 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 3 times with 1 mL of 1 × PBS and 1 mL of 0.04 × PBSK. Then, 0.5 mL of 0.04 × PBSK was added on 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 the addition of cortisol.} .. The results are shown in FIG.

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

In FIGS. 5 to 7, the dotted line represents the device characteristics before the addition of cortisol, and the solid line represents the device characteristics after the addition of cortisol. 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 the 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.

From the results shown in FIGS. 5 to 8, it was shown that the use of an aptamer-immobilized device in a high-concentration potassium ion-containing solution 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 aptamer to 1 × PBSK so that the aptamer is 100 nM, aptamer is added to a solution in which 1 × PBS and 1 × PBSK are mixed in equal amounts. The aptamer-immobilized device 3 was constructed in the same manner as in Example 1 except that the aptamer was added so as to be 100 nM.

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

[5] Detection of uncharged molecules-3
[Example 5-1]
Using device 1, the gate voltage shift ΔV _g before and after the addition of 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 the addition of cortisol was evaluated by the same method as in Example 2 except that the device 3 was used instead of the device 1. The results are shown in FIG.

[Example 5-3]
_{The gate voltage shift ΔV g} before and after the addition of cortisol was evaluated by the same method as in Example 2 except that the device 4 was used instead of the 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 by the same method as in Example 2 except that the device 2 was used instead of the 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 Except that the aptamer was added to 0.1 × PBSK so as to be 100 nM instead of being added to 1 × PBSK so that the aptamer was 100 nM. , The aptamer immobilization device 5 was constructed in the same manner as in Example 1.

[Comparative Example 5] Construction of aptamer-immobilized semiconductor sensing device 6 The aptamer was added to 10 × PBSK so that the aptamer was 100 nM instead of being added to 1 × PBSK so that the aptamer was 100 nM. The 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 the addition of 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 the addition of cortisol was evaluated in the same manner as in Example 2 except that the device 5 was used instead of the device 1. The results are shown in FIG.

[Comparative Example 6]
_{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 the device 6 was used instead of the device 1. The results are shown in FIG.

1 Silicon substrate 2 Insulation layer 3 First organic monolayer 4 Gate electrode 5 Source electrode 6 Drain electrode 7 Dope region 8 Reference part 9 Detection part 10 Template part 11 Aptamer (probe molecule)
12 uncharged molecule

Claims (7)

An aptamer-immobilized semiconductor sensing device including a field-effect transistor having an aptamer-immobilized detector as a probe molecule.
An aptamer-immobilized semiconductor sensing device in which the aptamer is immobilized on a detection unit in a state where a higher-order structure is formed, and then the higher-order structure is eliminated. The aptamer-immobilized semiconductor sensing device according to claim 1, wherein the aptamer is a DNA aptamer. The aptamer-immobilized semiconductor sensing device according to claim 1 or 2, wherein the higher-order structure is a G-quartet structure. In the detection unit, a first insulating layer containing a silicon oxide or an inorganic oxide is formed on the semiconductor as a reaction gate insulating portion, and an organic single molecule having a reactive functional group is formed on the first insulating layer. A first organic monomolecular film composed of a film is formed, and an aptamer in a state in which a higher-order structure is formed as a probe molecule is directly or crosslinked via the reactive functional group on the first organic monomolecular film. The aptamer immobilization according to any one of claims 1 to 3, comprising an aptamer / organic monomolecular film / insulating layer / semiconductor structure, which is obtained by binding using and then eliminating the higher-order structure. Semiconductor sensing device. A second insulating layer containing a silicon oxide or an inorganic oxide is further formed on the semiconductor as a reference gate insulating portion, and reacts with either the aptamer or the uncharged molecule on the second insulating layer. The aptamer-immobilized semiconductor sensing device according to claim 4, further comprising an organic monomolecular film / insulating layer / semiconductor structure as a reference unit, which forms a second organic monomolecular film composed of non-organic molecules. The step of interacting an aptamer immobilized on the aptamer-immobilized semiconductor sensing device according to any one of claims 1 to 5 with an uncharged molecule,
A method for detecting an uncharged molecule, which comprises a step of detecting a change in surface potential on a gate electrode due to the interaction. The method for detecting an uncharged molecule according to claim 6, wherein the uncharged molecule is a compound having a steroid skeleton.

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