Method for detecting uncharged molecules and semiconductor sensing device with immobilized probe molecules

Abstract

This invention provides a method for detecting uncharged molecules using a semiconductor sensing device in which probe molecules are immobilized by an immobilization method that does not generate nonspecific bonds. [Solution] A method for detecting uncharged molecules using a semiconductor sensing device on which probe molecules are immobilized, wherein the semiconductor sensing device on which the probe molecules are immobilized comprises a field-effect transistor having a detection unit on which probe molecules are immobilized, the detection unit having a first insulating layer formed on a semiconductor, a first organic monolayer made of an organic monolayer having reactive functional groups formed on the first insulating layer, and a probe molecule in which functional groups not originally present are introduced to the reactive functional groups of the first organic monolayer, and the probe molecule is bonded to the reactive functional groups directly or via a crosslinking molecule by bonding with functional groups not originally present on the probe molecule.

Description

Technical Field

[0001] The present invention relates to a method for detecting uncharged molecules and a probe molecule-immobilized semiconductor sensing device.

Background Art

[0002] The field-effect transistor (FET) is a very promising tool for detecting biomolecules. When using an FET, since the change in the charge density on the gate surface accompanying the adsorption of biomolecules 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 biomolecules using an FET has been widely conducted.

[0003] FET biosensors are expected to be applied in various fields such as medicine, food, and environment because of their simple operation, small size, and low cost. An FET biosensor is a device that measures the change in the surface potential on the gate electrode caused by the charge of a biomolecule captured by a probe molecule within the device length, which is the charge detection range. Therefore, by studying the method of immobilizing the probe molecule, the target molecule has been specifically and efficiently captured at the interface. In a silicon-based semiconductor biosensor, a method has been adopted in which an amino group originally possessed by the probe molecule or an introduced amino group is directly bonded to an organic film that modifies the surface of the SiO2 insulating film, or bonded via a crosslinking agent (Patent Document 1).

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] However, when using single-stranded DNA, which has attracted attention in recent years, as a probe, it was thought that the amino groups present in the bases (adenine, guanine, cytosine) within the constituent nucleotides would undergo nonspecific binding, resulting in disruption of the three-dimensional structure of the nucleic acid molecule and a decrease in its affinity for the target molecule. This raised concerns that a reduction in the number of probes effective in capturing the target molecule would lead to a decrease in the sensitivity of the sensor.

[0006] The present invention has been made in view of the above circumstances, and aims to provide a method for detecting uncharged molecules using a semiconductor sensing device in which probe molecules are immobilized by an immobilization method that does not generate nonspecific bonds. [Means for solving the problem]

[0007] As a result of diligent research to achieve the above objective, the present inventors have discovered that by introducing a functional group not present in the probe molecule into the probe molecule, and immobilizing the probe molecule on the detection unit through a bond that the functional group can form, the 3D structure of the probe molecule is not disrupted, and the affinity to the target molecule is maintained, thereby enabling highly sensitive detection of the target molecule. This led to the present invention.

[0008] Therefore, the present invention provides a method for detecting uncharged molecules and a probe molecule-immobilized semiconductor sensing device. 1. A method for detecting uncharged molecules, comprising the steps of: interacting an uncharged molecule with a probe molecule immobilized on a semiconductor sensing device that interacts with the uncharged molecule; and detecting a change in the potential of a gate channel region due to the interaction, The semiconductor sensing device on which the probe molecule is immobilized comprises a field-effect transistor having a detection unit on which the probe molecule is immobilized, The detection unit comprises a probe molecule / organic monolayer / insulating layer / semiconductor structure, wherein a first insulating layer containing silicon oxide or inorganic oxide is formed on a semiconductor as a reaction gate insulating layer, a first organic monolayer made of an organic monolayer having reactive functional groups is formed on the first insulating layer, and a probe molecule in which a functional group not originally present is introduced to the reactive functional group of the first organic monolayer is bonded directly via the reactive functional group or via a crosslinking molecule by bonding with the functional group not originally present on the probe molecule. A method for detecting uncharged molecules. 2. A method for detecting an uncharged molecule, wherein the probe molecule is a nucleic acid aptamer. 3. A method for detecting an uncharged molecule of type 1 or 2, wherein the bond formed by a functional group not originally present in the probe molecule is a disulfide bond or a thiol-maleimide bond. 4. A method for detecting one of the uncharged molecules (1-3) whose reactive functional group is an amino group in the first organic monolayer. 5. A method for detecting any of the uncharged molecules 1 to 4, wherein the crosslinking molecule is N-succinimidyl 3-(2-pyridyldithio)propionate or sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate. 6. A probe molecule immobilized semiconductor sensing device comprising a field-effect transistor having a detection unit on which probe molecules are immobilized, A probe molecule immobilized semiconductor sensing device comprising a probe molecule / organic monolayer / insulating layer / semiconductor structure, wherein the detection unit has a first insulating layer formed on a semiconductor as a reaction gate insulating layer containing silicon oxide or an inorganic oxide, a first organic monolayer formed on the first insulating layer consisting of an organic monolayer having reactive functional groups, and a probe molecule in which a functional group not originally present is introduced to the reactive functional group of the first organic monolayer, is bonded via a crosslinking molecule by bonding with the functional group not originally present on the probe molecule. [Effects of the Invention]

[0009] According to the method of the present invention, it is possible to detect uncharged molecules with higher sensitivity. [Brief explanation of the drawing]

[0010] [Figure 1] This is a cross-sectional view of the semiconductor sensing device of the present invention, where (A) is a field-effect transistor, (B) is a state in which an organic monolayer is formed on the insulating layer of the gate electrode of the field-effect transistor, and (C) is a state in which probe molecules are immobilized on the organic monolayer. [Figure 2] The image shows an example of an on-chip device unit configuration, where (A) is a partial plan view and (B) is an enlarged cross-sectional view thereof. [Figure 3] This is a conceptual diagram of the detection of uncharged molecules using the semiconductor sensing device of the present invention. [Figure 4] This graph shows the evaluation results of the amount of shift in semiconductor characteristics before and after cortisol addition, as measured in Example 1. [Figure 5] This graph shows the evaluation results of the amount of shift in semiconductor characteristics before and after substance addition, as measured in Example 2. [Figure 6] This graph shows the evaluation results of the amount of shift in semiconductor properties before and after cortisol addition, as measured in Example 3. [Figure 7] This graph shows the evaluation results of the amount of shift in semiconductor characteristics before and after cortisol addition, as measured in Example 4. [Modes for carrying out the invention]

[0011] The present invention provides a method for detecting uncharged molecules, comprising the steps of: interacting an uncharged molecule with a probe molecule immobilized on a probe molecule-immobilized semiconductor sensing device that interacts with the uncharged molecule; and detecting the potential change in the gate channel region due to the interaction.

[0012] The aforementioned probe molecule immobilized semiconductor sensing device comprises a FET having a detection unit immobilized via bonding by the probe molecule, in which a functional group that the probe molecule does not originally possess has been introduced.

[0013] The FETs applicable to the device of the present invention are not particularly limited, and conventionally known configurations such as ion-sensitive FETs and extended-gate FETs can be used. Furthermore, the semiconductor used in the FET is not particularly limited, and in addition to silicon, oxide semiconductors such as zinc oxide and indium oxide, molybdenum disulfide, graphene, and two-dimensional materials such as MXene can be used.

[0014] In the present invention, the detection portion of the FET preferably comprises a probe molecule / organic monolayer / insulating layer / semiconductor structure, wherein a first insulating layer containing silicon oxide or an inorganic oxide is formed on a semiconductor as a reaction gate insulating portion, a first organic monolayer made of an organic monolayer having reactive functional groups is formed on the first insulating layer, and a probe molecule in which a functional group not originally present is introduced to the reactive functional group of the first organic monolayer is bonded directly via the reactive functional group or via a crosslinking molecule by bonding with the functional group not originally present on the probe molecule.

[0015] In the detection unit, the insulating layer / semiconductor structure portion can utilize an FET in which an insulating layer containing silicon oxide or inorganic oxide is formed on a semiconductor as a reaction gate insulating portion, and the configuration can be one of those conventionally known. The insulating layer is preferably silicon oxide. The FET may be n-type or p-type. An example of such an FET is shown in Figure 1(A). In Figure 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.

[0016] Then, as shown in Figure 1(B), a first organic monolayer 3 is formed on the insulating layer 2. In this invention, the basic principle is to detect the change in surface potential associated with the bonding reaction between probe molecules and target molecules on the surface of the insulating layer as an electrical signal. The thickness of the insulating layer is preferably 30 to 300 nm, and particularly preferably 50 to 150 nm.

[0017] The first organic monolayer consists of an organic monolayer having a reactive functional group. The organic monolayer having the reactive functional group is preferably a monolayer of an alkoxysilane represented by the following formula (1). [ka]

[0018] In formula (1), R is an amino group, an aminooxy group, or a thiol group, but an amino group is preferred.

[0019] In formula (1), R 1 This is a linear alkanediyl group having 3 to 22 carbon atoms. The linear alkanediyl group is preferably one with 3 to 18 carbon atoms, and more preferably one with 3 to 8 carbon atoms. A shorter carbon chain is preferable because it weakens the hydrophobicity of the organic monolayer, thereby suppressing nonspecific adsorption caused by hydrophobic interactions of the target molecule.

[0020] R 1Specific examples of linear alkanediyl groups 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, and tride Examples include the can-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. Of these, those with 3 to 18 carbon atoms are preferred, and those with 3 to 8 carbon atoms are more preferred.

[0021] In formula (1), R 2 ~R 4 Each of these is 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, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl groups. Of these, methyl or ethyl groups are preferred. Examples of the alkoxyalkyl group include methoxymethyl, ethoxymethyl, 2-methoxyethyl, and 2-ethoxyethyl groups. Of these, alkoxyalkyl groups having 2 to 3 carbon atoms are preferred. 2 ~R 4 Particularly preferred are methyl groups, ethyl groups, and 2-methoxyethyl groups.

[0022] As the alkoxysilane in which R is an amino group, a carboxyl group, or a thiol group, commercially available products can be used. Furthermore, an alkoxysilane in which R is an aminooxy group can be synthesized according to the following scheme. [ka] (In the formula, R 1 ~R4 is the same as above. R 5 is R 1 is a linear alkanediyl group with 2 fewer carbon atoms than that of R. )

[0023] 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 hexachloroplatinic(IV) acid is added to a mixture of trialkoxyhydrosilane and O-alkenylhydroxyamine, and the mixture is reacted at 10 to 200 °C for 1 to 1200 hours, more preferably at 60 to 120 °C for 12 to 48 hours. For the film-forming operation, it is preferable to use a material obtained by removing excess trialkoxyhydrosilane by an operation such as distillation.

[0024] 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 by optimizing it, for example, a film in which the single molecules are most densely packed by the self-assembly function of the organic molecules is formed. 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 a dry room 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 and allowed to stand 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.

[0025] Examples of the organic solvent include toluene, methanol, ethanol, etc., and toluene, methanol, etc. are particularly preferable.

[0026] On the semiconductor of the FET, a second insulating layer containing silicon oxide or an inorganic oxide can be formed as a reference gate insulating layer. On this second insulating layer, a monolayer composed of organic molecules that do not react with either the probe molecule or the uncharged target molecule can be formed as a second organic monolayer, and this monolayer / insulating layer / semiconductor structure can be used as the reference layer. Furthermore, if the reaction gate insulating layer and the reference gate insulating layer are separated to the extent that they do not affect each other in potential change measurement, the first insulating layer of the reaction gate insulating layer and the second insulating layer of the reference gate insulating layer can be provided in the same layer.

[0027] Figure 2 shows an example of a unit configuration for an on-chip device in which an organic monolayer / insulating layer / semiconductor structure is applied to the detection unit 9 and the reference unit 8. In Figure 2, 1 is the silicon substrate, 2 is the insulating layer, and 10 is the template unit. The unit configuration of this device is not limited to the illustrated configuration, and the detection unit and the reference unit do not necessarily have to be arranged in a one-to-one relationship. The number and combination of detection units and reference units can be changed as needed. In addition, each detection unit and reference unit can be formed in size from a few to several tens of micrometers.

[0028] 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 preferred. When an alkoxysilane monolayer is used as the organic monolayer, the second insulating layer is preferably made of silicon oxide.

[0029] The second organic monolayer is preferably a self-assembled film 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). [ka]

[0030] In formula (2), R 6The linear alkyl group 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 is preferably one having 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-heneicosyl group, n-docosyl group, and the like.

[0031] In formula (2), R 7 ~R 9 Each of these is 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. Of these, methyl group or ethyl group is preferred. Examples of the alkoxyalkyl group include methoxymethyl group, ethoxymethyl group, 2-methoxyethyl group, 2-ethoxyethyl group, etc. Of these, alkoxyalkyl groups having 2 to 3 carbon atoms are preferred.

[0032] Specifically, 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, and 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) 13Si(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(CF 2)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)2S i(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 Examples include (CH2)2Si(OC2H5)3.

[0033] Furthermore, the first and second organic monolayers can be formed at desired positions by patterning. Patterning of organic monolayers is particularly effective for forming on-chip integrated devices. For example, a first monolayer composed of organic molecules having reactive functional groups is formed on the insulating layer surface of the detection section to immobilize probe molecules, while a second organic monolayer composed of organic molecules that do not react with either probe molecules or target molecules is formed in the reference section and the non-gate section (template section) in a positionally selective manner by patterning, in order to avoid nonspecific adsorption of target molecules.

[0034] As a reference layer, it is also possible to use a second organic monolayer similar to the first organic monolayer, with a compound that does not interact with the target molecule immobilized on it. In other words, a compound that does not interact with the target molecule / organic monolayer / insulating layer / semiconductor structure can also be used as the reference layer. In this case, the reference layer can be formed according to the same method as the organic monolayer formation method and compound immobilization method described later for the detection layer.

[0035] In the semiconductor sensing device, probe molecules are immobilized on the first organic monolayer of the detection unit. For example, as shown in Figure 1(C), probe molecules 11 are bonded to the first organic monolayer 3.

[0036] The probe molecule can be appropriately selected according to the target to be detected. The probe molecule is not particularly limited, but examples include compounds that bind to target molecules such as nucleic acids, antibodies, enzymes, glycans, and dye molecules.

[0037] Nucleic acids used as probe molecules include nucleic acid aptamers. A nucleic acid aptamer is a molecule containing nucleotide residues, and may consist only of nucleotide residues or contain nucleotide residues. Examples of nucleotides include ribonucleotides, deoxyribonucleotides, and their derivatives. The aptamer may contain deoxyribonucleotides and / or their derivatives (DNA aptamer), ribonucleotides and / or their derivatives (RNA aptamer), or both (DNA / RNA aptamer). DNA aptamers are preferred as the aptamer. Furthermore, the aptamer may be a single-stranded aptamer or a double-stranded aptamer.

[0038] The nucleotide may contain either a natural base or a non-natural base (artificial base) as its base. Examples of natural bases include adenine, cytosine, guanine, thymine, uracil, and modified bases thereof. Examples of modifications include methylation, fluorination, amination, and thiolation. Examples of non-natural bases include 2'-fluoropyrimidine and 2'-O-methylpyrimidine, and specifically, 2'-fluorouracil, 2'-aminouracil, 2'-O-methyluracil, and 2'-thiouracil.

[0039] The nucleotide may be a modified nucleotide. Examples of the modified nucleotide 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, and 2'-thiolated cytosine nucleotide. The aptamer may also contain non-nucleotides such as PNA (peptide nucleic acid) and LNA (locked nucleic acid).

[0040] The number of bases in the aptamer is not particularly limited, but its lower limit is preferably around 25, more preferably around 35, and even more preferably around 40. On the other hand, its upper limit is preferably around 200, more preferably around 120, and even more preferably around 80.

[0041] Antibodies used as probe molecules include immunoglobulins such as IgG, IgM, IgA, IgD, and IgE, as well as antibody fragments (Fab, F(ab')2).

[0042] The immobilization of the probe molecule is achieved through bonding via functional groups that the probe molecule does not inherently possess. The immobilization process will be explained below using a nucleic acid aptamer as the probe molecule as an example.

[0043] Since nucleic acid aptamers typically do not contain sulfur molecules, they can be immobilized on a first organic monolayer by, for example, introducing an -SH group into a nucleic acid aptamer and using a crosslinking agent that can react with the functional groups of the first organic monolayer and is capable of forming a bond with the -SH group. The introduction of an -SH group into a nucleic acid aptamer can be carried out by conventionally known methods.

[0044] The crosslinking molecule capable of forming a bond with the -SH group can be appropriately selected depending on the functional group of the first organic monolayer. For example, if the functional group of the first organic monolayer is an amino group, the crosslinking molecule may be N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), 4-(maleimidomethyl)cyclohexane-1-carboxylic acid N-succinimidyl (SMCC), 4-succinimidyloxycarbonyl-α-methyl-α(2-pyridyldithio)toluene (SMPT), N-α-maleimoidaceto-oxysuccinimidester (AMAS), N-β-maleimidopropyl-oxysuccinimidester (BMPS), N-γ-maleimidobutyryl-oxysuccinimidester (GMBS), or N-ε-maleidocaproyl-oxysuccinimidester (EMCS) succinimidyl 4 Various crosslinking molecules in which the succinimidyl group is replaced with a sulfosuccinimidyl group are preferred, such as -(p-maleimidophenyl)butyrate (SMPB), succinimidyl 6-((β-maleimidopropionamide)hexanoate) (SMPH), N-(iodoacetoxy)succinimidide (SIA), succinimidyl 3-(bromoacetamide)propionate (SBAP), succinimidyl (4-iodoacetyl)aminobenzoate (SIAB), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxy-(6-amidecaproate) (LC-SMCC), and sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Sulfo-SMCC).

[0045] The crosslinking agent is bonded to the first organic monolayer by reacting with the functional groups of the first organic monolayer. For example, the reaction can be carried out in a buffer solution containing 0.1 to 100 mM of the crosslinking agent at 10 to 50°C for 1 minute to 24 hours.

[0046] Next, if a crosslinking agent having a disulfide bond in its molecule, such as SPDP, is used, the disulfide bond is cleaved to generate an -SH group. The cleavage of the disulfide bond can be carried out by known methods, for example, by using reducing agents such as dithiothreitol (DTT), 2-mercaptoethanol (MCH), or tris(2-carboxyethyl)phosphine hydrochloride (TCEP). Specifically, one method is to react the crosslinking agent in a buffer containing 0.1 to 1000 mM of the reducing agent at 10 to 50°C for 1 minute to 24 hours. If a crosslinking agent having a functional group that reacts with the -SH group, such as Sulfo-SMCC, is used, the crosslinking agent should be bonded to the first organic monolayer before proceeding with the immobilization of the probe molecule.

[0047] Finally, the probe molecules can be immobilized by reacting them in a solution containing the probe molecules, preferably at 10-50°C for 1 minute to 24 hours, and more preferably at 10-35°C for 1 minute to 60 minutes. The solution can preferably be physiological saline, phosphate-buffered saline, Tris-buffered saline, MES-buffered saline, MOPS-buffered saline, PIPES-buffered saline, HEPES-buffered saline, or a mixture of these with potassium ions. The pH of the solution is preferably 5-10, more preferably 6-8. The concentration of the probe molecules is preferably 0.001-10 μM, more preferably 0.01-0.1 μM. The probe molecules are immobilized by disulfide bonds or thiol-maleimide bonds using the crosslinking agent.

[0048] Figure 3 shows a conceptual diagram of a method for detecting uncharged molecules based on probe molecule-uncharged molecule interaction using the probe molecule immobilized semiconductor sensing device of the present invention. In this detection method, uncharged molecules are made to interact with probe molecules directly immobilized on an organic monolayer, and the resulting change in the surface potential of the insulating layer is detected as an electrical signal. In Figure 3, 12 represents an uncharged molecule. Other components are given the same reference numerals as in Figure 1, and their descriptions are omitted.

[0049] To interact a probe molecule immobilized on the device with an uncharged molecule, a solution containing the uncharged molecule can be placed on the gate electrode, diluted as needed. While a general solution used for detecting uncharged molecules can be used as the solution, one that satisfies physiological conditions is particularly preferred. For example, physiological saline, phosphate-buffered saline, Tris-buffered saline, MES-buffered saline, MOPS-buffered saline, PIPES-buffered saline, HEPES-buffered saline, etc., are preferably used. The pH of the solution is preferably 5 to 10, and more preferably 6 to 8.

[0050] Furthermore, Ca 2+ Mg 2+ Ions such as ethylenediaminetetraacetic acid (EDTA) and glycol etherdiaminetetraacetic acid (EGTA) as chelating agents, surfactants such as Tween® 20, Triton® X-100, and Nonidet® P-40 may also be added. When the ions are added, their concentration is preferably 0.1 to 10 mM, and more preferably 0.5 to 5 mM. When the chelating agents are added, their concentration is preferably 0.1 to 10 mM, and more preferably 0.5 to 5 mM. When surfactants are added, their concentration is preferably 0.001 to 10% by volume, and more preferably 0.05 to 5% by volume.

[0051] The temperature at which the immobilized probe molecule interacts with the uncharged molecule 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.

[0052] In this invention, the uncharged molecules to be detected are not particularly limited as long as they have the property of interacting with the probe molecule. Examples include compounds having a steroid skeleton, peptides, amino acid derivatives, aromatic compounds, sugars, and the like.

[0053] Examples of compounds having the aforementioned steroid skeleton include cortisol, cholesterol, progesterone, 11-deonesicorticosterone, corticosterone, aldosterone, cortisone, dehydroepiandrosterone, dehydroepiandrosterone sulfate, dihydrotestosterone, androsterone, epiandrosterone, testosterone, 17β-estradiol, estrone, and estriol.

[0054] Examples of the aforementioned peptides include calcitonin, thyroid-stimulating hormone-releasing hormone, vasopressin, adrenocorticotropic hormone, gonadotropin-releasing hormone, growth hormone, oxytocin, glucagon, and secretin.

[0055] Examples of the aforementioned amino acid derivatives include melatonin, thyroid hormones, and catecholamines.

[0056] Examples of the aforementioned 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, tetrafen, pyrene, picene, pentaphene, perylene, helicene, coronene, and the like.

[0057] Examples of the aforementioned sugars include monosaccharides such as glucose, allose, talose, growth, altrose, mannose, galactose, idose, sedoheptulose, coliose, psicose, fructose, sorbose, tagatose, ribose, lyxose, xylose, arabinose, apiose, ribulose, xylulose, erythrose, threose, erythrulose, and glyceraldehyde, as well as oligosaccharides or polysaccharides composed of these monosaccharides.

[0058] The detectable concentration of uncharged molecules varies depending on the type, but is usually around 0.01 nM to 10 mM, with 1 nM to 1 mM being preferable. [Examples]

[0059] The present invention will be specifically described below with reference to examples and comparative examples, but the present invention is not limited to the following examples.

[0060] [Manufacturing Example 1] A semiconductor sensing device was constructed that incorporates an organic monolayer / insulating layer / semiconductor structure as the detection unit. Silicon oxide was used as the insulating layer. For the first organic monolayer of the detection unit, aminopropyltriethoxysilane was used to form the monolayer. The device fabrication method is described below.

[0061] The photoresist film was removed from a 10 μm long, 1000 μm wide n-type FET manufactured by Toppan Printing Co., Ltd. by ultrasonic treatment with acetone. Subsequently, ultrasonic cleaning was performed using a methanol / toluene mixed solvent (volume ratio 1:1). To introduce hydroxyl groups to the gate surface and create an active site, the area was immersed in piranha solution (30% hydrogen peroxide:concentrated sulfuric acid = 1:4, 120°C) for 10 minutes. A monolayer was formed on the gate electrode by immersing the device in toluene containing 1% by mass of aminopropyltriethoxysilane (Sigma-Aldrich) and allowing it to stand at 60°C for 7 minutes under an argon atmosphere. The FET with the monolayer was ultrasonically cleaned using a methanol / toluene mixed solvent (volume ratio 1:1) and rinsed with ethanol. After cleaning the substrate, it was allowed to stand at 160°C for 60 minutes under an argon atmosphere.

[0062] [Example 1] The response to cortisol addition was evaluated as follows using a device in which a 3'-terminal thiol group modified aptamer was immobilized via SPDP on the gate electrode of a detection unit modified with an amino monolayer, via a disulfide bond.

[0063] First, SPDP was reacted as a crosslinking molecule to bridge the amino group of the organic monolayer with the 3'-terminated thiol-group modified aptamer. The reaction was carried out by placing the device in a holder and immersing the detection unit of the device, which had a monolayer with an amino group at the end, in 20 μL of phosphate buffer (12 mM NaH2PO4·2H2O, 51 mM Na2HPO4·12H2O, pH 7.4, hereinafter referred to as 1×PB) containing 1 mM SPDP for 1 hour at room temperature. Subsequently, the 2-mercaptopyridine moiety was removed and a thiol group was formed by cleaving the disulfide bond in the SPDP structure. The cleavage of the disulfide bond was carried out by immersing the SPDP-modified device detection unit in 20 μL of 1×PB containing 10 mM DTT for 1 hour at room temperature. The aptamer, which is a single-stranded DNA, was immobilized as a bioreceptor by forming a disulfide bond with a thiol group derived from SPDP. The aforementioned immobilization reaction involves immobilizing the gate electrode portion, which has a thiol group formed within the SPDP structure, in a buffer solution (12 mM KH2PO4, 51 mM K2HPO4, pH 7.4, hereafter 1 × PB) containing a 100 nM 3'-terminal thiol-modified aptamer (55 bases), in which all sodium ions are replaced with potassium ions. kThe substrate was immersed for 1 hour in a mixed solution (volume ratio 1:1) of the following: After cleaning the substrate, it was subjected to a capping treatment with PB containing 100 μM 2-mercaptoethanol (MCH) for 1 hour.

[0064] Subsequently, the detection unit of the device is set to 1 × PB k 0.04 × PB diluted 25 times k After immersion in 200 μL for 3 minutes, the current-voltage curve of the aptamer-immobilized device was measured at room temperature using a single-junction Ag / AgCl reference electrode and a digital source meter (Keithley 2612). The measurement conditions were a gate voltage of -3V to 0.5V and a drain voltage of 0.1V.

[0065] Next, 20 μL of phosphate-buffered saline containing 1 μM cortisol (137 mM NaCl, 2.682 mM KCl, 8.1 mM Na2HPO4·12H2O, 1.469 mM KH2PO4, pH 7.4, hereafter referred to as 1×PBS) was added to the gate surface of the aptamer immobilization device, and after standing for 30 minutes, 3 mL of 1×PB and 0.04×PB were added. k Rinsing was performed using 3 mL. Afterwards, 0.04 × PB was applied to the gate surface. k After adding 200 μL and allowing it to stand for 3 minutes, the current-voltage curve of the cortisol adsorption device was measured, and the gate voltage shift ΔV before and after cortisol addition was measured. g I evaluated it.

[0066] As a control experiment, the response before and after cortisol addition was evaluated as follows in a FET sensor in which an aptamer was immobilized using a bond between an amino group and an aldehyde group, compared to an aptamer immobilized using a disulfide bond. First, glutaraldehyde was reacted with the gate electrode of the detection unit modified with an amino monolayer. The reaction was carried out by immersing the detection unit of a device with a monolayer formed at the end of an amino group in 10 μL of 1×PBS containing 2.5% by mass of glutaraldehyde for 30 minutes at room temperature. After that, the device was placed in a holder, and the gate electrode modified with glutaraldehyde was treated with 1×PB / 1×PB containing a 3'-terminated amino group modified aptamer (55 bases). kThe substrate was immersed in a mixed solution (volume ratio 1:1) for 1 hour. After cleaning the substrate, it was capped with 1×PBS containing 10 mM ethanolamine for 1 hour.

[0067] Subsequently, the detection unit of the device is set to 0.04 × PB k After immersion in 200 μL for 3 minutes, the current-voltage curve of the aptamer-immobilized device was measured at room temperature using a single-junction Ag / AgCl reference electrode and a digital source meter (Keithley 2612). The measurement conditions were a gate voltage of -3V to 0.5V and a drain voltage of 0.1V. Subsequently, 20 μL of 1×PBS containing 1 μM cortisol was added to the gate surface of the aptamer-immobilized device, and after standing for 30 minutes, 3 mL of 1×PB and 0.04×PB were added. k Rinsing was performed using 3 mL. Then, 200 μL of PB was added to the gate surface and allowed to stand for 3 minutes. After that, the current-voltage curve of the cortisol adsorption device was measured, and the gate voltage shift ΔV before and after cortisol addition was measured. g I evaluated it.

[0068] Figure 4 shows the evaluation results of the amount of semiconductor property shift before and after the addition of 1 μM cortisol to aptamer-immobilized devices fabricated using each crosslinking agent. When cortisol is added to an aptamer-immobilized device using disulfide bonds with the crosslinking agent SPDP, the gate voltage shift ΔV in the current-voltage curve, which is a semiconductor property, is observed. g The voltage became +10mV. On the other hand, when cortisol was added to an aptamer immobilized device using the bonding between amino groups and aldehyde groups by the crosslinking agent glutaraldehyde, the gate voltage shift ΔV in the change of the current-voltage curve was g The response was increased to +8mV. Furthermore, the standard deviation of the response decreased when disulfide bonds were used. Therefore, an increase in the response to cortisol was confirmed by aptamer immobilization via disulfide bonds.

[0069] [Example 2] In detection using an aptamer-immobilized device fabricated using disulfide bonds, cortisol specificity was evaluated as follows.

[0070] First, SPDP was reacted as a crosslinking molecule to bridge the amino group of the organic monolayer with the 3'-terminated thiol-group modified aptamer. The reaction was carried out by placing the device in a holder and immersing the detection part of the device, which had a monolayer with an amino group at the end, in 20 μL of 1×PB containing 1 mM SPDP for 1 hour at room temperature. Subsequently, the 2-mercaptopyridine moiety was removed and a thiol group was formed by cleaving the disulfide bond in the SPDP structure. The reaction was carried out by immersing the SPDP-modified device detection part in 20 μL of 1×PB containing 10 mM DTT for 1 hour at room temperature. An aptamer, which is a single-stranded DNA, was immobilized as a bioreceptor by forming a disulfide bond with a thiol group derived from SPDP. The reaction was carried out by immersing the gate electrode portion, which had a thiol group formed in the SPDP structure, in 1×PB / 1×PB containing 100 nM 3'-terminated thiol-group modified aptamer (55 bases). k The substrate was immersed in a mixed solution (volume ratio 1:1) for 1 hour. After cleaning the substrate, it was capped with 100 μM MCH-containing PB for 1 hour.

[0071] Subsequently, the detection unit of the device is set to 0.04 × PB k After immersion in 200 μL for 3 minutes, the current-voltage curve of the aptamer-immobilized device was measured at room temperature using a digital source meter (Keithley 2612) with a double-junction Ag / AgCl reference electrode. The measurement conditions were a gate voltage of -3V to 0.5V and a drain voltage of 0.1V.

[0072] Next, 20 μL of 1×PBS containing 1 μM cortisol was added to the gate surface of the aptamer-immobilized device, and after standing for 30 minutes, 3 mL of 1×PB and 0.04×PB were added. k Rinsing was performed using 3 mL. Afterwards, 0.04 × PB was applied to the gate surface. k After adding 200 μL and allowing it to stand for 3 minutes, the current-voltage curve of the cortisol adsorption device was measured, and the gate voltage shift ΔV before and after cortisol addition was measured. g I evaluated it.

[0073] As a control experiment, SPDP was reacted with an aptamer modified with a 3'-terminated thiol group to an amino group in an organic monolayer as a crosslinking molecule. The reaction was carried out by placing the device in a holder and immersing the detection part of the device, which had a monolayer with an amino group at the end, in 20 μL of 1×PB containing 1 mM SPDP for 1 hour at room temperature. Subsequently, the 2-mercaptopyridine moiety was removed and a thiol group was formed by cleaving the disulfide bond in the SPDP structure. The reaction was carried out by immersing the detection part of the SPDP-modified device in 20 μL of 1×PB containing 10 mM DTT for 1 hour at room temperature. An aptamer, which is a single-stranded DNA, was immobilized as a bioreceptor by forming a disulfide bond with a thiol group derived from SPDP. The reaction was carried out by immersing the gate electrode portion, which had a thiol group formed in the SPDP structure, in 1×PB / 1×PB containing 100 nM 3'-terminated thiol-modified aptamer (55 bases). k The substrate was immersed in a mixed solution (volume ratio 1:1) for 1 hour. After cleaning the substrate, it was capped with 100 μM MCH-containing PB for 1 hour.

[0074] Subsequently, the detection unit of the device is set to 0.04 × PB k After immersion in 200 μL for 3 minutes, the current-voltage curve of the aptamer-immobilized device was measured at room temperature using a digital source meter (Keithley 2612) with a double-junction Ag / AgCl reference electrode. The measurement conditions were a gate voltage of -3V to 0.5V and a drain voltage of 0.1V.

[0075] Next, 20 μL of 1×PBS containing 1 μM cortisone or 0.1 U / μL α-amylase was added to the gate surface of the aptamer-immobilized device, and after standing for 30 minutes, 3 mL of 1×PB and 0.04×PB were added. k Rinsing was performed using 3 mL. Afterwards, 0.04 × PB was applied to the gate surface. k After adding 200 μL and allowing it to stand for 3 minutes, the current-voltage curve of each device was measured, and the gate voltage shift ΔV before and after the addition of each molecule was measured. g I evaluated it.

[0076] Figure 5 shows the evaluation results of the amount of shift in semiconductor properties before and after the addition of 1 μM cortisol, 1 μM cortisone, or 0.1 U / μL α-amylase to aptamer-immobilized devices. When 1 μM cortisol is added, the gate voltage shift ΔV in the current-voltage curve, which is a semiconductor property, is observed. g The voltage became +14mV. On the other hand, when 1 μM cortisone or 0.1 U / μL α-amylase was added, the gate voltage shift ΔV in the change of the current-voltage curve was... g Almost no such detection was observed. Therefore, the specificity of cortisol detection in aptamer-immobilized devices fabricated using disulfide bonds was demonstrated.

[0077] [Example 3] The quantitative detection capability of cortisol using an aptamer-immobilized device fabricated using disulfide bonds was evaluated as follows.

[0078] First, SPDP was reacted as a crosslinking molecule to bridge the amino group of the organic monolayer with the 3'-terminated thiol-group modified aptamer. The reaction was carried out by placing the device in a holder and immersing the detection part of the device, which had a monolayer with an amino group at the end, in 20 μL of 1×PB containing 1 mM SPDP for 1 hour at room temperature. Subsequently, the 2-mercaptopyridine moiety was removed and a thiol group was formed by cleaving the disulfide bond in the SPDP structure. The reaction was carried out by immersing the SPDP-modified device detection part in 20 μL of 1×PB containing 10 mM DTT for 1 hour at room temperature. An aptamer, which is a single-stranded DNA, was immobilized as a bioreceptor by forming a disulfide bond with a thiol group derived from SPDP. The reaction was carried out by immersing the gate electrode portion, which had a thiol group formed in the SPDP structure, in 1×PB / 1×PB containing 100 nM 3'-terminated thiol-group modified aptamer (55 bases). k The substrate was immersed in a mixed solution (volume ratio 1:1) for 1 hour. After cleaning the substrate, it was capped with 100 μM MCH-containing PB for 1 hour.

[0079] Subsequently, the detection unit of the device is set to 0.04 × PBk After immersion in 200 μL for 3 minutes, the current-voltage curve of the aptamer-immobilized device was measured at room temperature using a digital source meter (Keithley 2612) with a double-junction Ag / AgCl reference electrode. The measurement conditions were a gate voltage of -3V to 0.5V and a drain voltage of 0.1V.

[0080] Next, 20 μL of 1×PBS containing 1-1000 nM cortisol was added to the gate surface, and after standing for 30 minutes, 3 mL of 1×PB and 0.04×PB were added. k Rinsing was performed using 3 mL. Afterwards, 0.04 × PB was applied to the gate surface. k After adding 200 μL and allowing it to stand for 3 minutes, the current-voltage curve of the cortisol adsorption device was measured, and the gate voltage shift ΔV before and after cortisol addition was measured. g I evaluated it.

[0081] Figure 6 shows the evaluation results of the amount of semiconductor characteristic shift before and after the addition of 1 to 1000 nM cortisol to aptamer-immobilized devices. After adding cortisol at various concentrations from 1 to 1000 nM to the aptamer-immobilized devices, the gate voltage shift ΔV due to cortisol addition was evaluated. g Upon obtaining the data, it was confirmed that the shift amount increased with increasing concentration. Therefore, the quantitative detection capability of cortisol in the aptamer-immobilized device fabricated using disulfide bonds was demonstrated.

[0082] [Example 4] The response to cortisol addition was evaluated as follows using a device in which a 3'-terminal thiol group modified aptamer was immobilized via a thiol-maleimide bond through Sulfo-SMCC on the gate electrode of a detector modified with an amino monolayer.

[0083] First, Sulfo-SMCC was reacted as a cross-linking molecule for cross-linking the amino group of the organic monolayer and the 3'-terminal thiol group-modified aptamer. The reaction was carried out by placing the device in a holder and immersing the detection part of the device with a monolayer having an amino group at the end in 20 μL of 1×PB containing 1 mM of Sulfo-SMCC at room temperature for 1 hour. The aptamer, which is single-stranded DNA as a bioreceptor, was immobilized by binding to the maleimide group of Sulfo-SMCC. The reaction was performed by immersing the gate electrode part that formed the maleimide group derived from the Sulfo-SMCC structure in a 1×PB / 1×PB k mixed solution (volume ratio 1:1) containing 100 nM of the 3'-terminal thiol group-modified aptamer (55 bases) for 1 hour. After washing the substrate, capping treatment with PB containing 100 μM of MCH was performed for 1 hour.

[0084] Thereafter, the detection part of the device was immersed in 1×PB k diluted 25-fold to 200 μL of 0.04×PB k for 3 minutes, and then at room temperature, the current-voltage curve of the aptamer-immobilized device was measured using a single-junction 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.

[0085] Subsequently, 20 μL of 1×PBS containing 1 μM of cortisol was added onto the gate surface of the aptamer-immobilized device, allowed to stand for 30 minutes, and then rinsed with 3 mL of 1×PB and 3 mL of 0.04×PB k . Thereafter, 200 μL of 0.04×PB k was added onto the gate surface, allowed to stand for 3 minutes, and then the current-voltage curve of the cortisol-adsorbed device was measured, and the gate voltage shift ΔV g before and after the addition of cortisol was evaluated.

[0086] The evaluation results of the shift amount of the semiconductor characteristics before and after the addition of 1 μM of cortisol to the aptamer-immobilized device are shown in Fig. 7. When 1 μM of cortisol was added, the gate voltage shift ΔV in the current-voltage curve, which is a semiconductor characteristicg The response was +12mV. Compared to the case in Example 1 where cortisol was added to the aptamer immobilized device using the bond between amino and aldehyde groups by the crosslinking agent glutaraldehyde, the standard deviation of the response was reduced. Therefore, cortisol detection was demonstrated in the aptamer immobilized device fabricated using the thiol-maleimide bond. [Explanation of Symbols]

[0087] 1. Silicon substrate 2. Insulating layer 3. First organic monolayer 4 gate 5 Source electrodes 6 Drain electrode 7 Doping Area 8 Reference section 9 Detection unit 10 Template section 11 Probe molecules 12 Uncharged molecules

Claims

1. A method for detecting uncharged molecules, comprising the steps of: interacting an uncharged molecule with a probe molecule immobilized on a probe molecule-immobilized semiconductor sensing device that interacts with the uncharged molecule; and detecting a change in the potential of a gate channel region due to the interaction, The semiconductor sensing device on which the probe molecule is immobilized comprises a field-effect transistor having a detection unit on which the probe molecule is immobilized, The detection unit comprises a probe molecule / organic monolayer / insulating layer / semiconductor structure, wherein a first insulating layer containing silicon oxide or inorganic oxide is formed on a semiconductor as a reaction gate insulating layer, a first organic monolayer made of an organic monolayer having reactive functional groups is formed on the first insulating layer, and a probe molecule in which a functional group not originally present is introduced to the reactive functional group of the first organic monolayer is bonded directly via the reactive functional group or via a crosslinking molecule by bonding with the functional group not originally present on the probe molecule. A method for detecting uncharged molecules.

2. The method for detecting an uncharged molecule according to claim 1, wherein the probe molecule is a nucleic acid aptamer.

3. The method for detecting an uncharged molecule according to claim 1, wherein the bond formed by a functional group not originally present in the probe molecule is a disulfide bond or a thiol-maleimide bond.

4. The method for detecting an uncharged molecule according to claim 1, wherein the reactive functional group of the first organic monolayer is an amino group.

5. The method for detecting an uncharged molecule according to claim 1, wherein the crosslinking molecule is N-succinimidyl 3-(2-pyridyldithio)propionate or sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate.

6. A probe molecule immobilized semiconductor sensing device comprising a field-effect transistor having a detection unit on which probe molecules are immobilized, The probe molecule immobilized semiconductor sensing device comprises a probe molecule / organic monolayer / insulating layer / semiconductor structure, wherein the detection unit has a first insulating layer formed on a semiconductor as a reaction gate insulating layer containing silicon oxide or an inorganic oxide, a first organic monolayer formed on the first insulating layer consisting of an organic monolayer having reactive functional groups, and a probe molecule in which a functional group not originally present is introduced to the reactive functional group of the first organic monolayer, and is bonded via a crosslinking molecule by bonding with the functional group not originally present on the probe molecule.

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