Membrane-type surface stress sensor and analysis method using same

Abstract

To provide a membrane-type surface stress sensor that takes a new form for binding targets.SOLUTION: A membrane-type surface stress sensor includes an aptamer, a membrane, and a sensor substrate. The aptamer is a nucleic acid molecule bound to a target and is immobilized to the membrane. The membrane is a membrane that deforms due to binding of the target to the aptamer. The sensor substrate has a support region that supports the membrane and includes a piezoresistive element. The piezoresistive element is an element that detects deformation of the membrane.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a membrane-type surface stress sensor and an analysis method using the same. [Background technology]

[0002] In a wide variety of fields such as food and medicine, target detection is important, and various methods have been proposed. In recent years, membrane-type surface stress sensors have been attracting attention (see Patent Document 1). The membrane-type surface stress sensor can analyze the presence or absence and amount of a target by, for example, binding a target to a film such as a silicon film, deforming the film, and measuring the fluctuation in electrical resistance due to the deformation. However, further improvements are required for the method of binding a target to the film, for example, from the viewpoint of improving analytical accuracy and expanding the range of applicable targets. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] International Publication No. 2011 / 148774 Summary of the Invention [Problem to be solved by the invention]

[0004] Therefore, an object of the present invention is to provide a membrane-type surface stress sensor having a new form for binding a target. [Means for solving the problem]

[0005] In order to achieve the above object, the membrane-type surface stress sensor of the present invention comprises: The present invention includes an aptamer, a membrane, and a sensor substrate, The aptamer is a nucleic acid molecule that binds to a target and is immobilized on the membrane; the membrane is a membrane that deforms upon binding of the target to the aptamer; the sensor substrate has a support region; the support region supports the membrane and includes a piezoresistive element; The piezoresistance element is an element for detecting deformation of the membrane. It is characterized by:

[0006] The method for analyzing a target of the present invention includes the steps of: A step of immersing the membrane-type surface stress sensor of the present invention in a sample liquid; applying a voltage to the membrane-type surface stress sensor in a liquid phase; and analyzing a target in the sample liquid by measuring a change in stress of the piezoresistance element in the film-type surface stress sensor. Effect of the Invention

[0007] According to the present invention, by immobilizing an aptamer on the membrane as a new form for binding a target, it is possible to expand the possibilities for application to targets different from those of conventional membrane-type surface stress sensors, and for modifications different from those of conventional membrane-type surface stress sensors. [Brief description of the drawings]

[0008] [Figure 1] FIG. 1 is a schematic diagram showing a general configuration of an MSS. [Diagram 2] FIG. 2 is a schematic diagram showing the structure of the MSS in Example 2 and a graph showing the voltage of the MSS. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] In the present invention, the term "membrane-type surface-stress sensor" is used hereinafter. The "MSS" is also called an MSS. In the so-called MSS, a membrane having binding properties to a target is supported by a support having a piezoresistance element. When the target binds to the membrane, the membrane is subjected to stress due to the binding, and the membrane is deformed (distortion occurs) due to the generation of distortion. Then, depending on the amount of deformation of the membrane, stress is generated in the piezoresistance element of the support supporting the membrane, and the resistance value of the piezoresistance element changes in proportion to the stress. Therefore, by applying a voltage to the MSS and measuring an electric signal accompanying the change in resistance value, it is possible to indirectly qualitatively determine the presence or absence of the target bound to the membrane, or to quantitatively analyze the amount of the target bound to the membrane. The present invention is characterized in that an aptamer that binds to a target is used in such an MSS, specifically, the aptamer is immobilized on the membrane to bind the target to the MSS. Therefore, in the present invention, other configurations are not particularly limited except for immobilizing the aptamer on the membrane, and existing configurations can be used, and future configurations that perform similar functions can also be used.

[0010] In the present invention, an "aptamer" is a nucleic acid molecule that has binding properties to a target. The aptamer can also be referred to as, for example, a nucleic acid molecule that specifically binds to a target. The constituent units of the aptamer are, for example, nucleotide residues and non-nucleotide residues. Examples of the nucleotide residues include deoxyribonucleotide residues and ribonucleotide residues, and the nucleotide residues may be modified or unmodified. Examples of the aptamer include DNA aptamers consisting of deoxyribonucleotide residues, RNA aptamers consisting of ribonucleotide residues, aptamers containing both, and aptamers containing modified nucleotide residues. The length of the aptamer is not particularly limited, and is, for example, 10 to 200 bases. The aptamer for the target may be, for example, an existing aptamer, or a newly obtained aptamer obtained by using, for example, the SELEX method, depending on the target.

[0011] In the present invention, the "target" is not particularly limited and can be set arbitrarily, and may be, for example, a substance that can come into contact with the aptamer in a liquid. Examples of the target include microorganisms such as bacteria such as anthrax, E. coli, Salmonella, and E. coli; viruses such as influenza virus; allergens; and the like. Examples of the allergens include grains such as wheat, eggs, meat, fish, shellfish, vegetables, fruits, milk, beans such as peanuts, and pollen such as cedar and cypress. The type of the target is not particularly limited and may be, for example, polymer compounds such as proteins, sugar chains, nucleic acids, and polymers; low molecular weight compounds; and the like.

[0012] In the present invention, the "liquid sample" may be any liquid. When the collected specimen is liquid, it may be used as a liquid sample as it is, or may be a liquid sample prepared by diluting, suspending, dispersing, or the like with a liquid solvent. When the collected specimen is solid, it may be a liquid sample prepared by dissolving, suspending, dispersing, or the like with a liquid solvent. When the collected specimen is gas, it may be a liquid sample prepared by concentrating aerosol in the gas, or may be a liquid sample prepared by dissolving, suspending, dispersing, or the like with a liquid solvent. The type of the liquid solvent is not particularly limited, and is, for example, a solvent that does not easily affect the binding between the aptamer and the target, and specific examples include water and a buffer solution. Examples of the collected specimen include food, blood, urine, saliva, body fluids, soil, wastewater, tap water, ponds, rivers, and air.

[0013] The following describes the embodiments of the present invention by way of examples. The present invention is not limited to the following embodiments. In addition, the descriptions of the embodiments can be mutually incorporated unless otherwise specified. Furthermore, the configurations of the embodiments can be combined unless otherwise specified.

[0014] [Embodiment 1] As described above, the MSS of this embodiment includes an aptamer, a membrane, and a sensor substrate, wherein the aptamer is a nucleic acid molecule that binds to a target and is immobilized on the membrane, the membrane being a membrane that deforms upon binding of the target to the aptamer, the sensor substrate having a support region that supports the membrane and has a piezoresistance element, and the piezoresistance element is an element that detects deformation of the membrane.

[0015] In the MSS of this embodiment, the film is also called an MSS film. As described above, the MSS film is not particularly limited as long as it is deformed by the binding of the target and the deformation applies stress to the piezoresistance element. The film is, for example, a thin film, and the thickness and the area of ​​each surface are not particularly limited, and are, for example, similar to the MSS film used in a commercially available MSS. The planar shape of the film is, for example, a circle, specifically, a perfect circle. The material of the film is not particularly limited, and is, for example, a silicon film, and a specific example is n-type Si(100).

[0016] In the MSS of this embodiment, the aptamer is immobilized on the MSS membrane. The aptamer may be immobilized, for example, on one surface of the MSS membrane, or on both surfaces. When the aptamer is immobilized on both surfaces of the MSS membrane, it is preferable that the aptamer on one surface and the aptamer on the other surface are, for example, the same aptamer that binds to the same target. In the following description, the surface of the MSS membrane may be, for example, one surface or both surfaces.

[0017] The method of immobilizing the aptamer on the MSS membrane is not particularly limited, and the aptamer may be directly or indirectly immobilized on the MSS membrane. In the former case, for example, the aptamer can be immobilized by covalent bonding or the like by chemically treating the MSS membrane and the aptamer. The direct immobilization method includes, for example, a method using photolithography, and U.S. Patent No. 5,424,186 and the like can be referred to as a specific example. Another direct immobilization method includes, for example, a method of synthesizing the sensor on the MSS membrane. This method includes, for example, the so-called spot method, and U.S. Patent No. 5,807,522 and the like can be referred to as a specific example. In the latter case, for example, the aptamer can be immobilized on the MSS membrane via a linker. The type of the linker is not limited at all, and examples thereof include a combination of biotin or a biotin derivative (hereinafter referred to as biotins) and avidin or an avidin derivative (hereinafter referred to as avidins). The biotin derivative is, for example, biocytin, and the avidin derivative is, for example, streptavidin. The length of the linker can be expressed, for example, by the length (main chain length) of the shortest molecular chain from a functional group on the MSS film (for example, an oxygen atom of a silanol group on a silicon film) to an affinity tag or aptamer such as avidin. The main chain length of the linker is 1 to 20, and is preferably 1 to 15, 1 to 13, 3 to 13, 5 to 13, 1 to 11, 3 to 11, 1 to 10, 3 to 10, 1 to 8, 3 to 8, 1 to 5, 1 to 3, 1, or 2, since it can improve the sensitivity of MSS. Examples of immobilization methods are given below, but the present invention is not limited to these.

[0018] As a first example, the biotins are bound to either the MSS membrane or the aptamer, and the avidins are bound to the other. By binding the biotins and the avidins, the aptamer can be indirectly immobilized on the MSS membrane.

[0019] In the first example, the aptamer is indirectly fixed to the MSS membrane by utilizing specific binding between avidins and biotins, i.e., the affinity of biotins to avidins, but the present invention is not limited thereto, and affinity tags other than avidins and biotins may be used. Examples of the affinity tag that can be used include His tag (His×6 tag)-nickel ion, glutathione-S-transferase-glutathione, maltose binding protein-maltose, and epitope tag (myc tag, FLAG tag, HA (hemagglutinin) tag)-antibody or antigen-binding fragment. The fact that other affinity tags may be used is the same in the second to fourth examples described below.

[0020] As a second example, the aptamer may be immobilized on the MSS film, for example, via an intervening film. The intervening film is formed on the MSS film, and the biotins are bound to either the intervening film or the aptamer, and the avidins are bound to the other, as in the first example, and the aptamer can be immobilized on the MSS film via the intervening film by the binding between the biotins and the avidins. The intervening film is, for example, a film of a metal such as gold, and can be formed by evaporating the metal onto the MSS film. The thickness of the intervening film is not particularly limited, and is, for example, 10 to 100 nm. The intervening film may be, for example, one layer or two or more layers. When the surface of the intervening film is made of gold, the intervening film is, for example, two layers, and it is preferable to form the gold film on the MSS film via a metal film (adhesive film) for adhesion, in order to improve the adhesiveness of the gold film. Examples of the metal of the adhesive film include titanium and chromium. The thickness of the adhesive film is, for example, 0.1 to 10 nm, and the thickness of the gold film is, for example, 0.1 to 100 nm. When the biotins are bound to the intervening film, for example, a self-assembled monolayer (SAM) of thiolalkane may be formed on the surface of the intervening film using a thiolalkane bound to the biotins, and the aptamer bound to the biotins may be brought into contact with the SAM, and the aptamer may be immobilized by the binding between the biotins and the avidins.

[0021] As a third example, there is a method of binding the streptavidins to the MSS film by binding an amino group and further binding glutaraldehyde. That is, a silane coupling agent having an amino group is reacted with the MSS film to bind the amino group on the MSS film. The reaction can be carried out, for example, by applying a solution containing a silane coupling agent having an amino group to the MSS film. Furthermore, a crosslinking agent capable of binding an amino group to the main chain or side chain of an amino acid, or a crosslinking agent such as glutaraldehyde capable of forming a linker between an amino group and the main chain or side chain of an amino acid is reacted with the MSS film, and one end of the crosslinking agent such as glutaraldehyde is bound to the amino group on the MSS film. Specifically, the surface of the MSS film after silane coupling is washed, and a solution containing a crosslinking agent is applied to the MSS film to bind the amino group to the crosslinking agent. The conditions of the crosslinking reaction can be appropriately determined according to, for example, the type of crosslinking agent. Next, the avidins are bound to the other end of the crosslinking agent such as glutaraldehyde. Specifically, the surface of the MSS membrane after crosslinking is washed, a solution containing avidins is applied, and the other end of the crosslinking agent is bound to the main chain or side chain of the amino acid of the avidins. Then, the aptamer bound to the biotin is contacted with the MSS membrane thus treated, and the aptamer can be immobilized by the binding between the biotins and the avidins.

[0022] The silane coupling agent is, for example, Y-Si(CH3) 3-n (OR) n In the case where the silane coupling agent has an amino group, n, R, and Y are, for example, as follows. The "n" is 2 or 3. Examples of R include alkyl groups such as methyl and ethyl groups; acyl groups such as acetyl and propyl groups; and the like. Y is a reactive functional group having an amino group at its terminal.

[0023] Examples of the silane coupling agent having an amino group include N-(2-aminoethyl)-3-aminopropylmethyldimethoxysilane (e.g., KBM-602 (manufactured by Shin-Etsu Silicone Co., Ltd.)), N-(2-aminoethyl)-3-aminopropyltrimethoxysilane (e.g., KBM-603 (manufactured by Shin-Etsu Silicone Co., Ltd.)), 3-aminopropyltrimethoxysilane (e.g., KBM-903 (manufactured by Shin-Etsu Silicone Co., Ltd.)), 3-aminopropyltriethoxysilane (e.g., KBE-903 (manufactured by Shin-Etsu Silicone Co., Ltd.)), 3-(2-aminoethylamino)propyltrimethoxysilane (e.g., GENIOSIL (registered trademark) GF 91 (manufactured by Wacker Asahi Kasei Silicone Co., Ltd.)), and 3-(2-aminoethylamino)propylmethyldimethoxysilane (e.g., GENIOSIL (registered trademark) GF 95 (manufactured by Wacker Asahi Kasei Silicone Co., Ltd.)).

[0024] The crosslinking agent can be appropriately determined depending on the functional group of the main chain or side chain of the amino acid to be bonded to the linker. Examples of the functional group include an amino group (-NH2), a thiol group (-SH), and a carboxyl group (-COOH). The amino group is, for example, present at the N-terminus of a protein or peptide or the side chain of lysine. The thiol group is, for example, present at the side chain of cysteine. The carboxyl group is, for example, present at the C-terminus of a protein or peptide or the side chain of aspartic acid or glutamic acid.

[0025] When an amino group in the main chain or side chain of the amino acid is used, examples of the crosslinking agent include crosslinking agents having aldehyde groups at both ends, such as glutaraldehyde; bis(sulfosuccinimidyl)suberate (BS3), disuccinimidyl glutarate (DSG), disuccinimidyl suberate (DSS), dithiobis(succinimidyl propionate), dithiobis(sulfosuccinimidyl propionate) (DSP), dithiobis(succinimidyl propionate) (DTSP), dithiobis(sulfosuccinimidyl propionate) (DTSSP), disuccinimidyl tartrate, crosslinkers having N-hydroxysuccinimide active esters (N-hydroxysuccinimide reactive groups) at both ends, such as ethylene glycol bis(succinimidyl succinate) (DST), ethylene glycol bis(succinimidyl succinate) (ESG), ethylene glycol bis(sulfosuccinimidyl succinate) (Sulfo-ESG), and PEGylated bis(sulfosuccinimidyl) (BS(PEG)5, BS(PEG)9, etc.); crosslinkers having imide ester reactive groups at both ends, such as dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), and dimethyl pimelimidate (DMS).

[0026] In the case where a thiol group in the side chain of the amino acid is used, examples of the crosslinking agent include N-(6-maleimidocaproyloxy)succinimide (EMCS), N-(6-maleimidocaproyloxy)sulfosuccinimide (Sulfo-EMCS), N-(8-maleimidocapryloxy)succinimide (HMCS), N-(8-maleimidocapryloxy)sulfosuccinimide (Suflo-HMCS), N-α-maleimidoacet-oxysuccinimide ester (AMAS), N-β-maleimidopropyl-oxysuccinimide ester (BMPS), N-γ-maleimidobutyryl-oxysuccinimide ester (GMBS), N-γ-maleimidobutyryl-oxysulfosuccinimide ester (Sulfo-GMBS), m-maleimidobenzoyl-N-hydroxysuccinimide ester(MBS), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester(Sulfo-MBS), succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate(SMCC), sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate(Sulfo-SMCC), succinimidyl 4-(p-maleimidophenyl) butyrate(SMPB), succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxy-(6-amidocaproate)(LC-SMCC), N-κ-maleimidoundecanoyl-oxysulfosuccinimideCrosslinkers having a maleimide group and an N-hydroxysuccinimide active ester at both ends of the molecule, such as succinimidyl iodoacetate (SIA), succinimidyl 3-(bromoacetamido) propionate (SBAP), succinimidyl (4-iodoacetyl) aminobenzoate (SIAB), sulfosuccinimidyl (4-iodoacetyl) aminobenzoate (Sulfo-SIAB), and other crosslinkers having an N-hydroxysuccinimide active ester and a haloacetyl reactive group at both ends; succinimidyl 3-(2-pyridyldithio) propionate (SPDP), succinimidyl 6-(3(2-pyridyldithio) propionamido) hexanoate (LC-SPDP), succinimidyl 6-(3(2-pyridyldithio) propionamido) Examples of such crosslinking agents include crosslinkers having an N-hydroxysuccinimide active ester and a pyridyldithiol reactive group at both ends, such as hexanoate (Sulfo-LC-SPDP), 4-succinimidyloxycarbonyl-alpha-methyl-α(2-pyridyldithio) toluene (SMPT), 2-Pyridyldithiol-tetraoxatetradecane-N-hydroxysuccinimide (PEG4-SPDP), and 2-Pyridyldithiol-tetraoxaoctatriacontane-N-hydroxysuccinimide (PEG12-SPDP).

[0027] When a carboxyl group in the main chain or side chain of the amino acid is utilized, examples of the crosslinking agent include dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), N-hydroxysulfosuccinimide (Sulfo-NHS), acetic anhydride, etc. DCC, EDC, NHS, Sulfo-NHS, and acetic anhydride directly bond an amino group and a carboxyl group, and therefore do not remain between the carboxyl group and the amino group, and a linker region (group) derived from the crosslinking agent is not generated.

[0028] The crosslinking agent is preferably a crosslinking agent that does not substantially cause self-condensation, since the length of the linker can be made substantially constant or fixed. The constant length of the linker means, for example, that in a linker of a plurality of aptamers, the length of the linker of each aptamer is substantially the same or the same. The length of the linker can be made substantially the same or the same, for example, by making the structure of the linker substantially the same or the same. In the third example, the sensitivity of the MSS can be improved by using such a crosslinking agent. The improvement in the sensitivity is presumed to be due to the following reasons. Note that the present invention is not limited to the following presumptions. When a target binds to an aptamer, a steric hindrance caused by the target occurs around the aptamer bound to the target. When the aptamer is immobilized at a different distance from the MSS membrane, the target is likely to come into contact with the aptamer present on the distal end side from the MSS membrane. For this reason, it is presumed that the target preferentially binds to the aptamer on the distal end side from the MSS membrane. In this case, even if steric hindrance occurs around the aptamer bound to the target due to the target, other aptamers are less likely to be affected by steric hindrance due to the target, since there are more aptamers present on the MSS membrane side than the aptamer bound to the target. Therefore, even if the target binds to the aptamer, the aptamers present in the surroundings are less likely to move in position due to the influence of steric hindrance. Therefore, when the aptamers are immobilized at different distances from the MSS membrane, the MSS membrane is relatively less likely to be distorted due to the movement of the positions of the surrounding aptamers. In other words, the surrounding aptamers are less likely to move due to the binding of the aptamer, and the distortion of the MSS membrane is less likely to be amplified. On the other hand, when the aptamers are immobilized at approximately the same distance from the MSS membrane, when the target binds to the aptamer, the surrounding aptamers are affected by steric hindrance due to the target. Therefore, there is a high possibility that the positions of the surrounding aptamers will move, and there is a relatively high possibility that the movement of the positions of the surrounding aptamers will cause distortion of the MSS membrane.That is, when the aptamers are immobilized at approximately the same distance from the MSS membrane, the binding of one aptamer to a target on the MSS membrane also causes the position of the surrounding aptamers to move, amplifying the distortion of the MSS membrane. Therefore, when the aptamers are immobilized at approximately the same distance from the MSS membrane, that is, when the length of the linker is approximately constant, it is estimated that the sensitivity of the MSS membrane is improved.

[0029] Specific examples of the crosslinking agent that does not substantially undergo self-condensation include a crosslinking agent having an N-hydroxysuccinimide active ester at both ends, a crosslinking agent having an imide ester reactive group at both ends, a crosslinking agent having a maleimide group and an N-hydroxysuccinimide active ester at both ends of the molecule, a crosslinking agent having an N-hydroxysuccinimide active ester and a haloacetyl reactive group at both ends, a crosslinking agent having an N-hydroxysuccinimide active ester and a pyridyldithiol reactive group at both ends, DCC, EDC, NHS, Sulfo-NHS, acetic anhydride, and the like.

[0030] The linker is represented, for example, by the following formula (1). In the following formula (1), M1 represents an atom bonded to a silane coupling agent on the MSS membrane, L1 represents a region (group) derived from the silane coupling agent, L2 represents a region (group) derived from the crosslinking agent, and L2 may or may not be present, and M2 represents an atom bonded to the crosslinking agent or NH in the affinity tag. In addition, NH represents an amine derived from the amino group of a silane coupling agent having an amino group. M1-L1-NH-L2-M2···(1)

[0031] L1 is, for example, (M1)-Si(CH3) 2-m (OR4) m -R1-(NH) or (M1)-Si(CH3) 2-m (OR4) mIt is represented by -R2-NH-R3-(NH). R1 ​​is a straight or branched alkyl group having 1 to 5 carbon atoms. R2 and R3 are, for example, each independently a straight or branched alkyl group having 1 to 5 carbon atoms, and may be the same or different. Examples of the alkyl group include a methyl group, an ethyl group, a propyl group, a butyl group, and a pentyl group. R4 is, for example, a hydrogen atom or a bond. m is 1 or 2.

[0032] When 3-aminopropyltriethoxysilane is used as the silane coupling agent, L1 is represented, for example, by (M1)-Si(OR4)2-(CH2)3-(NH). R4 is, for example, a hydrogen atom or a bond. When glutaraldehyde is used as the crosslinking agent, L2 is, for example, represented by (NH)=CH-C3H6-CH=(CH(CHO)-C2H4-CH). n It is represented by =CH(CHO)-C2H4-C=(M2).

[0033] The length of the linker can be expressed, for example, by the length (main chain length) of the shortest molecular chain from a functional group on the MSS film (for example, an oxygen atom of a silanol group on a silicon film) to an affinity tag such as avidin or an aptamer. The main chain length of the linker is 1 to 20, and is preferably 1 to 15, 1 to 13, 3 to 13, 5 to 13, 1 to 11, 3 to 11, 1 to 10, 3 to 10, 1 to 8, 3 to 8, 1 to 5, 1 to 3, 1, or 2, since this can improve the sensitivity of the MSS.

[0034] In the third example, avidins-biotins binding is used, but the third example is not limited thereto, and a linker may be directly bound to the hydroxyl or phosphate group of the aptamer. In this case, the aptamer can be immobilized on the MSS membrane by amidating the phosphate group at the 3' end and reacting it with the linker.

[0035] As a fourth example, there is a method in which a methacryl group (-C(=O)-C(CH3)=CH2) is bound to the MSS film, and the streptavidins are bound to the MSS film via an amino acid or its derivative (hereinafter referred to as "amino acid derivative"). That is, a silane coupling agent having a methacryl group is reacted with the MSS film to bind an amino group to the MSS film. The reaction can be carried out, for example, by applying a solution containing a silane coupling agent having a methacryl group to the MSS film. Furthermore, after reacting an amino acid derivative such as N-acetylcysteine ​​with the MSS film, a crosslinker capable of forming a linker between the main chain or side chain of the amino acid derivative and the main chain or side chain of the amino acid of the avidins is reacted with the main chain or side chain of the amino acid derivative, and one end of the crosslinker is bound to the amino acid derivative on the MSS film. Specifically, the surface of the MSS film after the amino acid derivative treatment is washed, and a solution containing a crosslinker is applied to the MSS film to bind the amino acid derivative and the crosslinker. The conditions of the crosslinking reaction can be appropriately determined according to, for example, the type of crosslinking agent. Next, the avidins are bound to the other end of the crosslinking agent. Specifically, the surface of the MSS membrane after crosslinking is washed, a solution containing avidins is applied, and the other end of the crosslinking agent is bound to the main chain or side chain of the amino acid of the avidins. Then, the aptamer bound to the biotin is contacted with the MSS membrane thus treated, and the aptamer can be immobilized by the binding between the biotins and the avidins. In the fourth example, the binding of avidins to biotins is utilized, but the fourth example is not limited thereto, and a linker may be directly bound to the hydroxyl group or phosphate group of the aptamer.

[0036] As described above, the silane coupling agent is, for example, Y-Si(CH3) 3-n (OR) nIn the case where the silane coupling agent has a methacryl group, n, R, and Y are, for example, as follows. The "n" is 2 or 3. Examples of R include alkyl groups such as methyl and ethyl groups; acyl groups such as acetyl and propyl groups; and the like. Y is a reactive functional group having a methacryl group at its terminal.

[0037] Examples of the silane coupling agent having a methacryl group include 3-(methacryloyloxy)propylmethyldimethoxysilane (e.g., KBM-502 (manufactured by Shin-Etsu Silicones)), 3-(methacryloyloxy)propyltrimethoxysilane (e.g., KBM-503 (manufactured by Shin-Etsu Silicones), GENIOSIL (registered trademark) GF31 (manufactured by Wacker Asahi Kasei Silicones)), 3-(methacryloyloxy)propylmethyldimethoxysilane (e.g., KBE-502 (manufactured by Shin-Etsu Silicones)), (3-methacryloyloxypropyl)triethoxysilane (e.g., KBE-503 (manufactured by Shin-Etsu Silicones)), and the like.

[0038] The amino acid or amino acid derivative has, for example, a functional group capable of reacting with a methacryl group and a carboxyl group. The functional group capable of reacting with a methacryl group can be, for example, a thiol group (-SH). The amino acid or amino acid derivative having a thiol group can be, for example, cysteine; cysteine ​​with a modified amino group such as N-acetylcysteine; and the like.

[0039] The crosslinking agent can be appropriately determined depending on, for example, the functional group of the amino acid derivative to be crosslinked and the functional group of the amino acid of the avidin to be crosslinked. As a specific example, when the two functional groups are amino groups, the crosslinking agent can be described in the third example when the amino group of the main chain or side chain of the amino acid is used. In addition, when one of the two functional groups is an amino group and the other is a thiol group, the crosslinking agent can be described in the third example when the thiol group of the side chain of the amino acid is used. Furthermore, when one of the two functional groups is an amino group and the other is a carboxyl group, the crosslinking agent can be described in the third example when the carboxyl group of the main chain or side chain of the amino acid is used.

[0040] The crosslinking agent is preferably one that does not substantially undergo self-condensation, since it can make the linker length constant. In the fourth example, by using such a crosslinking agent, the sensitivity of MSS can be improved by the same mechanism as that described in the third example. Specific examples of crosslinking agents that do not substantially undergo self-condensation include, for example, a crosslinking agent having an N-hydroxysuccinimide active ester at both ends, a crosslinking agent having an imide ester reactive group at both ends, a crosslinking agent having a maleimide group and an N-hydroxysuccinimide active ester at both ends of the molecule, a crosslinking agent having an N-hydroxysuccinimide active ester and a haloacetyl reactive group at both ends, a crosslinking agent having an N-hydroxysuccinimide active ester and a pyridyldithiol reactive group at both ends, DCC, EDC, NHS, Sulfo-NHS, acetic anhydride, and the like.

[0041] The linker is represented, for example, by the following formula (2): In the following formula (2), M1 represents an atom bonded to a silane coupling agent on the MSS membrane, L1 represents a region (group) derived from the silane coupling agent, A represents an amino acid derivative, L2 represents a region (group) derived from a crosslinking agent, L2 may or may not be present, and M2 represents an atom bonded to a crosslinking agent or NH in the affinity tag. M1-L1-A-L2-M2···(2)

[0042] L1 is, for example, (M1)-Si(CH3) 2-m (OR4) m -R5-C(=O)-CH l (CH3) 2-l -(A). R4 is, for example, a hydrogen atom or a bond. R5 is a straight or branched alkyl group having 1 to 5 carbon atoms. Examples of the alkyl group include a methyl group, an ethyl group, a propyl group, a butyl group, and a pentyl group. m is 1 or 2. l is 0 or 1.

[0043] When 3-(methacryloyloxy)propyltrimethoxysilane is used as the silane coupling agent, L1 is represented, for example, by (M1)-Si(OR4)2-(CH2)3-OC(=O)-C(CH3)2-(A). R4 is, for example, a hydrogen atom or a bond. When N-acetylcysteine ​​is used as the amino acid derivative, A is represented, for example, by (L1)-S-CH2-CH(NH-COCH3)-C(=O)-(M2). When acetic anhydride is used as the crosslinking agent, L2 is, for example, absent.

[0044] The length of the linker can be expressed, for example, by the length (main chain length) of the shortest molecular chain from a functional group on the MSS membrane (for example, a silanol group on a silicon membrane) to an affinity tag such as avidin. The main chain length of the linker is 1 to 20, and is preferably 1 to 15, 1 to 13, 1 to 11, 1 to 10, 1 to 8, 1 to 5, 1 to 3, 1, or 2, since this can improve the sensitivity of MSS.

[0045] In the fourth example, avidins-biotins binding is used, but the fourth example is not limited thereto, and a linker may be directly bound to the hydroxyl or phosphate group of the aptamer. In this case, the aptamer can be immobilized on the MSS membrane by amidating the phosphate group at the 3' end and reacting it with the linker.

[0046] The site at which the aptamer is immobilized on the MSS membrane is not particularly limited, and examples thereof include the 3' end or the 5' end.

[0047] In the MSS of this embodiment, the sensor substrate has a support region that supports the MSS membrane, and the support region has a piezoresistance element. The sensor substrate supports the MSS membrane by the support region. The MSS membrane has, for example, one or both opposing surfaces to which the aptamer is immobilized as described above, and is supported by the sensor substrate at its side. For example, the sensor substrate preferably partially supports the MSS membrane, and more specifically, preferably partially supports the side surface of the MSS membrane. The number of points (support portions) in the MSS membrane that are supported by the support region of the sensor substrate is not particularly limited, and is, for example, four points. Note that this is merely an example and is not limiting in any way.

[0048] In the sensor substrate, the support region is, for example, a silicon film, and any region of the silicon film can be made p-type by doping with an impurity, so that the p-typed region (p-type Si) can function as the piezoresistance element. The support region has the piezoresistance element, for example, at a location supporting the MSS film or in the vicinity thereof. The sensor substrate may be, for example, entirely made of silicon, or only the support region may be a silicon film, and materials other than those of the support region including the piezoresistance element are not particularly limited.

[0049] In the MSS of this embodiment, for example, the sensor substrate has a circuit for applying a voltage. When the support region supports the MSS film at multiple points and has piezoresistance elements at and near the supporting points, for example, the circuit can be a Wheatstone bridge circuit including multiple piezoresistance elements in the support region. The MSS of this embodiment can measure an electrical signal associated with a change in resistance value in the piezoresistance elements, as described above, by applying a voltage to the Wheatstone bridge circuit.

[0050] In the MSS of this embodiment, for example, the sensor substrate may have a plurality of the support regions, and each of the plurality of support regions may support the MSS membrane. In the MSS of this embodiment, the number of the support regions and the number of the MSS membranes supported are not particularly limited, and each may be one, or may be two or more. When the MSS of this embodiment has a plurality of the MSS membranes, the plurality of MSS membranes may be, for example, MSS membranes to which aptamers for the same target are immobilized, or MSS membranes to which aptamers for different targets are immobilized, or may be both. Here, the "aptamer for the same target" may be, for example, an aptamer of the same sequence for the same target, or an aptamer of different sequences for the same target.

[0051] When the MSS of the present embodiment has a plurality of MSS membranes on which aptamers for the same target are immobilized, for example, multiple analyses for the same target can be performed simultaneously with one MSS. Also, when the MSS of the present embodiment has a plurality of MSS membranes on which aptamers for different targets are immobilized, for example, analyses for different targets can be performed simultaneously with one MSS.

[0052] The MSS of the present embodiment may be in a form in which the MSS film is disposed on the sensor substrate during use, and may be in a form in which the sensor substrate and the MSS film are separate and independent before use. In the latter case, the MSS of the present invention may be, for example, a kit including the sensor substrate and the MSS film separately and independently.

[0053] The MSS of this embodiment can measure, for example, in an analysis method described below, a change in resistance value associated with a change in stress of the piezoresistance element in the MSS as an electronic signal using an existing measurement module.

[0054] [Embodiment 2] The target analysis method of this embodiment is characterized by including the steps of immersing the membrane-type surface stress sensor (MSS) of the present invention in a sample liquid, applying a voltage to the MSS in the liquid phase, and analyzing the target in the sample liquid by measuring the stress change of the piezoresistance element in the MSS. The analysis method of the present invention is characterized by using the MSS to which the aptamer is immobilized as described above, and other steps and conditions are not particularly limited.

[0055] In the immersion step, the immersion of the MSS may be performed, for example, so long as the support region including the piezoresistance element in the sensor substrate and the MSS film supported thereon can be immersed in the sample liquid. The conditions for immersing the MSS in the sample liquid are not particularly limited, and examples thereof include a temperature of 20 to 35° C. for 0.1 to 120 minutes and a temperature of 50 to 60° C. for 0.1 to 120 minutes. When the MSS has multiple MSS films, for example, the multiple MSS films in the MSS may be immersed in the same sample liquid at the same time.

[0056] In the application step, as described above, a voltage is applied to the membrane-type surface stress sensor in a liquid phase. The conditions for applying the voltage are not particularly limited, and may be, for example, the same conditions as those for commercially available MSS. The liquid phase may be, for example, the sample liquid in the immersion step, or another solvent. In the latter case, the MSS after the immersion step may be taken out of the sample liquid and immersed in a new solvent, and a voltage may be applied. In the case where the MSS is washed after the immersion step in the sample liquid to remove substances that did not bind to the aptamer in the sample, it is preferable to immerse the MSS in a new solvent in this way and apply a voltage. The solvent is not particularly limited, and examples thereof include buffer solutions such as PBS and Tris-HCl, and water.

[0057] As described above, the analysis step analyzes the target in the sample liquid by measuring the stress change of the piezoresistance element in the MSS. The stress change can be measured, for example, by measuring an electric signal, and a commercially available measurement module (e.g., MSS-8RM, NANOSENSOR) can be used. EXAMPLES

[0058] [Example 1] We confirmed that it is possible to immobilize aptamers on the MSS membrane of a commercially available MSS and analyze the targets.

[0059] (1) Immobilization of aptamers by nonspecific adsorption A commercially available MSS (product name: SD-MSS-1K2G, NANOSENSOR) was used. The configuration of the MSS is shown in a top view in Fig. 1. As shown in Fig. 1, the MSS has a sensor substrate 10 having an electrode 11, an aluminum wire 12, an MSS film 13, and a piezoresistance element 14, the MSS film 13 is connected to the aluminum wire 12 via the piezoresistance element 14, and the aluminum wire 12 is connected to the electrode 11.

[0060] The aluminum wire on the sensor board was waterproofed by applying acrylic resin (product name: Mr.COLOR 62, manufactured by GSI Creos) and epoxy resin (product name: PM 165-R Hi, manufactured by Cemedine). The sensor board after the treatment was then connected to a board with a connector (product name: IFB-FFC-(0.5)4P-B, manufactured by AITENDO) so that the electrodes could be inserted. Furthermore, the entire connector of the board with a connector was waterproofed by applying and filling the same resin as above to the exposed metal parts and gaps leading to the metal parts.

[0061] Next, 1 μl of streptavidin solution was dropped onto the back surface (the surface opposite to the surface on which the aluminum wires were formed) of the MSS film of the sensor substrate, and the substrate was left to stand for 1 hour under a water vapor atmosphere (100% (relative humidity)) at room temperature (about 25° C.). The streptavidin solution was prepared by suspending streptavidin in 1×PBS (pH 7.4) so ​​that the streptavidin concentration was 2%. Next, after washing the back surface with the PBS, 3 μl of the streptavidin solution was dropped onto the front surface of the MSS film, the substrate was left to stand under the same conditions, and then washed with the PBS. The sensor substrate was then dried at room temperature for 10 minutes.

[0062] In this manner, the two sensor substrates were treated, and an aptamer was further bound to one of the sensor substrates to form the MSS of Example 1A, and polyT was further bound to the other sensor substrate to form the MSS of Reference Example 1A.

[0063] That is, 3 μl of the aptamer solution was dropped onto the surface of one of the sensor substrates, and the substrate was left to stand for 1 hour at room temperature under a water vapor atmosphere (100% (relative humidity)). The aptamer solution was prepared by suspending a thrombin aptamer (SEQ ID NO: 1: GGTTGGTGTGGTTGGTTTTT-biotin-3') with a biotin tag added to the 3' end in the PBS to a final concentration of 1 μmol / l. Since the thrombin aptamer has a biotin tag, if streptavidin is bound to the surface of the MSS membrane, the thrombin aptamer will be fixed to the surface of the MSS membrane by the binding between the biotin and the streptavidin. This was designated as the MSS of Example 1A.

[0064] 3 μl of polyT solution was dropped onto the surface of the other sensor substrate, and the substrate was left to stand for 1 hour at room temperature under a water vapor atmosphere (100% (relative humidity)). The polyT solution was prepared by suspending polyT DNA (SEQ ID NO: 2: TTTTTTTTTTTTTTTTTTTT-biotin-3') with a biotin tag added to the 3' end in the PBS to a final concentration of 1 μmol / l. Since the polyT has a biotin tag, if streptavidin is bound to the surface of the MSS membrane, the polyT will be fixed to the surface of the MSS membrane by the binding between the biotin and the streptavidin. This was designated as MSS in Reference Example 1.

[0065] (2) Immobilization of aptamers by gold deposition Unless otherwise specified, the same treatment as in (1) above was performed. That is, for the commercially available MSS sensor substrate, after waterproofing treatment was performed on the aluminum wires on the sensor substrate, the electrodes of the sensor substrate were masked, titanium was evaporated onto the entire surface of the sensor substrate including the MSS film, and then gold was evaporated. A titanium thin film with a thickness of about 5 nm was formed by the titanium evaporation, and a gold thin film with a thickness of about 100 nm was formed by the gold evaporation. The sensor substrate was then washed with ethanol.

[0066] Next, the entire sensor substrate was immersed in a 100 μmol / l BiotinSAM ethanol solution (Dojindo Laboratories), left to stand at room temperature for 1 hour, and then washed with ethanol. Then, similar to (1) above, it was connected to the connector-equipped substrate, and the entire connector was waterproofed.

[0067] Next, 1 μl of 0.5% streptavidin solution was dropped onto one surface (the surface on which the aluminum wire was formed) of the MSS film of the sensor substrate, and the sensor substrate was left to stand for 0.5 hours under a water vapor atmosphere (100% (relative humidity)) at room temperature. After washing with the PBS, the sensor substrate was dried at room temperature for 10 minutes.

[0068] In this manner, the two sensor substrates were treated, and an aptamer was further bound to one of the sensor substrates to form the MSS of the embodiment, and polyT was further bound to the other sensor substrate to form the MSS of the reference example.

[0069] That is, 3 μl of the aptamer solution was dropped onto the surface of one of the sensor substrates, and the sensor substrate was left standing for 1 hour at room temperature under a water vapor atmosphere (100% (relative humidity)). The aptamer solution was prepared by suspending the thrombin aptamer in the PBS so that the final concentration was 5 μmol / l. After the standing, the sensor substrate was further immersed in PBS containing 1% BSA, left standing at room temperature for 50 minutes, and washed with the PBS. Since the thrombin aptamer has a biotin tag, if streptavidin is bound to the surface of the MSS membrane, the thrombin aptamer will be fixed to the surface of the MSS membrane by the binding between the biotin and the streptavidin. This was the MSS of Example 2.

[0070] 3 μl of polyT solution was dropped onto the surface of the other sensor substrate, and the substrate was left to stand for 1 hour at room temperature under a water vapor atmosphere (100% (relative humidity)). The polyT solution was prepared by suspending the polyT DNA in the PBS to a final concentration of 5 μmol / l. After standing, the substrate was further immersed in PBS containing 1% BSA, left at room temperature for 50 minutes, and washed with the PBS. Since the polyT has a biotin tag, if streptavidin is bound to the surface of the MSS membrane, the polyT will be fixed to the surface of the MSS membrane by the binding between the biotin and the streptavidin. This was designated as MSS of Reference Example 2.

[0071] (3) Immobilization of aptamers by silane coupling method After the sensor substrate of the commercially available MSS was washed with ethanol, the end where the MSS film was located was immersed in a silane coupling solution and left at room temperature for 20 minutes. The silane coupling agent was composed of 8 ml of ethanol, 200 μl of acetic acid, 10 μl of APTMS (3-aminopropyltriethoxysilane), and 1.8 ml of pure water. The immersed end of the sensor substrate was washed with pure water and treated at 110° C. for 1.5 hours.

[0072] As in (1) above, after the aluminum wires on the sensor board were waterproofed, the sensor board was connected to the board with connector, and the entire connector was waterproofed.

[0073] Next, 1 μl of a 14% glutaraldehyde solution was dropped onto one surface (the surface on which the aluminum wire was formed) of the MSS film of the sensor substrate, and the sensor substrate was left standing at room temperature for 0.75 hours in a water vapor atmosphere (100% (relative humidity)). Next, after washing with the PBS, the sensor substrate was dried at room temperature for 10 minutes. Furthermore, 1 μl of a 0.5% streptavidin solution was dropped onto the same surface of the MSS film of the sensor substrate, and the sensor substrate was left standing at room temperature for 0.5 hours in a water vapor atmosphere (100% (relative humidity)). Next, the surface of the end of the sensor substrate on which the MSS film was disposed was washed with 0.1 mol / l Tris-HCl (pH 8), and the end was further immersed in 0.1 mol / l Tris-HCl (pH 8) and left standing at room temperature for 15 minutes. Then, the end was washed with the PBS.

[0074] In this manner, the two sensor substrates were treated, and an aptamer was further bound to one of the sensor substrates to form the MSS of the embodiment, and polyT was further bound to the other sensor substrate to form the MSS of the reference example.

[0075] That is, 3 μl of the aptamer solution was dropped onto the surface of one of the sensor substrates, and the sensor substrate was left standing for 1 hour at room temperature under a water vapor atmosphere (100% (relative humidity)). The aptamer solution was prepared by suspending the thrombin aptamer in the PBS so that the final concentration was 5 μmol / l. After the standing, the sensor substrate was further immersed in PBS containing 1% BSA, left standing at room temperature for 30 minutes, and washed with the PBS. Since the thrombin aptamer has a biotin tag, if streptavidin is bound to the surface of the MSS membrane, the thrombin aptamer will be fixed to the surface of the MSS membrane by the binding between the biotin and the streptavidin. This was the MSS of Example 3.

[0076] 3 μl of polyT solution was dropped onto the surface of the other sensor substrate, and the substrate was left to stand for 1 hour at room temperature under a water vapor atmosphere (100% (relative humidity)). The polyT solution was prepared by suspending the polyT DNA in the PBS to a final concentration of 5 μmol / l. After standing, the substrate was further immersed in PBS containing 1% BSA, left at room temperature for 30 minutes, and washed with the PBS. Since the polyT has a biotin tag, if streptavidin is bound to the surface of the MSS membrane, the polyT will be fixed to the surface of the MSS membrane by the binding between the biotin and the streptavidin. This was designated as MSS of Reference Example 3.

[0077] (4) Detection of electrical signals The MSS of Example 1 and Reference Example 1, the MSS of Example 2 and Reference Example 2, and the MSS of Example 3 and Reference Example 3 were each set, and simultaneously immersed in a sample solution, voltage was applied, and the voltage change associated with the stress change was measured. Specifically, first, the end of the MSS including the MSS membrane was immersed in the PBS, voltage was applied to the MSS, and the MSS was left until the voltage signal stabilized. Then, at the point of measurement time of 1400 seconds when the voltage signal was sufficiently stabilized, the immersion of the MSS was switched to a thrombin solution, and the voltage signal was continuously measured. The thrombin solution was prepared by mixing a thrombin reagent (trade name: αThrombin, Human, Funakoshi Co., Ltd.) with the PBS to a final concentration of 240 nmol / l.

[0078] Then, for each MSS, the voltage signal was converted to voltage, and the difference (Vs-Vt) between the stable voltage (Vs) after immersion in the sample solution and the lowest voltage (Vt) after immersion in the thrombin solution was calculated, and this was taken as the voltage drop value (μV) due to immersion in the thrombin solution. These results are shown in Table 1 below.

[0079] [Table 1]

[0080] For each of the MSSs of Example 1, Example 2, and Example 3, a rapid voltage drop was confirmed after immersion in the thrombin solution. As shown in Table 1, the voltage drop in each Example was significantly larger (large voltage drop) than the voltage drop in the corresponding Reference Example. From this result, it can be seen that the MSS of the Example was immersed in the target thrombin solution, and thrombin bound to the MSS membrane of the MSS via the aptamer, causing a stress change. As shown in Table 1, a decrease in the measured value of the electric signal was observed in each Reference Example after switching to the thrombin solution, but this is due to the influence of glycerin contained in the thrombin reagent used (the influence of nonspecific adsorption of glycerin to the MSS membrane, etc.), and can be ignored by background correction. Also, since each Example showed a larger voltage drop than the corresponding Reference Example, it can be said that in the Examples, unlike the Reference Example, specific binding with the target thrombin occurred. From the above results, it was confirmed that the target can be analyzed by using the MSS with the aptamer immobilized thereon.

[0081] [Example 2] It was confirmed that the sensitivity of MSS was improved by immobilizing the aptamer at approximately the same distance from the MSS membrane.

[0082] (1) Preparation of MSS As an example of MSS, the MSS shown in FIG. 2(A) was prepared. First, the sensor substrate of the commercially available MSS of Example 1(1) was washed with ethanol, and then the end where the MSS film was located was rinsed with about 100 μl of a silane coupling solution and left at room temperature for 1.5 hours. The silane coupling agent was composed of 8 ml of ethanol, 200 μl of acetic acid, 100 μl of APTMS (trimethoxysilyl 3-propyl methacrylate (3-(methacryloyloxy)propyl trimethoxysilane), and 1.8 ml of pure water. The sensor substrate was then washed with ethanol and dried at room temperature for 5 minutes.

[0083] Next, 1 μl of N-acetylcysteine ​​solution was dropped onto one surface of the MSS film of the sensor substrate (the surface on which the aluminum wire was formed), and UV (Nitride Corporation, NS365L-6SMG) was irradiated for several minutes. The irradiation was continued at room temperature under a water vapor atmosphere (100% (relative humidity)) until the substrate was dried. Thereafter, the sensor substrate was washed with pure water and dried at room temperature.

[0084] Next, the sensor portion of the sensor substrate was immersed in an acetic anhydride solution (10% acetic anhydride, 90% acetonitrile) and reacted for 0.5 hours at 60° C. After the reaction, the sensor substrate was washed with acetonitrile.

[0085] Next, the sensor substrate was dried at room temperature for 10 minutes. Furthermore, 1 μl of 0.5% streptavidin solution was dropped onto the same surface of the MSS film of the sensor substrate, and the sensor substrate was left to stand for 1.5 hours at room temperature under a water vapor atmosphere (100% (relative humidity)). After the standing, the sensor substrate was washed with the PBS.

[0086] As in Example 1(1), the aluminum wires on the sensor board were waterproofed, and then the sensor board was connected to the board with connector, and the entire connector was waterproofed.

[0087] In this manner, the two sensor substrates were treated, and an aptamer was further bound to one of the sensor substrates to produce the MSS of the embodiment (Example 2-1), and polyT was further bound to the other sensor substrate to produce the MSS of the reference example (Reference Example 2-1).

[0088] That is, 3 μl of the aptamer solution was dropped onto the surface of one of the sensor substrates, and the sensor substrate was left standing at room temperature for 1 hour under a water vapor atmosphere (100% (relative humidity)). The aptamer solution was prepared by suspending the thrombin aptamer in the PBS so as to give a final concentration of 5 μmol / l. After the standing, the sensor substrate was further immersed in PBS containing 1% BSA, left standing at room temperature for 30 minutes, and washed with the PBS. Since the thrombin aptamer has a biotin tag, if streptavidin is bound to the surface of the MSS membrane, the thrombin aptamer will be immobilized on the surface of the MSS membrane by the binding between the biotin and the streptavidin, as shown in FIG. 2(A). This was the MSS of Example 2-1. The MSS of Example 2-1 corresponds to the MSS in which the aptamer is immobilized at approximately the same distance from the MSS membrane.

[0089] 3 μl of polyT solution was dropped onto the surface of the other sensor substrate, and the substrate was left to stand for 1 hour at room temperature under a water vapor atmosphere (100% (relative humidity)). The polyT solution was prepared by suspending the polyT DNA in the PBS to a final concentration of 5 μmol / l. After standing, the substrate was further immersed in PBS containing 1% BSA, left at room temperature for 30 minutes, and washed with the PBS. Since the polyT has a biotin tag, if streptavidin is bound to the surface of the MSS membrane, the polyT will be fixed to the surface of the MSS membrane by the binding between the biotin and the streptavidin. This was designated as MSS in Reference Example 2-1.

[0090] Furthermore, in the same manner as in Example 1(1), an MSS of an example (Example 2-2) and an MSS of a reference example (Reference Example 2-2) were prepared.

[0091] (2) Detection of electrical signals The MSS of Example 2-1 and Reference Example 2-1, and the MSS of Example 2-2 and Reference Example 2-2 were each set, and simultaneously immersed in a sample solution, voltage was applied, and the voltage change associated with the stress change was measured. Specifically, first, the end of the MSS including the MSS membrane was immersed in the PBS, voltage was applied to the MSS, and the MSS was left until the voltage signal stabilized. Then, at the measurement time of 1200 seconds or 2100 seconds when the voltage signal was sufficiently stabilized, the immersion of the MSS was switched to a thrombin solution, and the voltage signal was continuously measured. The thrombin solution was prepared by mixing the thrombin reagent with the PBS so that the final concentration was about 200 nmol / l. The results are shown in FIG. 2.

[0092] FIG. 2 is a schematic diagram showing the structure of the MSS and a graph showing the voltage of the MSS. In FIG. 2, (A) is a diagram showing the structure of the MSS of Example 2-1, (B) is a graph showing the results of Example 2-1 and Reference Example 2-1, and (C) is a graph showing the results of Example 2-2 and Reference Example 2-2. In FIG. 2(B) and (C), the horizontal axis indicates the time after the end is started to be immersed in PBS, and the vertical axis indicates the voltage. As shown in FIG. 2(B) and (C), both the MSS of Example 2-1 and Example 2-2 showed significantly higher voltage values ​​than Reference Example 2-1 and Reference Example 2-2, and it was confirmed that the voltage changes depending on the presence or absence of a target. That is, it was found that the MSS of the present invention can detect a target. In addition, when the difference between the voltage values ​​of the MSS of Example 2-1 and the MSS of Reference Example 2-1 was compared with the difference between the voltage values ​​of the MSS of Example 2-2 and the MSS of Reference Example 2-2, the former was about twice as large as the latter, i.e., the MSS of Example 2-1 exhibited twice the sensitivity as the MSS of Example 2-2.

[0093] Also, as shown in Table 1, the MSS of Example 1 prepared by the nonspecific adsorption method shows about twice the sensitivity of the MSS of Example 3 prepared by the silane coupling method. Therefore, it can be said that the MSS of Example 2-1 shows about four times the sensitivity of the MSS of Example 3 in Table 1. Furthermore, in the MSS of Example 3 in Table 1, streptavidin is immobilized on the MSS membrane using a silane coupling agent, but since glutaraldehyde is used as a crosslinking agent, the aptamer is immobilized at different distances from the MSS membrane. Therefore, it was presumed that the difference in sensitivity between the MSS of Example 2-1 and the MSS of Example 3 in Table 1 depends on whether the aptamer is immobilized at a constant distance from the MSS membrane. Furthermore, since the main chain length of the linker in the MSS of Example 2-1 is 11, it was presumed that the sensitivity of the MSS can be improved by immobilizing the aptamer at a substantially constant distance from the MSS membrane and setting the linker length at about 11.

[0094] From the above, it was found that the sensitivity of MSS was improved by immobilizing aptamers at approximately the same distance from the MSS membrane.

[0095] Although the present invention has been described above with reference to the embodiments and examples, the present invention is not limited to the above-mentioned embodiments and examples. Various modifications that can be understood by those skilled in the art can be made to the configuration and details of the present invention within the scope of the present invention.

[0096] This application claims priority based on Japanese Patent Application No. 2019-128311, filed on July 10, 2019, and Japanese Patent Application No. 2020-056897, filed on March 26, 2020, the disclosures of which are incorporated herein in their entirety.

[0097] <Additional Notes> Some or all of the above-described embodiments and examples may be described as follows, but are not limited to the following: (Appendix 1) The present invention includes an aptamer, a membrane, and a sensor substrate, The aptamer is a nucleic acid molecule that binds to a target and is immobilized on the membrane; the membrane is a membrane that deforms upon binding of the target to the aptamer; the sensor substrate has a support region; the support region supports the membrane and includes a piezoresistive element; The piezoresistance element is an element for detecting deformation of the membrane. A membrane-type surface stress sensor. (Appendix 2) 2. The membrane-type surface stress sensor according to claim 1, wherein the membrane is a silicon membrane. (Appendix 3) 3. The membrane-type surface stress sensor according to claim 1 or 2, wherein the support region partially supports the membrane. (Appendix 4) 4. The membrane-type surface stress sensor according to any one of claims 1 to 3, wherein the aptamer is immobilized on one surface of the membrane. (Appendix 5) 4. The membrane-type surface stress sensor according to any one of claims 1 to 3, wherein the aptamer is immobilized on both sides of the membrane. (Appendix 6) 6. The membrane-type surface stress sensor according to any one of claims 1 to 5, wherein the aptamer is immobilized on the membrane via a conjugate of avidin or an avidin derivative and biotin or a biotin derivative. (Appendix 7) 7. A membrane-type surface stress sensor according to any one of claims 1 to 6, comprising a metal film on a surface of the membrane, and the aptamer is immobilized on the surface of the membrane via the metal film. (Appendix 8) The membrane-type surface stress sensor according to any one of claims 1 to 7, wherein the aptamer is fixed to the membrane surface via a linker. (Appendix 9) 9. The membrane-type surface stress sensor according to claim 8, wherein the length of the linker in each aptamer is approximately constant. (Appendix 10) 10. The membrane-type surface stress sensor according to claim 8 or 9, wherein the linker includes a silane coupling agent (a region derived from a silane coupling agent). (Appendix 11) The membrane-type surface stress sensor according to any one of appendixes 8 to 10, wherein the linker includes a crosslinker (a region derived from a crosslinker). (Appendix 12) 12. The membrane-type surface stress sensor according to any one of claims 8 to 11, wherein the linker has a main chain length of 1 to 15. (Appendix 13) 13. The membrane-type surface stress sensor according to any one of claims 1 to 12, wherein the aptamer is immobilized on the surface of the membrane via a silane coupling agent (a region derived from a silane coupling agent). (Appendix 14) the sensor substrate having a plurality of support regions; 14. The membrane-type surface stress sensor according to any one of claims 1 to 13, wherein each of the plurality of support regions supports the membrane. (Appendix 15) 15. The membrane-type surface stress sensor of claim 14, wherein the plurality of membrane-type surface stress sensors include sensors having aptamers immobilized thereon for different targets. (Appendix 16) the sensor substrate has a circuit; the support region includes a plurality of piezo-resistive elements; 16. The membrane-type surface stress sensor according to any one of claims 1 to 15, wherein the circuit is a Wheatstone bridge circuit including the plurality of piezoresistance elements. (Appendix 17) A step of immersing a membrane-type surface stress sensor according to any one of appendices 1 to 16 in a sample liquid; applying a voltage to the membrane-type surface stress sensor in a liquid phase; and analyzing the target in the sample liquid by measuring a stress change in the piezoresistance element in the film-type surface stress sensor. (Appendix 18) In the application step, the liquid phase is the sample liquid, The analysis method according to claim 17, wherein the voltage application step is carried out directly after the immersion step. [Industrial Applicability]

[0098] According to the present invention, by immobilizing an aptamer on the membrane as a new form for binding a target, it is possible to expand the possibilities for application to targets different from those of conventional membrane-type surface stress sensors, and for modifications different from those of conventional membrane-type surface stress sensors. [Explanation of symbols]

[0099] 10 Sensor board 11 electrodes 12 Aluminum Wire 13 MSS membrane 14 Piezoresistive element

Claims

1. The present invention includes an aptamer, a membrane, and a sensor substrate, The aptamer is a nucleic acid molecule that binds to a target and is immobilized on the membrane via a linker; The linker comprises a cross-linking agent; The crosslinking agent is a crosslinking agent that does not substantially undergo self-condensation, the length of the linker in each aptamer is approximately constant due to the reaction with a crosslinker that does not substantially cause self-condensation; the membrane is a membrane that deforms upon binding of the target to the aptamer; the sensor substrate has a support region; the support region supports the membrane and includes a piezoresistive element; The piezoresistance element is an element for detecting deformation of the membrane. A membrane-type surface stress sensor.

2. The membrane-type surface stress sensor according to claim 1 , wherein the membrane is a silicon membrane.

3. The membrane-type surface stress sensor according to claim 1 , wherein the support region partially supports the membrane.

4. The membrane-type surface stress sensor according to claim 1 , wherein the linker includes a silane coupling agent.

5. 5. The film-type surface stress sensor according to claim 4, wherein the silane coupling agent is a silane coupling agent having a methacryl group.

6. A membrane-type surface stress sensor as described in claim 4 or 5, wherein the aptamer is immobilized on the surface of the membrane via the silane coupling agent.

7. The membrane-type surface stress sensor according to claim 1 , wherein the linker comprises at least one of an amino acid and a derivative thereof.

8. The membrane-type surface stress sensor according to claim 1 , wherein the crosslinking agent that does not substantially undergo self-condensation includes acetic anhydride.

9. The membrane-type surface stress sensor according to claim 1 , wherein the linker comprises an affinity tag.

10. the main chain length of the linker is 1 to 15; The membrane-type surface stress sensor according to claim 9, wherein the main chain length is the length of the shortest molecular chain between the functional group on the membrane and the affinity tag, or the length of the shortest molecular chain between the functional group on the membrane and the aptamer.

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