Biochip and Detection Method

Abstract

A biochip capable of highly sensitively detecting a substance to be measured and a method for detecting the substance to be measured using the biochip are disclosed. On the surface of a substrate, a biochip immobilizes a selective binding substance capable of selectively binding to a substance to be measured via a polymer containing a unit represented by formula (Ia) or formula (Ib). In formula (Ia) and formula (Ib), R 1 represents an alkylene group having 1 to 4 carbon atoms, and R 2 represents R 3 , OR 4 or NHR 5 , R 3 and R 5 each independently represent a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, R 4 represents an alkyl group having 1 to 4 carbon atoms, R 6 represents an alkylene group having 1 to 2 carbon atoms, and R 7 and R 8 each independently represent a hydrogen atom or an alkyl group having 1 to 4 carbon atoms.

Description

Technical Field

[0001] The present invention relates to a biochip in which a substance that selectively binds to a substance to be measured (referred to as a "selective binding substance" in this specification) is immobilized on the surface of a substrate, and a method for detecting a substance to be measured using the biochip. Background Art

[0002] A biochip in which a selective binding substance such as nucleic acid or protein is immobilized on a substrate can detect fluorescence after selective binding to a substance to be measured, and perform molecular recognition and diagnosis based on the intensity change and pattern thereof. In accurate molecular recognition and diagnosis using a biochip, high detection sensitivity for a substance to be measured is desired as the performance of the biochip. Specifically, it is desired that the signal value is high during fluorescence detection of a substance to be measured, and the ratio of the signal value to the noise value (hereinafter referred to as "SN ratio") is high.

[0003] For a biochip, the detection sensitivity of a substance to be measured sometimes varies depending on the method of immobilizing a selective binding substance such as nucleic acid or protein on a substrate. For example, there are reports that a biochip with high detection sensitivity can be manufactured by immobilizing a selective binding substance on the surface of a substrate via a polymer (Patent Documents 1 and 2). As the polymer used at this time, "amine-containing polymers" having an amino group in the chemical structure such as polyethyleneimine, polyallylamine, and poly-L-lysine are often used. In Patent Document 1, a biochip in which a selective binding substance is immobilized on the surface of a substrate via polyethyleneimine, which is one kind of amine-containing polymer, is disclosed.

[0004] Prior Art Documents

[0005] Patent Documents

[0006] Patent Document 1: International Publication No. 01 / 70641

[0007] Patent Document 2: International Publication No. 01 / 70851 Summary of the Invention

[0008] Problems to be Solved by the Invention

[0009] In order to develop a biochip capable of detecting with a high signal-to-noise ratio (SN ratio), the inventors selected the immobilization of a selective binding substance to a substrate with reference to Patent Document 1 and fabricated a biochip. Specifically, a biochip was fabricated in which a selective binding substance was immobilized on the surface of a substrate via polyethyleneimine, which is a kind of polymer containing an amino group (Comparative Example 2 described later). Although its signal value was higher than that of a biochip in which a selective binding substance was directly condensed and immobilized on the substrate surface without using a polymer (Comparative Example 1 described later), its noise value also increased, and no increase in the SN ratio was observed. Therefore, as another polymer containing an amino group, the immobilization of a selective binding substance via polyallylamine was investigated (Comparative Example 3 described later). Although its signal value was further increased compared to the biochip in which the selective binding substance was immobilized via polyethyleneimine, its noise value also further increased, and the increase in the SN ratio was insufficient. That is, it was shown that simply immobilizing a selective binding substance via a polymer containing an amino group does not necessarily lead to an increase in the signal value and the SN ratio. In order to obtain a biochip having excellent signal values and SN ratios, there is a problem that it is necessary to study the structure of the polymer containing an amino group, particularly the side chain structure of the polymer.

[0010] Method for solving the problem

[0011] To overcome the above problems, the inventors studied the polymer structure suitable for biochips and found that by using a polymer with a modified side chain structure, a biochip with high signal values and a high SN ratio can be obtained.

[0012] That is, the present invention provides the following (1) to (8).

[0013] (1) A biochip, wherein a selective binding substance capable of selectively binding to a substance to be measured is immobilized on the surface of a substrate via a polymer containing a unit represented by formula (Ia) or formula (Ib).

[0014]

[0015] In formula (Ia) and formula (Ib), R 1 represents an alkylene group having 1 to 4 carbon atoms, and R 2 represents R 3 , OR 4 or NHR 5 , R 3 and R 5 each independently represent a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, R 4 represents an alkyl group having 1 to 4 carbon atoms, R 6 represents an alkylene group having 1 to 2 carbon atoms, and R 7 and R 8Each independently represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms.

[0016] (2) The biochip according to (1), wherein the polymer is represented by the above formula (Ia) (wherein R 1 and R 2 have the same definitions as those described in (1)).

[0017] (3) The biochip according to (1), wherein the polymer is represented by the above formula (Ib) (wherein R 6 , R 7 and R 8 have the same definitions as those described in (1)).

[0018] (4) The biochip according to any one of (1) to (3), wherein the polymer is a copolymer.

[0019] (5) The biochip according to (4), wherein the copolymer is a copolymer with a unit represented by the formula (II).

[0020]

[0021] In the formula, R 1 represents an alkylene group having 1 to 4 carbon atoms.

[0022] (6) The biochip according to any one of (1) to (5), wherein the number average molecular weight of the polymer is 300 to 1,000,000.

[0023] (7) The biochip according to any one of (1) to (6), wherein the selective binding substance is a nucleic acid or a protein.

[0024] (8) A method for detecting a substance to be measured, which uses the biochip according to (1) to (7) in which a selective binding substance capable of selectively binding to the substance to be measured is immobilized on the surface of a substrate, and the method includes: bringing a sample containing the substance to be measured into contact with the surface of the substrate, and detecting the formed complex with the selective binding substance.

[0025] Effects of the Invention

[0026] By immobilizing the selective binding substance on the substrate via a polymer containing a unit represented by the formula (Ia) or (Ib), particularly a copolymer of a unit represented by the formula (Ia) or (Ib) and a unit represented by the formula (II), a biochip capable of detecting a substance to be measured with high sensitivity can be provided. By using the biochip of the present invention, more accurate molecular recognition and diagnosis can be performed. Detailed Embodiments

[0027] The biochip of the present invention is characterized in that a polymer containing units represented by formula (Ia) or formula (Ib) is used, and a substance having a selective binding property to a substance to be measured is immobilized on a substrate.

[0028] R in formula (Ia) and formula (Ib) 1 represents an alkylene group having 1 to 4 carbon atoms. R 1 is preferably methylene having 1 carbon atom and ethylene having 2 carbon atoms, and more preferably methylene having 1 carbon atom.

[0029] R in formula (Ia) 2 represents R 3 , OR 4 or NHR 5 . R 3 and R 5 each independently represent a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, and R 4 represents an alkyl group having 1 to 4 carbon atoms. R 3 is preferably methyl having 1 carbon atom and ethyl having 2 carbon atoms, and more preferably methyl having 1 carbon atom. R 4 is preferably methyl having 1 carbon atom and ethyl having 2 carbon atoms, and more preferably methyl having 1 carbon atom. R 5 is preferably a hydrogen atom.

[0030] R in formula (Ib) 6 represents an alkylene group having 1 to 2 carbon atoms. R 6 is preferably ethylene having 2 carbon atoms.

[0031] R in formula (Ib) 7 and R 8 each independently represent a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. R 7 and R 8 are preferably a hydrogen atom and methyl having 1 carbon atom, and more preferably a hydrogen atom.

[0032] The above-mentioned polymer only needs to contain the units represented by formula (Ia) or (Ib), and may be a heteropolymer containing units other than the units represented by formula (Ia) or (Ib). When the polymer is a heteropolymer and is immobilized on the substrate surface by covalent bonds, the units other than the units represented by formula (Ia) or (Ib) preferably have reactive functional groups. Examples of the reactive functional group include an amino group, a carboxyl group, a hydroxyl group, a halogenated group, a toluenesulfonyl group, an epoxy group, an acyl group, and an azide group, but an amino group is preferred. In the case of a heteropolymer, the ratio of the units represented by formula (Ia) or (Ib) (hereinafter, for convenience, they are sometimes collectively referred to as formula (I)) to all the units is preferably 5 mol% or more and 95 mol% or less, more preferably 10 mol% or more and 90 mol% or less, and still more preferably 30 mol% or more and 70 mol% or less.

[0033] When the polymer containing the units represented by formula (Ia) or (Ib) is a heteropolymer, it is preferably a heteropolymer with the units represented by formula (II). R in formula (II) 1 is the same as R in formula (Ia) or (Ib) 1 and represents an alkylene group having 1 to 4 carbon atoms. In addition, the ratio of the units represented by formula (I) in the heteropolymer to the total of the units represented by formula (I) and the units represented by formula (II) (hereinafter referred to as (m / (m + n))) is preferably 5 mol% or more and 95 mol% or less, more preferably 10 mol% or more and 90 mol% or less, and still more preferably 30 mol% or more and 70 mol% or less.

[0034] As a method for producing the polymer containing the units represented by formula (I), as shown in the following diagram, there can be mentioned a method (A) in which the monomer represented by formula (III) is used as a raw material, polymerized, and then the side-chain amino group is modified; a method (B) in which the monomer represented by formula (III) is also used as a raw material, the side-chain amino group is modified, and then polymerization is carried out, but the former method (A) is preferred.

[0035]

[0036] R in formula (III) 1 is the same as R in formula (Ia) and (Ib) 1 and represents an alkylene group having 1 to 4 carbon atoms. The monomer represented by formula (III) can be obtained from reagent manufacturers (for example, Merck, Enamine, etc.).

[0037] The polymerization of the monomer represented by the formula (III) in the above method (A) can be carried out by radical polymerization. For example, as long as 0.1 to 30 mol% of an azo-based radical polymerization initiator (e.g., 2,2'-azobis(2-amidinopropane) dihydrochloride, etc.) is added to the monomer in a monomer solution of 10 to 80% by weight, and the reaction is carried out at room temperature to 80 °C for 3 to 100 hours. In addition, R in the formula (III) 1 The homopolymer of the monomer in which R is a methylene group having 1 carbon atom can also be obtained from reagent manufacturers (e.g., Nitto Boehringer Medical Co., Ltd., etc.).

[0038] As a method for modifying the side-chain amino group after the polymerization of the monomer represented by the formula (III), a method of reacting with a reagent for amino modification can be cited as follows.

[0039] In R in the formula (Ia) 2 is R 3 and R 3 is a hydrogen atom, a formylating reagent such as 1-formylpiperidine can be used as the reagent for amino modification. When R 3 is an alkyl group having 1 to 4 carbon atoms, acid anhydrides such as acetic anhydride, propionic anhydride, butyric anhydride, and valeric anhydride (R 3 CO2COR 3 ) can be used as the reagent for amino modification.

[0040] In R in the formula (Ia) 2 is OR 4 , a carbonic acid diester such as dimethyl carbonate, diethyl carbonate, dipropyl carbonate, and dibutyl carbonate (R 4 OCO2R 4 ) can be used as the reagent for amino modification.

[0041] In R in the formula (Ia) 2 is NHR 5 , an isocyanate derivative (R 5 -NCO) can be used as the reagent for amino modification.

[0042] In R in the formula (Ib) 6 is a methylene group having 1 carbon atom, bromoacetamide derivatives such as 2-bromoacetamide, 2-bromo-N-methylacetamide, 2-bromo-N-ethylacetamide, 2-bromo-N-propylacetamide, 2-bromo-N-isopropylacetamide, 2-bromo-N-butylacetamide, 2-bromo-N,N-dimethylacetamide, 2-bromo-N,N-diethylacetamide, etc. (BrCH2CONR 7 R 8 ) can be used as the reagent for amino modification.

[0043] R in formula (Ib) 6 In the case where it is an ethylene group having 2 carbon atoms, as a reagent for amino modification, acrylamide derivatives such as acrylamide, N-methylacrylamide, N-ethylacrylamide, N-propylacrylamide, N-isopropylacrylamide, N-butylacrylamide, N,N-dimethylacrylamide, N,N-diethylacrylamide, etc. (CH2=CHCONR 7 R 8 ) can be used.

[0044] For the reaction of the above-mentioned reagent for amino modification with the polymer of the monomer represented by formula (III), for example, as long as the reagent for amino modification is added to a 10 to 50 wt% polymer solution and the reaction is carried out at room temperature to 80 °C for 3 to 100 hours.

[0045] The side chain modification of the monomer represented by formula (III) in the above method (B) can be carried out in the same manner as above using a reagent for amino modification. In addition, the polymerization of the modified monomer can also be carried out under the same general conditions as above.

[0046] For the production of the heteropolymer of the unit represented by formula (Ia) or formula (Ib) and the unit represented by formula (II), it is only necessary to modify a part of the side chain amino groups in the above-mentioned side chain amino group modification step. It is particularly preferred to use the method of modifying only a part of the side chain amino groups of the polymer of the monomer represented by formula (III) in the above method (A). In this case, the ratio of the unit represented by formula (Ia) or formula (Ib) to the unit represented by formula (II) can be adjusted by adjusting the molar ratio of the side chain amino groups in the polymer to the reagent for amino modification used. The ratio of the unit represented by formula (Ia) or formula (Ib) to the unit represented by formula (II) can be calculated by 1 1H-NMR measurement.

[0047] The molecular weight of the above polymer in the biochip of the present invention is preferably 300 to 1,000,000 in terms of number average molecular weight, more preferably 1,000 to 100,000, and still more preferably 1,600 to 25,000. The molecular weight of the above polymer can be calculated, for example, by gel permeation chromatography (GPC) method using polyethylene glycol as a standard.

[0048] The material of the substrate of the biochip of the present invention can be any one of resin, glass, metal, and silicon wafer, but is preferably resin from the viewpoints of ease of surface treatment and mass productivity.

[0049] Examples of the resin used as the substrate material include, for example, polyacrylate, polymethacrylate, polycarbonate, polystyrene, polyvinyl acetate, polyester, etc., and polyacrylate and polymethacrylate are preferred. Among them, examples of polymethacrylate include polyalkyl methacrylate (PAMA) such as polymethyl methacrylate (PMMA), polyethyl methacrylate (PEMA), or polypropyl methacrylate, but PMMA is preferred.

[0050] In addition, as the resin, known copolymers can also be used. Examples include acrylonitrile / styrene copolymer (AS resin), acrylonitrile / butadiene / styrene copolymer (ABS resin), acrylonitrile / ethylene-propylene-diene / styrene copolymer (AES resin), methyl methacrylate / acrylonitrile / butadiene / styrene copolymer (MABS resin) as a copolymer containing polymethacrylate, methyl methacrylate / butadiene / styrene copolymer (MBS resin), methyl methacrylate / styrene copolymer (MS resin), etc.

[0051] The immobilization mode of the above polymer on the substrate surface can be physical adsorption or covalent bond, but from the viewpoint of suppressing the peeling and dissolution of the above polymer from the substrate during substrate washing, a covalent bond is desired.

[0052] As a method for immobilizing the above polymer on the substrate surface by physical adsorption, a method can be cited in which a polymer solution obtained by dissolving the polymer in an organic solvent at a concentration of 0.05 to 10% by weight is prepared, coated on the surface of the substrate by a known method such as dipping or spraying, and then dried at room temperature or under heating. As the organic solvent, a single solvent such as ethanol, methanol, tert-butanol, benzene, toluene, tetrahydrofuran, di ane, dichloromethane, chloroform, acetone, methyl ethyl ketone, etc., or a mixed solvent thereof is used. When the substrate material is resin, the method of using ethanol or methanol as the organic solvent does not modify the substrate and is easy to dry, so it is preferred.

[0053] As a method for immobilizing the above polymer on the substrate surface by covalent bond, a method can be used in which functional groups present in the polymer react with functional groups present in the substrate surface to form a covalent bond. Examples of the functional groups present in the substrate surface include amino group, carboxyl group, hydroxyl group, halogenated group, etc. As the mode of the covalent bond, any of amide bond, ester bond, ether bond, etc. can be used, but from the viewpoints of ease of bond formation and firmness, an amide bond is preferred.

[0054] As a method for immobilizing the above polymer on the substrate surface via an amide bond, a method of reacting the amino group present in the polymer with the carboxyl group present on the substrate surface can be used, or a method of reacting the carboxyl group present in the polymer with the amino group present on the substrate surface can be used. However, a method of reacting the amino group present in the polymer with the carboxyl group present on the substrate surface is more preferred.

[0055] The immobilization of the polymer having an amino group by a condensation reaction with the carboxyl group present on the substrate surface can use 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (alias: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (DMT-MM) and other condensing agents to directly react the carboxyl group on the substrate surface with the amino group in the polymer, or the carboxyl group on the substrate surface can be temporarily converted into an activated ester using N-hydroxysuccinimide (NHS) or the like and then reacted with the amino group in the polymer.

[0056] In the case where there are no functional groups capable of reacting with the polymer on the substrate surface, functional groups capable of reacting with the polymer can be generated on the substrate surface by a method suitable for the material of the substrate.

[0057] As a method for generating functional groups on the surface of a resin substrate, a method of immersing the substrate in a solution containing a compound having a target functional group can be cited; a method of oxidizing the resin on the substrate surface by exposure to UV, radiation, ozone, or plasma, etc. As a method for generating a carboxyl group among the functional groups on the surface of a resin substrate, a method of hydrolyzing the substrate surface with an alkali, an acid, etc. can be cited; a method of ultrasonically treating the substrate surface in warm water; a method of irradiating the substrate surface with UV or ozone; a method of exposing the substrate surface to oxygen plasma, argon plasma, or radiation, etc. In the case where the resin is a resin having an ester structure in the side chain such as polyacrylate or polymethacrylate, by immersing the substrate in an alkali or an acid and hydrolyzing the ester structure on the resin surface, a carboxyl group can be generated on the substrate surface. As a specific example, it is sufficient to immerse the resin substrate in an aqueous solution of sodium hydroxide or sulfuric acid (preferred concentration is 1N to 20N), preferably at a temperature of 30°C to 80°C, and maintain for 1 hour to 100 hours. In the case of a resin having no ester structure in the side chain, by plasma treatment in the presence of oxygen, the carbon atoms on the resin surface are oxidized, so that a carboxyl group can be generated on the substrate surface.

[0058] As a method for generating a functional group on the surface of a glass substrate, a method of reacting a silane coupling agent having a target functional group can be mentioned, etc. In addition, a method of reacting a functional group introduced by reaction with a silane coupling agent with a compound having another functional group to convert it into another functional group can also be used. As a method for generating a carboxyl group on the surface of a glass substrate in the functional group, a method of reacting a dicarboxylic anhydride such as succinic anhydride with an amino group generated by reacting a silane coupling agent such as 3-aminopropyltriethoxysilane with a silanol group on the substrate surface can be used.

[0059] As a method for generating a functional group on the surface of a metal substrate, a method of reacting a silane coupling agent having a target functional group, a method of reacting an alkanethiol having a target functional group, etc. can be mentioned. As a method for generating a carboxyl group on the surface of a metal substrate in the functional group, in addition to the above method for generating a carboxyl group on the surface of a glass substrate, a method of reacting an alkanethiol having a carboxyl group such as 5-carboxy-1-pentanethiol with the substrate can also be used.

[0060] As a method for generating a functional group on the surface of a silicon wafer substrate, the same method as that for generating a functional group on the surface of a glass substrate can be used.

[0061] In the biochip of the present invention, a selective binding substance is immobilized on the surface of a substrate via the above polymer. Here, the so-called "selective binding substance" refers to a substance that can selectively bind directly or indirectly to a substance to be measured. Representative examples include nucleic acids, proteins, saccharides, and other antigenic compounds. As nucleic acids, in addition to DNA and RNA, PNA can also be used. A single-stranded nucleic acid having a specific base sequence binds by selectively hybridizing with a single-stranded nucleic acid having the complementary base sequence of the base sequence or a part thereof, and thus is equivalent to a selective binding substance. In addition, as proteins, antigen-binding fragments of antibodies such as antibodies, Fab fragments, and F(ab’)2 fragments, and various antigens can be mentioned. Antibodies, their antigen-binding fragments selectively bind to corresponding antigens, and antigens selectively bind to corresponding antibodies, and thus are equivalent to selective binding substances. As saccharides, polysaccharides are preferred, and various antigens can be mentioned. In addition, substances having antigenicity other than proteins and saccharides can also be immobilized. The selective binding substance used in the present invention can be a commercially available product, and in addition, it can also be a substance obtained from living cells or the like. As the selective binding substance, nucleic acids are particularly preferred. Among nucleic acids, oligonucleic acids having a length of 10 bases to 100 bases can be easily artificially synthesized using a synthesizer, and in addition, the amino modification of the nucleic acid terminus is easy, so that the immobilization on the substrate surface becomes easy, and thus it is preferred. In addition, from the viewpoint of hybridization stability, a length of 20 to 100 bases is more preferred. As the functional group capable of modifying the terminus of a nucleic acid, in addition to an amino group, a formyl group, an epoxy group, a carboxyl group, a hydroxyl group, or a thiol group (thioalkyl group) can also be preferably used. Among them, an amino group is preferred. The method of binding these functional groups to the terminus of a nucleic acid is well-known. For example, binding an amino group to the terminus of a nucleic acid can be carried out by binding a phosphoramidite reagent containing an amino group (Reference: International Publication No. 2013 / 024694).

[0062] In the biochip of the present invention, the selective binding substance is immobilized on the surface of the substrate via a covalent bond with the polymer bound to the substrate by the method as described above. The covalent bond between the selective binding substance and the polymer can be directly bound to the reactive functional group in the polymer or can be bound using a known crosslinking agent. Examples of known crosslinking agents include homobifunctional linkers such as 1,4-phenylene diisocyanate, 1,4-butanediol diglycidyl ether, 1,4-phenylene diisothiocyanate, or heterobifunctional linkers such as N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), N-succinimidyl maleimidodiacetate (AMAS). More specifically, for example, the covalent bond between the selective binding substance and the polymer can be formed by immersing the substrate immobilized with the polymer in a dimethyl sulfoxide solution of 1,4-phenylene diisothiocyanate (preferably at a concentration of 10 to 50% by weight), preferably at a temperature of 0°C to 40°C, for 1 hour to 10 hours, and then contacting the substrate with an aqueous solution of the selective binding substance (preferably at a concentration of 0.1 to 10% by weight), preferably at a temperature of 0°C to 40°C, for 1 hour to 100 hours.

[0063] The substance to be measured in the biochip of the present invention is a substance that can be selectively bound by the above selective binding substance, and examples thereof include nucleic acids, proteins, and peptides. As nucleic acids, in addition to free DNA, genomic DNA, messenger RNA, and microRNA, artificially synthesized nucleic acids can also be used. As proteins, antibodies, antigens, cytokines, and allergens can be mentioned. In addition, the substance to be measured can be a substance that has been previously labeled with a fluorescent substance or the like.

[0064] The measurement of the substance to be measured using the biochip of the present invention can be carried out by a known method. It generally includes the following steps: a step of forming a complex with a selective binding substance by bringing a sample containing the substance to be measured into contact with the surface of the substrate; and a step of detecting the formed complex. In addition, usually, the surface of the substrate is washed before this detection step. The sample only needs to contain or may contain the substance to be measured. Specifically, it is body fluids such as blood, plasma, and serum, and buffer solutions containing the substance to be measured. The detection method of the above complex is not particularly limited, and it can be carried out by known methods such as fluorescence and chemiluminescence. When the substance to be measured is labeled with a fluorescent substance or the like, it can be used for the detection of the above complex, or the above complex can be labeled with other fluorescent substances or the like and then detected. In addition, when measuring the signal value derived from the above complex from the region where the selective binding substance is immobilized, the noise value derived from the selective binding substance non-specifically bound to the substrate can be measured together from the region where the selective binding substance is not immobilized and used for the determination of the noise level.

[0065] Examples

[0066] Examples are shown below, but the present invention is not limited by these examples.

[0067] Example 1

[0068] A biochip using a partially methoxycarbonylated polyallylamine-bonded PMMA substrate (substrates 2 to 6)

[0069] (1) Synthesis of partially methoxycarbonylated polyallylamine (Formula (IV))

[0070] 100 mL (263 mmol) of a 15 wt% aqueous solution of polyallylamine (number average molecular weight 15,000, Nittobo Medical PAA-15C) was added to a flask, and the temperature was maintained at 50 °C. 2.4 g of dimethyl carbonate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., 26.3 mmol) was added dropwise to this aqueous solution over 15 minutes. After the addition was completed, the reaction was continued for 12 hours while maintaining the temperature at 50 °C. After the reaction, the polymer solution was placed in a dialysis membrane (Spectra pore 3, cut-off molecular weight 3,500), and by-products were removed by dialysis in water. After purification, water was removed by freeze-drying to obtain 10 mol% methoxycarbonylated polyallylamine. Measured in heavy water 1 1H-NMR, and the methoxycarbonylation ratio (m / (m + n)) was confirmed to be 10 mol% from the area ratio of the peak (2.91 ppm) derived from the methoxycarbonylated allylamine unit and the peak (2.45 ppm) derived from the unmodified allylamine unit.

[0071] By the same method, 30 mol% methoxycarbonylated polyallylamine was obtained by using 7.1 g of dimethyl carbonate (manufactured by FUJIFILM Wako Pure Chemical Corporation, 78.9 mmol), 50 mol% methoxycarbonylated polyallylamine was obtained by using 11.9 g of dimethyl carbonate (manufactured by FUJIFILM Wako Pure Chemical Corporation, 132 mmol), 70 mol% methoxycarbonylated polyallylamine was obtained by using 16.6 g of dimethyl carbonate (manufactured by FUJIFILM Wako Pure Chemical Corporation, 184 mmol), and 90 mol% methoxycarbonylated polyallylamine was obtained by using 21.3 g of dimethyl carbonate (manufactured by FUJIFILM Wako Pure Chemical Corporation, 237 mmol).

[0072]

[0073] In the formula, m and n independently represent positive numbers. Therefore, m / (m + n) and n / (m + n) represent the molar ratios of the respective units.

[0074] (2) Fabrication of NHS-esterified PMMA substrate (substrate 1)

[0075] A flat plate (75 mm × 25 mm × 1 mm) made of polymethyl methacrylate (PMMA) was immersed in a 10 N aqueous sodium hydroxide solution at 70 °C for 15 hours. Then, it was washed in the order of pure water, 0.1 N HCl aqueous solution, and pure water. By such an operation, the side chains of PMMA on the substrate surface were hydrolyzed to generate carboxyl groups.

[0076] Next, 100 mg of N-hydroxysuccinimide (NHS) and 350 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) were dissolved in 400 mL of 2-morpholinoethanesulfonic acid monohydrate (MES) buffer solution (adjusted to pH 5.0 with 0.1 N sodium hydroxide). The above-mentioned hydrolyzed PMMA substrate was immersed in their mixed solution and stirred with a magnetic stirrer for 1 hour to obtain an NHS-esterified PMMA substrate (substrate 1).

[0077] (3) Fabrication of partially methoxycarbonylated polyallylamine-bonded PMMA substrates (substrates 2 to 6)

[0078] The NHS-esterified PMMA substrate (substrate 1) obtained in (2) above was immersed in 400 mL of a boric acid buffer solution (100 mM, adjusted to pH 10 with 1N sodium hydroxide) in which the 10 mol% methoxycarbonylated polyallylamine obtained in (1) above was dissolved to a concentration of 1 wt%, and stirred with a magnetic stirrer for 1 hour. After stirring, it was immersed in 400 mL of a dimethyl sulfoxide solution containing 100 mg of 1,4-phenylene diisothiocyanate as a crosslinking agent, and stirred with a magnetic stirrer for 1 hour to obtain a 10 mol% methoxycarbonylated polyallylamine-bonded PMMA substrate (substrate 2).

[0079] By the same method, a 30 mol% methoxycarbonylated polyallylamine-bonded PMMA substrate (substrate 3) was obtained by using 30 mol% methoxycarbonylated polyallylamine, a 50 mol% methoxycarbonylated polyallylamine-bonded PMMA substrate (substrate 4) was obtained by using 50 mol% methoxycarbonylated polyallylamine, a 70 mol% methoxycarbonylated polyallylamine-bonded PMMA substrate (substrate 5) was obtained by using 70 mol% methoxycarbonylated polyallylamine, and a 90 mol% methoxycarbonylated polyallylamine-bonded PMMA substrate (substrate 6) was obtained by using 90 mol% methoxycarbonylated polyallylamine.

[0080] (4) Immobilization of probe DNA on the substrate

[0081] As the probe DNA, a DNA consisting of the base sequence of SEQ ID NO: 1 below was synthesized.

[0082] 5’-AACTATACAACCTACTACCTCA-3’ (SEQ ID NO: 1: 23 bases, 5’-terminal aminated).

[0083] This DNA was dissolved in pure water at a concentration of 100 μM to prepare a stock solution. The stock solution was diluted 5-fold with PBS (prepared by dissolving 8 g of NaCl, 2.9 g of Na2HPO4·12H2O, 0.2 g of KCl, and 0.2 g of K2PO4 in pure water and diluting to 1 L. pH 7.4) to prepare a spotting solution. Approximately 40 μl of the spotting solution was taken out, and using a spotting robot (GTMASStamp-2, Nippon Laser & Electronics Co., Ltd.), 24 DNAs were spotted at the central part of each substrate (substrates 2 - 6) prepared in (3) above. After spotting, the substrates were placed in a sealed plastic container and incubated at 37 °C and 100% humidity for about 20 hours to immobilize the probe DNA. After incubation, the substrates were washed with pure water.

[0084] (5) Hybridization to the substrate immobilized with probe DNA

[0085] For total RNA derived from prostate tissue (Thermo Fisher Scientific), miRNA was fluorescently labeled using the 3D-Gene (registered trademark) miRNA Labeling kit (Toray Industries, Inc.) based on the experimental method specified by the company to prepare a stock solution. The stock solution was made into a hybridization solution that was 50-fold diluted with a solution of 1 wt% BSA (bovine serum albumin), 5× SSC (so-called 5× SSC is obtained by dissolving 43.8 g of NaCl and trisodium citrate hydrate in 22.1 g of pure water and diluting to 200 ml. In addition, a solution obtained by dissolving 43.8 g of NaCl and trisodium citrate hydrate in 22.1 g of pure water and diluting to 1 l is designated as 1× SSC, its 10-fold concentrated solution is designated as 10× SSC, and its 5-fold diluted solution is designated as 0.2× SSC), 0.1 wt% SDS (sodium dodecyl sulfate), and 0.01 wt% salmon sperm DNA (each concentration is the final concentration). 100 μl of the hybridization solution was dropped onto each substrate (substrates 2 to 6) immobilized with probe DNA, and a cover glass was placed on it. In addition, the periphery of the cover glass was sealed with paper tape to prevent the hybridization solution from drying. It was placed in a plastic container and incubated at 35°C and 100% humidity for 12 hours for hybridization. After incubation, the cover glass was peeled off and then washed and dried.

[0086] (6) Fluorescence measurement of the hybridized biochip

[0087] The substrate that had undergone hybridization was placed in the “‘3D-Gene’ (registered trademark) Scanner” (Toray Industries, Inc.), and measurement was performed in a state where the excitation light was set to 635 nm, the laser output was 100%, and PMT was 30. The results are shown in Table 1.

[0088] Example 2

[0089] A biochip using a substrate made of partially acetylated polyallylamine-bonded PMMA (substrates 7 to 9)

[0090] (1) Synthesis of partially acetylated polyallylamine (Formula (V))

[0091] 100 mL (263 mmol) of an aqueous solution of polyallylamine (number average molecular weight 15,000, Nitto Boehringer Medical PAA-15C) at 15 wt% was added to a flask, the temperature was maintained at 50 °C, and 8.1 g (78.9 mmol) of acetic anhydride (manufactured by Fujifilm Wako Pure Chemical Corporation) was added dropwise to this aqueous solution over 15 minutes. After the addition was completed, the reaction was continued for 12 hours while maintaining the temperature at 50 °C. After the reaction, an aqueous sodium hydroxide solution (manufactured by Fujifilm Wako Pure Chemical Corporation, 8 equivalents) was added until the pH reached 11, and the polymer solution was placed in a dialysis membrane (Spectra pore 3, cut-off molecular weight 3,500), and by dialysis operation in water, by-products were removed. After purification, water was removed by freeze-drying to obtain 30 mol% acetylated polyallylamine. Measured in heavy water 1 1H-NMR, and from the ratio of the areas of the peaks (2.87 ppm) derived from the acetylated allylamine units and the peaks (2.45 ppm) derived from the unmodified allylamine units, the acetylation ratio (m / (m + n)) was confirmed to be 30 mol%.

[0092] By the same method, 50 mol% acetylated polyallylamine was obtained by using 13.5 g (132 mmol) of acetic anhydride, and 70 mol% acetylated polyallylamine was obtained by using 18.8 g (184 mmol) of acetic anhydride.

[0093]

[0094] In the formula, m and n each independently represent a positive number. Therefore, m / (m + n) and n / (m + n) represent the molar ratios of the respective units.

[0095] (2) Preparation of partially acetylated polyallylamine-bonded PMMA substrates (substrates 7 - 9)

[0096] By the same method as in (2) and (3) of Example 1, a 30 mol% acetylated polyallylamine-bonded PMMA substrate (substrate 7) was obtained by using 30 mol% acetylated polyallylamine, a 50 mol% acetylated polyallylamine-bonded PMMA substrate (substrate 8) was obtained by using 50 mol% acetylated polyallylamine, and a 70 mol% acetylated polyallylamine-bonded PMMA substrate (substrate 9) was obtained by using 70 mol% acetylated polyallylamine.

[0097] (3) Immobilization and evaluation of probe DNA on the substrate

[0098] For each substrate obtained in (2) above, the probe DNA was immobilized in the same manner as in (4) of Example 1 to obtain a biochip. For the obtained biochip, fluorescence measurement after hybridization was performed in the same manner as in (5) and (6) of Example 1. The results are shown in Table 1.

[0099] Example 3

[0100] Biochip using a substrate made of partially carbamylated polyallylamine-bound PMMA (substrates 10 to 12)

[0101] (1) Synthesis of partially carbamylated polyallylamine (Formula (VI))

[0102] 100 mL (263 mol) of a 15 wt% aqueous solution of polyallylamine (number average molecular weight 15,000, Nittobo Medical PAA-15C) was placed in a flask, and while cooling with ice, 21.9 g (263 mol) of concentrated hydrochloric acid (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added dropwise. Subsequently, the temperature was raised to 50 °C, and 68.4 g (78.9 mmol) of a 7.5 wt% aqueous solution of sodium cyanate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added dropwise, and the reaction was carried out for 24 hours. After the reaction, an aqueous sodium hydroxide solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., 8 equivalents) was added until the pH reached 11, and the polymer solution was placed in a dialysis membrane (Spectra pore 3, cut-off molecular weight 3,500), and by dialysis operation in water, by-products were removed. After purification, water was removed by freeze-drying to obtain 30 mol% carbamylated polyallylamine. Measured in heavy water 1 1H-NMR, and from the ratio of the area of the peak (2.88 ppm) derived from the carbamylated allylamine unit to the peak (2.51 ppm) derived from the unmodified allylamine unit, the carbamylation ratio (m / (m + n)) was confirmed to be 30 mol%.

[0103] By the same method, 50 mol% carbamylated polyallylamine was obtained by using 114.4 g (132 mmol) of a 7.5 wt% aqueous solution of sodium cyanate, and 70 mol% carbamylated polyallylamine was obtained by using 159.5 g (184 mmol) of a 7.5 wt% aqueous solution of sodium cyanate.

[0104]

[0105] In the formula, m and n each independently represent a positive number. Therefore, m / (m + n) and n / (m + n) represent the molar ratios of the respective units.

[0106] (2) Fabrication of Partially Carbamoylated Polyallylamine-Bound PMMA Substrates (Substrates 10 - 12)

[0107] By the same method as in (2) and (3) of Example 1, using 30 mol% carbamoylated polyallylamine, a 30 mol% carbamoylated polyallylamine-bound PMMA substrate (substrate 10) was obtained. Using 50 mol% carbamoylated polyallylamine, a 50 mol% carbamoylated polyallylamine-bound PMMA substrate (substrate 11) was obtained. Using 70 mol% carbamoylated polyallylamine, a 70 mol% carbamoylated polyallylamine-bound PMMA substrate (substrate 12) was obtained.

[0108] (3) Immobilization and Evaluation of Probe DNA on the Substrate

[0109] For each of the substrates obtained in (2) above, probe DNA was immobilized in the same manner as in (4) of Example 1 to obtain a biochip. For the obtained biochip, fluorescence measurement after hybridization was performed in the same manner as in (5) and (6) of Example 1. The results are shown in Table 1.

[0110] Comparative Example 1

[0111] Biochip Using NHS-Esterified PMMA Substrate (Substrate 1)

[0112] For the NHS-esterified PMMA substrate (substrate 1) obtained in (2) of Example 1, probe DNA was immobilized in the same manner as in (4) of Example 1 to obtain a biochip. For the obtained biochip, fluorescence measurement after hybridization was performed in the same manner as in (5) and (6) of Example 1. The results are shown in Table 1.

[0113] Comparative Example 2

[0114] Polyethyleneimine-Bound PMMA Substrate (Substrate 13)

[0115] (1) Fabrication of Polyethyleneimine-Bound PMMA Substrate (Substrate 13)

[0116] By the same method as in (2) and (3) of Example 1, using polyethyleneimine (number average molecular weight 10,000, Nippon Shokubai SP-200), a polyethyleneimine-bound PMMA substrate (substrate 13) was obtained.

[0117] (2) Immobilization and Evaluation of Probe DNA on the Substrate

[0118] For the substrate obtained in the above (1), the immobilization of the probe DNA was carried out in the same manner as in (4) of Example 1 to obtain a biochip. For the obtained biochip, the fluorescence measurement after hybridization was carried out in the same manner as in (5) and (6) of Example 1. The results are shown in Table 1.

[0119] Comparative Example 3

[0120] A biochip using a polyallylamine-bonded PMMA substrate (substrate 14)

[0121] (1) Fabrication of a polyallylamine-bonded PMMA substrate (substrate 14)

[0122] By the same method as in (2) and (3) of Example 1, a polyallylamine-bonded PMMA substrate (substrate 14) was obtained by using polyallylamine (number average molecular weight 15,000, Nittobo Medical PAA-15C).

[0123] (2) Immobilization and evaluation of probe DNA on the substrate

[0124] For the substrate obtained in the above (1), the immobilization of the probe DNA was carried out in the same manner as in (4) of Example 1 to obtain a biochip. For the obtained biochip, the fluorescence measurement after hybridization was carried out in the same manner as in (5) and (6) of Example 1. The results are shown in Table 1.

[0125] Comparative Example 4

[0126] A biochip using a partially carboxymethylated polyallylamine-bonded PMMA substrate (substrate 15)

[0127] (1) Synthesis of partially carboxymethylated polyallylamine (Formula (VII))

[0128] 100 mL (263 mol) of a 15 wt% aqueous solution of polyallylamine (number average molecular weight 15,000, Nittobo Medical PAA-15C) was added to a flask, heated to 50 °C, and 9.1 g of bromoacetic acid (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., 65.8 mmol) was added dropwise, followed by reaction for 24 hours. After the reaction, an aqueous sodium hydroxide solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., 8 equivalents) was added until the pH reached 11, and the polymer solution was placed in a dialysis membrane (Spectra pore 3, molecular weight cut-off 3,500) and the by-products were removed by dialysis in water. After purification, the water was removed by freeze-drying to obtain 25 mol% carboxymethylated polyallylamine. Measured in heavy water 1The carboxymethylation ratio (m / (m + n)) was confirmed to be 25 mol% by the ratio of the peak area of the carboxymethylated allylamine unit (2.61 ppm) to the peak area of the unmodified allylamine unit (2.51 ppm) in the 1H-NMR spectrum.

[0129]

[0130] In the formula, m and n each independently represent a positive number. Therefore, m / (m + n) and n / (m + n) represent the molar ratios of the respective units.

[0131] (2) Preparation of a partially carboxymethylated polyallylamine-bonded PMMA substrate (substrate 15)

[0132] A 25 mol% carboxymethylated polyallylamine-bonded PMMA substrate (substrate 15) was obtained by the same method as in (2) and (3) of Example 1, using 25 mol% carboxymethylated polyallylamine.

[0133] (3) Immobilization and evaluation of probe DNA on the substrate

[0134] For the substrate obtained in (2) above, probe DNA was immobilized in the same manner as in (4) of Example 1 to obtain a biochip. For the obtained biochip, fluorescence measurement after hybridization was performed in the same manner as in (5) and (6) of Example 1. The results are shown in Table 1.

[0135] Table 1

[0136]

[0137] Table 1 shows the fluorescence value (signal value) of the portion of the substrate where the probe DNA is immobilized, the fluorescence value (noise value) of the portion where the probe DNA is not immobilized, and the ratio of the noise value to the signal value (SN ratio) after hybridization. The fluorescence detected here is derived from the fluorescent dye contained in the labeling kit and the RNA fluorescently labeled by the labeling kit, and the higher the binding amount, the higher the value. The signal value corresponds to the amount of the fluorescently labeled RNA having a sequence complementary to the probe DNA that binds to the probe DNA by hybridization. On the other hand, the noise value corresponds to the amount of the fluorescent dye and the fluorescently labeled RNA that non-specifically bind to the substrate. Regarding the SN ratio, since the more the RNA binds by hybridization to the probe DNA and the less the non-specific binding of the RNA and the fluorescent dye to the substrate, the higher the value, it is used as an index of the detection sensitivity of the biochip.

[0138] Substrates (Example 1, Substrates 2 to 6) of partially methoxycarbonylated polyallylamine obtained by modifying the side-chain amino groups of polyallylamine showed a high SN ratio not only compared to substrates without polymer binding (Comparative Example 1, Substrate 1), but also compared to substrates bound with polyethyleneimine (Comparative Example 2, Substrate 13) and substrates bound with polyallylamine (Comparative Example 3, Substrate 14). It is speculated that this is due to the effect of suppressing the non-specific adsorption of fluorescent dyes, which is the cause of the increase in noise values, through the modification of methoxycarbonyl, and the result of the improved hybridization efficiency through the regulation of the density of reactive functional groups on the substrate surface.

[0139] The same effect was also observed in substrates (Example 2, Substrates 7 to 9) of partially acetylated polyallylamine and substrates (Example 3, Substrates 10 to 12) of partially carbamylated polyallylamine. On the other hand, it was not observed in substrates bound with partially carboxymethylated polyallylamine (Comparative Example 4, Substrate 15). From the above results, it can be seen that the type of functional group modified on the polymer is important for obtaining a biochip with excellent signal values and SN ratios.

[0140] Example 4

[0141] Biochips using PMMA substrates (Substrates 16 to 18) bound with partially propanamide-modified polyallylamine

[0142] (1) Synthesis of partially propanamide-modified polyallylamine (Formula (VIII))

[0143] 100 mL (263 mmol) of a 15 wt% aqueous solution of polyallylamine (number-average molecular weight 15,000, Nittobo Medical PAA-15C) was added to a flask, and the temperature was maintained at 50 °C. An aqueous solution obtained by dissolving 5.6 g of acrylamide (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., 78.9 mmol) in 50 mL of distilled water was added dropwise to this aqueous solution over 15 minutes. After the addition was completed, the reaction was continued for 12 hours while maintaining the temperature at 50 °C. After the reaction, the polymer solution was placed in a dialysis membrane (Spectrum, pore size 3, molecular weight cut-off 3,500), and by-products were removed by dialysis in water. After purification, water was removed by freeze-drying to obtain polyallylamine modified with 30 mol% propanamide. Measured by 1 1H-NMR, and the ratio of the area of the peak (2.62 ppm) derived from the allylamine unit modified with propanamide to the peak (2.45 ppm) derived from the unmodified allylamine unit confirmed that the ratio of propanamide modification (m / (m + n)) was 30 mol%.

[0144] By the same method, 50 mol% propanamide-modified polyallylamine was obtained by using 9.4 g of acrylamide (manufactured by FUJIFILM Wako Pure Chemical Corporation, 132 mmol), and 70 mol% propanamide-modified polyallylamine was obtained by using 13.1 g of acrylamide (manufactured by FUJIFILM Wako Pure Chemical Corporation, 184 mmol).

[0145]

[0146] In the formula, m and n independently represent positive numbers. Therefore, m / (m + n) and n / (m + n) represent the molar ratios of the respective units.

[0147] (2) Preparation of NHS-esterified PMMA substrate (substrate 1)

[0148] An NHS-esterified PMMA substrate (substrate 1) was prepared in the same manner as in (2) of Example 1.

[0149] (3) Preparation of partially propanamide-modified polyallylamine-bonded PMMA substrates (substrates 16 to 18)

[0150] The NHS-esterified PMMA substrate (substrate 1) prepared in (2) was immersed in 400 mL of a boric acid buffer solution (100 mM, adjusted to pH 10 with 1 N sodium hydroxide) in which 30 mol% propanamide-modified polyallylamine obtained in the above (1) was dissolved to a concentration of 1 wt%, and stirred with a magnetic stirrer for 1 hour. After stirring, it was immersed in 400 mL of a dimethyl sulfoxide solution containing 100 mg of 1,4-phenylene diisothiocyanate as a crosslinking agent, and stirred with a magnetic stirrer for 1 hour to obtain a 30 mol% propanamide-modified polyallylamine-bonded PMMA substrate (substrate 16).

[0151] By the same method, a 50 mol% propanamide-modified polyallylamine-bonded PMMA substrate (substrate 17) was obtained by using 50 mol% propanamide-modified polyallylamine, and a 70 mol% propanamide-modified polyallylamine-bonded PMMA substrate (substrate 18) was obtained by using 70 mol% propanamide-modified polyallylamine.

[0152] (4) Immobilization of probe DNA on the substrate

[0153] In the same manner as in (4) of Example 1, DNA composed of the base sequence of SEQ ID NO: 1 (23 bases, 5'-terminal aminoated) was used as the probe DNA, and immobilized on each substrate by the same method as in Example 1.

[0154] (5) Hybridization to the substrate immobilized with probe DNA

[0155] Hybridization to the substrate immobilized with probe DNA was carried out by the same method as in (5) of Example 1.

[0156] (6) Fluorescence measurement of the hybridized biochip

[0157] Fluorescence measurement of the hybridized biochip was carried out by the same method as in (6) of Example 1. The results are shown in Table 2.

[0158] Example 5

[0159] A biochip using a substrate (substrate 19) made of PMMA bound with partially N,N-dimethylpropionamide-modified polyallylamine

[0160] (1) Synthesis of partially N,N-dimethylpropionamide-modified polyallylamine (Formula (IX))

[0161] 100 mL (263 mmol) of a 15 wt% aqueous solution of polyallylamine (number average molecular weight 15,000, Nittobo Medical PAA-15C) was placed in a flask, the temperature was maintained at 50 °C, and an aqueous solution in which 13.1 g of N,N-dimethylacrylamide (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., 132 mmol) was dissolved in 50 mL of distilled water was added dropwise to this aqueous solution over 15 minutes. After the addition was completed, the reaction was continued for 12 hours while maintaining the temperature at 50 °C. After the reaction, the polymer solution was placed in a dialysis membrane (Spectrum, pore size 3, molecular weight cut-off 3,500), and by-products were removed by dialysis in water. After purification, water was removed by freeze-drying to obtain 50 mol% N,N-dimethylpropionamide-modified polyallylamine. Measured in heavy water 1 1H-NMR, and the ratio of the peak area of the allylamine unit modified with N,N-dimethylpropionamide (2.62 ppm) to the peak area of the unmodified allylamine unit (2.45 ppm) confirmed that the modification ratio of N,N-dimethylpropionamide (m / (m + n)) was 50 mol%.

[0162]

[0163] In the formula, m and n each independently represent a positive number. Therefore, m / (m + n) and n / (m + n) represent the molar ratios of the respective units.

[0164] (2) Production of a substrate (substrate 19) made of PMMA bound with partially N,N-dimethylpropionamide-modified polyallylamine

[0165] By the same method as in (2) and (3) of Example 4, 50 mol% N,N-dimethylpropionamide-modified polyallylamine was obtained by modifying polyallylamine with 50 mol% N,N-dimethylpropionamide, and a substrate (substrate 19) made of PMMA bound with 50 mol% N,N-dimethylpropionamide-modified polyallylamine was obtained.

[0166] (3) Immobilization and evaluation of probe DNA on the substrate

[0167] For each substrate obtained in the above (2), probe DNA was immobilized by the same operation as in (4) of Example 4 to obtain a biochip. For the obtained biochip, fluorescence measurement after hybridization was performed by the same operation as in (5) and (6) of Example 4. The results are shown in Table 2.

[0168] Example 6

[0169] A biochip using a substrate (substrate 20) made of PMMA bound with partially N,N-diethylpropionamide-modified polyallylamine

[0170] (1) Synthesis of partially N,N-diethylpropionamide-modified polyallylamine (Formula (X))

[0171] 100 mL (263 mmol) of a 15 wt% aqueous solution of polyallylamine (number average molecular weight: 15,000, Nittobo Medical PAA-15C) was added to a flask, and the temperature was maintained at 50 °C. An aqueous solution prepared by dissolving 16.8 g (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., 132 mmol) of N,N-diethylacrylamide in 50 mL of distilled water was added dropwise to this aqueous solution over 15 minutes. After the dropwise addition, the reaction was continued for 12 hours while maintaining the temperature at 50 °C. After the reaction, the polymer solution was placed in a dialysis membrane (Spectrum Laboratories, Inc., pore size 3, molecular weight cut-off 3,500), and by-products were removed by dialysis in water. After purification, water was removed by freeze-drying to obtain 50 mol% N,N-diethylpropionamide-modified polyallylamine. 1H-NMR was measured in heavy water. 1 From the ratio of the areas of the peak (2.64 ppm) derived from the allylamine unit modified with N,N-diethylpropionamide and the peak (2.45 ppm) derived from the unmodified allylamine unit, the modification ratio (m / (m + n)) of N,N-diethylpropionamide was confirmed to be 50 mol%.

[0172]

[0173] In the formula, m and n independently represent positive numbers. Therefore, m / (m + n) and n / (m + n) represent the molar ratios of the respective units.

[0174] (2) Fabrication of Substrate (Substrate 20) with PMMA Bound to Partially N,N - Diethylpropanamide - Modified Polyallylamine

[0175] By the same method as in (2) and (3) of Example 4, a substrate (substrate 20) with PMMA bound to 50 mol% N,N - diethylpropanamide - modified polyallylamine was obtained by using 50 mol% N,N - diethylpropanamide - modified polyallylamine.

[0176] (3) Immobilization and Evaluation of Probe DNA on the Substrate

[0177] For each substrate obtained in (2) above, probe DNA was immobilized by operating in the same manner as in (4) of Example 4 to obtain a biochip. For the obtained biochip, fluorescence measurement after hybridization was carried out by operating in the same manner as in (5) and (6) of Example 4. The results are shown in Table 2.

[0178] Example 7

[0179] Biochip Using Substrate (Substrate 21) with PMMA Bound to Partially N - Isopropylpropanamide - Modified Polyallylamine

[0180] (1) Synthesis of Partially N - Isopropylpropanamide - Modified Polyallylamine (Formula (XI))

[0181] 100 mL (263 mmol) of a 15 wt% aqueous solution of polyallylamine (number - average molecular weight 15,000, Nittobo Medical PAA - 15C) was added to a flask, and the temperature was maintained at 50°C. An aqueous solution obtained by dissolving 14.9 g of N - isopropylacrylamide (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., 132 mmol) in 50 mL of distilled water was added dropwise to this aqueous solution over 15 minutes. After the addition was completed, the reaction was continued for 12 hours while maintaining the temperature at 50°C. After the reaction, the polymer solution was placed in a dialysis membrane (Spectra pore 3, cut - off molecular weight 3,500), and by dialysis operation in water, by - products were removed. After purification, water was removed by freeze - drying to obtain 50 mol% N - isopropylpropanamide - modified polyallylamine. Measured by 1 1H - NMR in heavy water, and the ratio of the area of the peak (2.60 ppm) derived from the allylamine unit modified with N - isopropylpropanamide to the peak (2.45 ppm) derived from the unmodified allylamine unit confirmed that the modification ratio (m / (m + n)) of N - isopropylpropanamide was 50 mol%.

[0182]

[0183] In the formula, m and n independently represent positive numbers. Therefore, m / (m + n) and n / (m + n) represent the molar ratios of the respective units.

[0184] (2) Production of a substrate (substrate 7) in which a partially N-isopropylacrylamide-modified polyallylamine is bound to PMMA

[0185] By the same method as in (2) and (3) of Example 4, a substrate (substrate 21) in which a 50 mol% N-isopropylacrylamide-modified polyallylamine is bound to PMMA was obtained by using a 50 mol% N-isopropylacrylamide-modified polyallylamine.

[0186] (3) Immobilization and evaluation of probe DNA on the substrate

[0187] For each of the substrates obtained in the above (2), probe DNA was immobilized in the same manner as in (4) of Example 4 to obtain a biochip. For the obtained biochip, fluorescence measurement after hybridization was performed in the same manner as in (5) and (6) of Example 4. The results are shown in Table 2. In addition, for easy comparison, the results of Comparative Examples 1 to 4 shown in Table 1 are also shown in Table 2.

[0188] Table 2

[0189]

[0190] Example 8

[0191] A biochip in which ovalbumin allergens are immobilized on a substrate (substrates 3 to 5) in which a partially methoxycarbonyl-modified polyallylamine is bound to PMMA

[0192] (1) Immobilization of ovalbumin allergens on the substrate

[0193] Ovalbumin allergens were obtained as a freeze-dried powder from GREER. The freeze-dried powder was dissolved in pure water so that the protein concentration became 1.0 mg / mL to prepare a spotting solution. Using a spotting robot (GTMASStamp-2, manufactured by Nippon Laser & Electronics Co., Ltd.), 6 × 4 = 24 ovalbumin allergens were spotted at the central part of each of the substrates (substrates 3 to 5) produced in (3) of Example 1. After spotting, the substrates were placed in a sealed plastic container and incubated at 37 °C and 100% humidity for about 20 hours to immobilize the ovalbumin allergens. After incubation, the substrates were washed with phosphate-buffered saline (0.05% Tween 20 (trade name)).

[0194] (2) Contact of ovalbumin allergy-positive human serum with the substrate on which ovalbumin allergens are immobilized

[0195] The human serum positive for egg white allergy was obtained from PlasmaLab, diluted 3-fold with phosphate buffered saline, and 50 μL of this dilution was dropped onto the substrate prepared in (1), and a notched cover glass (manufactured by Matsunami Glass Industry Co., Ltd.: 24 mm × 25 mm, notch size 20 μm) was placed on it for sealing. After reacting at 37°C for 2 hours, the notched cover glass was removed, and the substrate was washed with phosphate buffered saline (0.05% Tween 20 (trade name)).

[0196] (3) Detection of egg white allergen-specific IgE antibody

[0197] A 1.0 mg / mL solution of Dylight-650 pigment-labeled anti-human IgE goat polyclonal antibody (manufactured by Novus biologicals) was diluted 1000-fold with phosphate buffered saline (0.05% Tween 20 (trade name)) containing 1 wt% bovine serum albumin. 50 μL of this dilution was dropped onto the substrate that had been in contact with human serum in (2), and a notched cover glass was placed on it and reacted at room temperature for 1 hour. Then, the notched cover glass was removed, and the substrate was washed with phosphate buffered saline (0.05% Tween 20 (trade name)).

[0198] The substrate was placed in the “‘3D-Gene’ (registered trademark) Scanner” (Toray Industries, Inc.), and measurement was performed in a state where the excitation light was set to 635 nm, the laser output was 100%, and PMT was 30. The results are shown in Table 3.

[0199] Example 9

[0200] A biochip immobilized with egg white allergen on a substrate made of partially ethoxycarbonyl-modified polyallylamine-bonded PMMA (substrates 22 - 24)

[0201] (1) Synthesis of partially ethoxycarbonyl-modified polyallylamine (chemical formula (XII))

[0202] 100 mL (263 mmol) of an aqueous solution of polyallylamine (number-average molecular weight 15,000, Nittobo Medical PAA-15C) at 15 wt% was added to a flask, the temperature was maintained at 50 °C, and 9.32 g (78.9 mmol) of diethyl carbonate (manufactured by Fujifilm Wako Pure Chemical Corporation) was added dropwise to this aqueous solution over 15 minutes. After the addition was completed, the reaction was continued for 12 hours while maintaining the temperature at 50 °C. After the reaction, the polymer solution was placed in a dialysis membrane (Spectrum, pore size 3, molecular weight cut-off 3,500), and by dialysis operation in water, by-products were removed. After purification, water was removed by freeze-drying to obtain 30 mol% ethoxycarbonylated polyallylamine. 1H-NMR was measured in heavy water, and the ratio of the area of the peak (2.87 ppm) derived from the ethoxycarbonylated allylamine unit to the peak (2.47 ppm) derived from the unmodified allylamine unit confirmed that the ratio of ethoxycarbonyl modification (m / (m + n)) was 30 mol%.

[0203] By the same method, 50 mol% ethoxycarbonyl-modified polyallylamine was obtained by using 15.6 g (132 mmol) of diethyl carbonate (manufactured by Fujifilm Wako Pure Chemical Corporation), and 70 mol% ethoxycarbonyl-modified polyallylamine was obtained by using 21.8 g (184 mmol) of diethyl carbonate (manufactured by Fujifilm Wako Pure Chemical Corporation).

[0204]

[0205] (2) Preparation of Partially Ethoxycarbonyl-Modified Polyallylamine-Bound PMMA Substrates (Substrates 22 to 24)

[0206] By the same method as in (2) and (3) of Example 1, a 30 mol% ethoxycarbonyl-modified polyallylamine-bound PMMA substrate (substrate 22) was obtained by using 30 mol% ethoxycarbonyl-modified polyallylamine, a 50 mol% ethoxycarbonyl-modified polyallylamine-bound PMMA substrate (substrate 23) was obtained by using 50 mol% ethoxycarbonyl-modified polyallylamine, and a 70 mol% ethoxycarbonyl-modified polyallylamine-bound PMMA substrate (substrate 24) was obtained by using 70 mol% ethoxycarbonyl-modified polyallylamine.

[0207] (3) Immobilization and Evaluation of Egg White Allergen on the Substrate

[0208] For each substrate obtained in the above (2), the immobilization of ovalbumin allergen was carried out in the same manner as in (1) of Example 8 to obtain a biochip. For the obtained biochip, the fluorescence measurement after contact with human serum positive for ovalbumin allergy was carried out in the same manner as in (2) and (3) of Example 8. The results are shown in Table 3.

[0209] Example 10

[0210] Biochip with ovalbumin allergen immobilized on a partially propoxycarbonyl-modified polyallylamine-bound PMMA substrate (substrates 25 to 27)

[0211] (1) Synthesis of partially propoxycarbonyl-modified polyallylamine (chemical formula (XIII))

[0212] 100 mL (263 mmol) of a 15 wt% aqueous solution of polyallylamine (number average molecular weight 15,000, Nittobo Medical PAA-15C) was added to a flask, the temperature was maintained at 50 °C, and 11.5 g (manufactured by Sigma-Aldrich, 78.9 mmol) of dipropyl carbonate was added dropwise to this aqueous solution over 15 minutes. After the addition was completed, the reaction was continued for 12 hours while maintaining the temperature at 50 °C. After the reaction, the polymer solution was placed in a dialysis membrane (Spectrum, pore size 3, molecular weight cut-off 3,500), and by-products were removed by dialysis in water. After purification, water was removed by freeze-drying to obtain 30 mol% propoxycarbonylated polyallylamine. 1H-NMR was measured in heavy water, and the ratio of the peak area of the propoxycarbonylated allylamine unit (2.85 ppm) to the peak area of the unmodified allylamine unit (2.50 ppm) confirmed that the ratio of propoxycarbonyl modification (m / (m + n)) was 30 mol%.

[0213] By the same method, 50 mol% propoxycarbonyl-modified polyallylamine was obtained by using 19.3 g (manufactured by Sigma-Aldrich, 132 mmol) of dipropyl carbonate, and 70 mol% propoxycarbonyl-modified polyallylamine was obtained by using 26.9 g (manufactured by Sigma-Aldrich, 184 mmol) of dipropyl carbonate.

[0214]

[0215] (2) Fabrication of a partially propoxycarbonyl-modified polyallylamine-bound PMMA substrate (substrates 25 to 27)

[0216] In the same manner as in (2) and (3) of Example 1, 30 mol% propoxycarbonyl-modified polyallylamine was used to obtain a 30 mol% propoxycarbonyl-modified polyallylamine-bonded PMMA substrate (substrate 25), 50 mol% propoxycarbonyl-modified polyallylamine was used to obtain a 50 mol% propoxycarbonyl-modified polyallylamine-bonded PMMA substrate (substrate 26), and 70 mol% propoxycarbonyl-modified polyallylamine was used to obtain a 70 mol% propoxycarbonyl-modified polyallylamine-bonded PMMA substrate (substrate 27).

[0217] (3) Immobilization and evaluation of ovalbumin allergen on the substrate

[0218] For each of the substrates obtained in (2) above, ovalbumin allergen was immobilized in the same manner as in (1) of Example 8 to obtain a biochip. For the obtained biochip, fluorescence measurement was performed in the same manner as in (2) and (3) of Example 8 after contact with ovalbumin allergy-positive human serum. The results are shown in Table 3.

[0219] Example 11

[0220] A biochip in which ovalbumin allergen was immobilized on a partially propanamide-modified polyallylamine-bonded PMMA substrate (substrate 17)

[0221] (1) Immobilization and evaluation of ovalbumin allergen on the substrate

[0222] For the substrate 17 obtained in (3) of Example 4, ovalbumin allergen was immobilized in the same manner as in (1) of Example 8 to obtain a biochip. For the obtained biochip, fluorescence measurement was performed in the same manner as in (2) and (3) of Example 8 after contact with ovalbumin allergy-positive human serum. The results are shown in Table 3.

[0223] Example 12

[0224] A biochip in which ovalbumin allergen was immobilized on a partially N,N-dimethylpropanamide-modified polyallylamine-bonded PMMA substrate (substrate 19)

[0225] (1) Immobilization and evaluation of ovalbumin allergen on the substrate

[0226] For the substrate 19 obtained in (3) of Example 5, ovalbumin allergen was immobilized in the same manner as in (1) of Example 8 to obtain a biochip. For the obtained biochip, fluorescence measurement was performed in the same manner as in (2) and (3) of Example 8 after contact with ovalbumin allergy-positive human serum. The results are shown in Table 3.

[0227] Example 13

[0228] A biochip in which ovalbumin allergen is immobilized on a substrate (substrate 20) of partially N,N - diethylpropionamide - modified polyallylamine - bound PMMA

[0229] (1) Immobilization and evaluation of ovalbumin allergen on the substrate

[0230] For the substrate 20 obtained in (3) of Example 6, ovalbumin allergen was immobilized in the same manner as in (1) of Example 8 to obtain a biochip. For the obtained biochip, fluorescence measurement was performed in the same manner as in (2) and (3) of Example 8 after contact with ovalbumin allergy - positive human serum. The results are shown in Table 3.

[0231] Example 14

[0232] A biochip in which ovalbumin allergen is immobilized on a substrate (substrate 21) of partially N - isopropylpropionamide - modified polyallylamine - bound PMMA

[0233] (1) Immobilization and evaluation of ovalbumin allergen on the substrate

[0234] For the substrate 21 obtained in (3) of Example 7, ovalbumin allergen was immobilized in the same manner as in (1) of Example 8 to obtain a biochip. For the obtained biochip, fluorescence measurement was performed in the same manner as in (2) and (3) of Example 8 after contact with ovalbumin allergy - positive human serum. The results are shown in Table 3.

[0235] Comparative Example 5

[0236] A biochip in which ovalbumin allergen is immobilized on an NHS - esterified PMMA substrate (substrate 1)

[0237] For the substrate 1 obtained in (2) of Example 1, ovalbumin allergen was immobilized in the same manner as in (1) of Example 8 to obtain a biochip. For the obtained biochip, fluorescence measurement was performed in the same manner as in (2) and (3) of Example 8 after contact with ovalbumin allergy - positive human serum. The results are shown in Table 3.

[0238] Comparative Example 6

[0239] A biochip in which ovalbumin allergen is immobilized on a polyallylamine - bound PMMA substrate (substrate 14)

[0240] For the substrate 14 obtained in (1) of Comparative Example 3, the immobilization of ovalbumin allergen was carried out in the same manner as in (1) of Example 8 to obtain a biochip. For the obtained biochip, fluorescence measurement was carried out in the same manner as in (2) and (3) of Example 8 after contact with human serum positive for ovalbumin allergy. The results are shown in Table 3.

[0241] Table 3

[0242]

[0243] Table 3 shows the fluorescence value (signal value) of the part of the substrate immobilized with ovalbumin allergen, the fluorescence value (noise value) of the part of the substrate not immobilized with ovalbumin allergen, and the ratio of the noise value to the signal value (SN ratio) after contact with human serum positive for ovalbumin allergy and a pigment-labeled anti-human IgE goat polyclonal antibody. The fluorescence detected here is derived from the pigment-labeled anti-human IgE goat polyclonal antibody, and the higher the binding amount of the pigment-labeled anti-human IgE goat polyclonal antibody, the higher the value shown. The signal value corresponds to the amount of the pigment-labeled anti-human IgE goat polyclonal antibody bound to the complex of human IgE antibody (substance to be measured) and ovalbumin allergen (selective binding substance). On the other hand, the noise value corresponds to the amount of the pigment-labeled anti-human IgE goat polyclonal antibody non-specifically bound to the substrate. Regarding the SN ratio, the more the pigment-labeled anti-human IgE goat polyclonal antibody binds to the above complex and the less the pigment-labeled anti-human IgE goat polyclonal antibody non-specifically binds to the substrate, the higher the value shown, so it is used as an index of the detection sensitivity of the biochip.

[0244] The substrate (Example 8, substrates 3 to 5) combined with partially methoxycarbonyl-modified polyallylamine obtained by modifying the side-chain amino group of polyallylamine showed a higher SN ratio not only compared with the substrate not combined with the polymer (Comparative Example 5, substrate 1) but also compared with the substrate combined with polyallylamine (Comparative Example 6, substrate 14). It is speculated that this is the result of the effect of suppressing the non-specific adsorption of the pigment-labeled anti-human IgE goat polyclonal antibody, which is the cause of the increase in the noise value, by the modification of methoxycarbonyl and the improvement of the formation efficiency of the above complex by adjusting the density of the reactive functional groups on the substrate surface.

[0245] The same effect was also observed in the substrates combined with partially ethoxycarbonyl-modified polyallylamine (Example 9, substrates 22 to 24), the substrates combined with partially propoxycarbonyl-modified polyallylamine (Example 10, substrates 25 to 27), partially propanamide-modified polyallylamine (Example 11, substrate 17), partially N,N-dimethylpropanamide-modified polyallylamine (Example 12, substrate 19), partially N,N-diethylpropanamide-modified polyallylamine (Example 13, substrate 20), and partially N-isopropylpropanamide-modified polyallylamine (Example 14, substrate 21). Furthermore, it was found that since the SN ratio varies depending on the type of functional group modified on the polymer, the type of functional group modified on the polymer is important for obtaining a biochip with excellent signal values and SN ratios.

[0246] Industrial applicability

[0247] According to the present invention, there is provided a biochip capable of highly sensitively detecting a substance to be measured. By using the biochip of the present invention, more accurate molecular recognition and diagnosis can be performed.

Claims

1. A biochip, wherein, On the surface of a substrate, a selective binding substance capable of selectively binding to a substance to be measured is immobilized via a polymer containing units represented by formula (Ia) or formula (Ib). In formula (Ia) and formula (Ib), R 1 represents an alkylene group having 1 to 4 carbon atoms, R 2 represents R 3 , OR 4 or NHR 5 , R 3 and R 5 each independently represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, R 4 represents an alkyl group having 1 to 4 carbon atoms, R 6 represents an alkylene group having 1 to 2 carbon atoms, R 7 and R 8 each independently represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms.

2. The biochip according to claim 1, wherein the polymer is represented by the formula (Ia), where R 1 and R 2 are the same as defined in claim 1.

3. The biochip according to claim 1, wherein the polymer is represented by the formula (Ib), where R 6 , R 7 and R 8 are the same as defined in claim 1.

4. The biochip according to any one of claims 1 to 3, wherein the polymer is a heteropolymer.

5. The biochip according to claim 4, wherein the heteropolymer is a heteropolymer with units represented by chemical formula (II). In the formula, R 1 represents an alkylene group having 1 to 4 carbon atoms.

6. The biochip according to any one of claims 1 to 3, wherein the number average molecular weight of the polymer is 300 to 1,000,000.

7. The biochip according to any one of claims 1 to 3, wherein the selective binding substance is a nucleic acid or a protein.

8. A detection method for determining a target substance, wherein, Using the biochip according to any one of claims 1 to 7, in which a selective binding substance capable of selectively binding to a substance to be measured is immobilized on the surface of a substrate, the method comprises: bringing a sample containing the substance to be measured into contact with the surface of the substrate, and detecting the complex formed with the selective binding substance.

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