Substrate for biochip, biochip, method for manufacturing biochip, and method for storing the same

Abstract

The present invention aims to provide a biochip substrate, a biochip, a method for manufacturing a biochip, and a method for storing the same. The present invention relates to a biochip and a method for manufacturing the same, which comprises a substrate having a hydrophilic reaction area and spots of immobilized substances including biological substances arranged in the reaction area, the reaction area being surrounded by a boundary capable of retaining a liquid therein, and the spots further containing a thickener and / or a surfactant.

Description

Technical Field

[0001] The present invention relates to a biochip used for detecting and analyzing biochemical reactions, etc. by using a biological substance or the like fixed to a substrate, a substrate used for the biochip, a method for manufacturing the biochip, and a method for storing the biochip. In particular, the present invention relates to a test diagnostic chip using an antigen-antibody reaction used for allergy tests, tumor marker tests, etc.

Background Art

[0002] In recent years, immunoplates for immunoassay in which an antibody or an antigen is immobilized on a plate, DNA chips in which nucleic acids are immobilized on a chip, etc. have been widely used. In these chips, biological substances such as proteins and nucleic acids, which are substances to be immobilized, are immobilized in a microarray form on the surface of the substrate in a spot shape.

[0003] As a method for immobilizing a protein or the like on a substrate, BSA is spotted on a polymer layer obtained by applying a vinyl polymer of polyethylene glycol monomethacrylate having a polyethylene glycol molecular weight of 350 on an acrylic resin substrate, and BSA is immobilized on the substrate by a photocrosslinking reaction with sodium 4,4'-diazidostilbene-2,2'-disulfonate (Patent Document 1), or N-[4-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenyl]oxirane and a peptide are spotted on a film obtained by applying a vinyl polymer of polyethylene glycol monomethacrylate having a polyethylene glycol molecular weight of 350 on an acrylic resin substrate to immobilize the peptide (Patent Document 2).

[0004] On the other hand, as a method for detecting and analyzing a biochemical reaction, a maleimide group is introduced in a spot shape into PEG3-OH alkanethiol bonded to a glass slide vapor-deposited with chromium and gold by 8-AOT and SSMCC, and a peptide is sequenced and immobilized in a spot shape through this, and a test sample is flowed through an SPR apparatus to detect the phosphorylation of cSrc kinase (Patent Document 3).

[0005] Patent Document 4 discloses a method in which a silicon or stainless steel microchip coated with diamond / diamond-like carbon is subjected to an activation treatment, an allergen is spotted thereon, unreacted groups remaining on the chip are inactivated, and then blocked with BSA before reacting with a test sample.

[0006] As a chip capable of detecting with a small amount of test sample, a chip is disclosed that includes a plurality of reaction chambers (wells) in a substrate, flow paths connecting to each of the wells, and an injection port for injecting a solution into the flow paths, enabling analysis of a plurality of test samples and reaction with a small amount of test sample (Patent Document 5). Further, a chip is disclosed that includes a spot on which a component that reacts with a component to be detected is fixed, disposed in a housing portion capable of housing a liquid test sample, a drainage portion for discharging the test sample and a cleaning liquid by centrifugal force, and a separation portion (Patent Document 6).

[0007] In addition, since protein chips with proteins immobilized on a plate are generally developed positioned on the extension line of DNA chips, studies have been made on immobilizing proteins or molecules that capture them (substances to be immobilized) on the surface of a glass substrate (Patent Document 7).

[0008] After immobilizing the substance to be immobilized on the substrate and reacting it with another bioactive substance (substance to be detected: protein, antigen, etc.) on the surface, and further reacting a labeled protein and finally detecting it with a detector or the like, if a substance to be detected other than the molecule is immobilized on a portion where the capture molecule is not fixed, it becomes noise during detection, causing a decrease in the signal-to-noise ratio (S / N ratio) and a decrease in detection accuracy. In particular, in serum or plasma used in clinical diagnosis and the like, there is a lot of non-specific adsorption of contaminating proteins, resulting in high noise and a tendency for the S / N ratio to be low.

[0009] Therefore, after immobilizing the antibody on the chip surface, in order to prevent non-specific adsorption of the antigen and the secondary antibody, coating with a non-specific adsorption inhibitor is performed. For example, a method of applying a vinyl polymer of polyethylene glycol monomethacrylate with a polyethylene glycol molecular weight of 350 on an acrylic resin substrate (Patent Document 2), or a method of bonding PEG3-OH alkanethiol to a glass slide vapor-deposited with chromium and gold (Patent Document 3) are disclosed.

[0010] On the other hand, on a saturated cyclic polyolefin resin substrate, a layer containing a polymer composed of 2-methacryloyloxyethyl phosphorylcholine, n-cyclohexyl methacrylate, and N-[2-[2-[2-(t-butoxycarbonylaminooxyacetylamino)ethoxy]ethoxy]ethyl]-methacrylamide is introduced, an aldehyde group or a maleimide group is provided on the surface, and after immobilizing the peptide, by deactivating the aldehyde group or maleimide group remaining on the substrate surface, a substrate for immobilizing a bioactive substance that suppresses non-specific adsorption and binding of the substance to be detected without coating with an adsorption inhibitor is disclosed (Patent Document 8).

Prior Art Documents

Patent Documents

[0011]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Patent Document 6

Patent Document 7

Patent Document 8

Summary of the Invention

Problems to be Solved by the Invention

[0012] However, the methods of Patent Documents 1 to 2 have the problem that the immobilization site of the substance to be immobilized cannot be controlled, resulting in low detection efficiency of the substance to be detected. Moreover, it is difficult to obtain a more uniform spot and the detection sensitivity is not high. In addition, the method of Patent Document 3 has the problem that it is difficult to perform inspection with a small amount of test sample. On the other hand, although the chip of Patent Document 4 may be able to perform inspection with a small amount of test sample, it requires a special substrate coated with diamond / diamond-like carbon. After the chip is subjected to an activation treatment, allergens are spotted, and then unreacted groups remaining on the chip need to be inactivated and further blocked with BSA, making the preparation of the chip complicated. Since the chips of Patent Documents 5 to 6 have a complex shape, there is a problem that the chip molding process becomes complicated, and there is also a concern that manufacturing defects are likely to occur.

[0013] Also, the methods of Patent Documents 2 to 3 require a step of forming a coating layer on the substrate to prevent non-specific adsorption between the substrate and the substance to be detected and to immobilize the biological substance. However, the step of forming the coating layer on the substrate is time-consuming and costly, and defective products are likely to occur, so it is not always satisfactory. On the other hand, the substrate of Patent Document 8 does not form a coating layer to prevent non-specific adsorption, but forms a layer for immobilizing the substance to be immobilized, and performs a hydrophobic treatment after immobilizing the substance to be immobilized to prevent non-specific adsorption, and the process is more complicated than that of Patent Documents 2 to 3.

[0014] Furthermore, since these chips immobilize biological substances, refrigeration or freezing is required for storing the chips. Therefore, there has been a demand for a biochip in which the above problems of the prior art are improved.

Means for Solving the Problems

[0015] In one aspect, the present invention is as follows, for example. [1-1] A substrate for a biochip having a resin surface including a hydrophilic reaction area. [1-2] The biochip substrate according to [1-1], wherein the reaction area is surrounded by a boundary capable of retaining a liquid within the reaction area, and a bond related to carbon of the resin is cleaved, and the cleaved portion is covered with a polar functional group formed by bonding with oxygen. [1-3] A biochip in which a substance to be immobilized containing a biological substance is immobilized on the reaction area of the biochip substrate according to [1-1] or [1-2]. [1-4] The biochip according to [1-3], wherein the substance to be immobilized is present in a spot containing a thickener and / or a surfactant present in the reaction area.

[0016] [1-5] A biochip having a substrate having a hydrophilic reaction area and a spot of a substance to be immobilized containing a biological substance disposed in the reaction area, wherein the reaction area is surrounded by a boundary capable of retaining a liquid inside thereof, and the spot further contains a thickener and / or a surfactant. [1-6] The biochip according to [1-5], wherein the substance to be immobilized is immobilized on the substrate via a coating layer that retains the substance to be immobilized. [1-7] The biochip according to [1-6], wherein the coating layer is made of a water-soluble polymer. [1-8] The biochip according to [1-7], wherein the water-soluble polymer is polyethylene glycol methacrylate.

[0017] [1-9] The biochip according to any one of [1-3] to [1-8], wherein the substance to be immobilized is immobilized on the reaction area by a photocrosslinking agent having at least two photoreactive groups in one molecule. [1-10] The biochip according to any one of [1-3] to [1-9], wherein the substance to be immobilized is a peptide, nucleic acid, sugar chain, or a mixture of one or more selected therefrom. [1-11] The biochip according to any one of [1-3] to [1-10], wherein the immobilized substance is immobilized on the immobilization carrier, and the immobilization carrier on which the immobilized substance is immobilized is fixed in the reaction area.

[0018] [1-12] The biochip according to any one of [1-4] to [1-11], wherein the thickener is gellan gum, xanthan gum, curdlan, pullulan, guar gum derivative, locust bean gum, carrageenan, pectin, tamarind gum, psyllium seed gum, dextran, glycerin, carboxymethyl cellulose, hydroxyethyl cellulose, hydroxymethylpropyl cellulose, lanolin, methyl cellulose, petrolatum, polyethylene glycol, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, polyvinyl pyrrolidone, polyvinyl alcohol, dextrin phosphate fatty ester, inulin phosphate fatty ester, or a mixture of two or more selected therefrom. [1-13] The biochip according to any one of [1-4] to [1-12], wherein the surfactant is Triton X-100, Tween 20, Tween 80, or a mixture of two or more selected therefrom. [1-14] The biochip according to any one of [1-3] to [1-13], wherein the substrate is a resin material of any one of polystyrene, polypropylene, polycarbonate, and acrylic resin.

[0019] [1-15] A step of hydrophilizing the resin surface of the biochip substrate The method for manufacturing a biochip substrate according to [1-1] or [1-2], comprising the above step. [1-16] The manufacturing method according to [1-15], wherein the hydrophilization treatment is selected from UV-ozone treatment, plasma treatment, corona treatment, and flame treatment. [1-17] The manufacturing method according to [1-15] or [1-16], further comprising a step of forming a boundary surrounding the reaction area. [1-18] A step of attaching, as grid-like spots, a liquid containing a substance to be immobilized, a photo-crosslinking agent having at least two photoreactive groups in one molecule, a thickening agent and / or a surfactant, to the reaction area of the substrate for a biochip described in [1-1] or [1-2]; A step of immobilizing the substance to be immobilized attached to the reaction area; A method for manufacturing a biochip according to any one of [1-3] to [1-4], [1-9] to [1-14], which includes the above.

[0020] [1-19] A method for manufacturing a biochip according to any one of [1-5] to [1-14], which includes: A step of forming a hydrophilic reaction area; A step of forming spots of the substance to be immobilized containing a biological substance in the reaction area; The method, wherein the spots contain a thickening agent and / or a surfactant. [1-20] The step of forming the spots includes: A step of attaching, as grid-like spots on the substrate, a liquid containing a substance to be immobilized, a photo-crosslinking agent having at least two photoreactive groups in one molecule, a thickening agent and / or a surfactant; A step of immobilizing the substance to be immobilized attached to the substrate; The manufacturing method according to [1-19], which includes the above.

[0021] [1-21] A package obtained by packaging the biochip according to any one of [1-3] to [1-14] in at least one of the following modes (a) to (d): (a) Vacuum packaging; (b) Packaging together with an inert gas; (c) Packaging together with an oxygen scavenger; and (d) Packaging with a packaging material having an oxygen scavenging function. [1-22] The package according to [1-21], in which a desiccant is enclosed.

[0022] In another aspect, the present invention is as follows, for example. [2-1] It has a substrate having a reaction area and a spot of an immobilized substance containing a biological substance disposed in the reaction area. The reaction area is surrounded by a boundary capable of retaining a liquid inside it. The biochip in which the immobilized substance is fixed on the substrate via a coat layer that retains the immobilized substance. [2-2] The biochip according to [2-1], wherein the spot further contains a thickening agent and / or a surfactant, and the immobilized substance is fixed on the substrate by a photocrosslinking agent having at least two photoreactive groups in one molecule. [2-3] The biochip according to [2-1] or [2-2], wherein the immobilized substance is a peptide, a nucleic acid, a sugar chain, or a mixture of one or more selected from these. [2-4] The biochip according to any one of [2-1] to [2-3], wherein the immobilized substance is immobilized on an immobilization carrier, and the immobilization carrier on which the immobilized substance is immobilized is fixed on the substrate.

[0023] [2-5] The thickening agent is gellan gum, xanthan gum, curdlan, pullulan, guar gum derivative, locust bean gum, carrageenan, pectin, tamarind gum, psyllium seed gum, dextran, glycerin, carboxymethyl cellulose, hydroxyethyl cellulose, hydroxymethylpropyl cellulose, lanolin, methyl cellulose, petrolatum, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, polyvinylpyrrolidone, polyvinyl alcohol, fatty acid ester of dextrin phosphate, fatty acid ester of inulin phosphate, or a mixture of two or more selected from these. The biochip according to any one of [2-2] to [2-4]. [2-6] The biochip according to any one of [2-2] to [2-5], wherein the surfactant is Triton X-100, Tween 20, Tween 80, or a mixture of two or more selected from these. The biochip according to any one of [2-1] to [2-6], wherein the coat layer is made of a water-soluble polymer. The biochip according to [2-7], wherein the water-soluble polymer is polyethylene glycol methacrylate.

[0024] The biochip according to any one of [2-1] to [2-8], wherein the substrate is formed of a light-transmissive material. The biochip according to [2-9], wherein the substrate is a resin material of any one of polystyrene, polypropylene, polycarbonate, and acrylic resin. A method for manufacturing the biochip according to any one of [2-1] to [2-10], comprising: a step of forming the reaction area; a step of providing a coat layer for holding the substance to be immobilized on the reaction area on the substrate; a step of forming spots of the substance to be immobilized containing a biological substance on the coat layer; and a method having the above steps. The manufacturing method according to [2-11], wherein the step of forming the spots includes: adhering a liquid containing a substance to be immobilized, a photo-crosslinking agent having at least two photo-reactive groups in one molecule, and a thickener and / or a surfactant as lattice-shaped spots on the substrate; a step of immobilizing the substance to be immobilized adhered on the substrate; and a method including the above steps.

[0025] A package obtained by packaging the biochip according to any one of [2-1] to [2-10] in at least one of the following modes (a) to (d): (a) Vacuum packaging; (b) Packaging together with an inert gas; (c) Packaging together with an oxygen scavenger; and (d) Packaging with a packaging material having an oxygen scavenging function. The package according to [2-13], further enclosed with a desiccant. [2-15] A method for storing a biochip, comprising the step of storing the biochip according to any one of [2-1] to [2-10] in at least one of the following modes (a) to (d). (a) After being put into a package, the inside of the package is evacuated and sealed for storage. (b) Put into a package, replaced with an inert gas and sealed for storage. (c) Sealed in a package together with an oxygen scavenger for storage, and (d) Packaged with a packaging material having an oxygen scavenging function for storage. [2-16] The storage method according to [2-15], wherein the biochip is stored together with a desiccant.

[0026] In another aspect, the present invention is as follows, for example. [3-1] A substrate for a biochip having a resin surface including a hydrophilic reaction area. [3-2] The substrate for a biochip according to [3-1], wherein the reaction area is surrounded by a boundary capable of retaining a liquid in the reaction area. [3-3] The substrate for a biochip according to [3-1] or [3-2], which is formed of a light-transmitting material. [3-4] The substrate for a biochip according to any one of [3-1] to [3-3], wherein the bond related to carbon of the resin in the reaction area is cleaved, and the cleaved portion is covered with a polar functional group formed by bonding with oxygen. [3-5] A biochip in which a substance to be immobilized containing a biological substance is immobilized on the reaction area of the substrate for a biochip according to any one of [3-1] to [3-4]. [3-6] The biochip according to [3-5], wherein the substance to be immobilized is immobilized on the reaction area by a photo-crosslinking agent. [3-7] The biochip according to [3-5] or [3-6], wherein the substance to be immobilized is present in a spot containing a thickening agent and / or a surfactant present in the reaction area. [3-8] The biochip according to any one of [3-5] to [3-7], wherein the substance to be immobilized is immobilized on an immobilization carrier.

[0027] Step of hydrophilizing the surface of the substrate for a biochip according to any one of [3-9] [3-1] to [3-4] A method for manufacturing a substrate for a biochip, comprising: [3-10] The manufacturing method according to [3-9], wherein the hydrophilization treatment is selected from UV-ozone treatment, plasma treatment, corona treatment, and flame treatment. [3-11] The manufacturing method according to [3-9] or [3-10], further comprising a step of forming a boundary surrounding the reaction area. [3-12] A step of adhering, as a lattice-shaped spot, a liquid containing a substance to be immobilized, a photo-crosslinking agent having at least two photoreactive groups in one molecule, a thickening agent and / or a surfactant, to the reaction area of the substrate for a biochip according to any one of [3-1] to [3-4]; A step of immobilizing the substance to be immobilized adhered to the reaction area; A method for manufacturing a biochip according to any one of [3-5] to [3-8], comprising:

[0028] [3-13] A package obtained by packaging the biochip according to any one of [3-5] to [3-8] in at least one of the following modes (a) to (d): (a) Vacuum packaging; (b) Packaging together with an inert gas; (c) Packaging together with an oxygen scavenger; and (d) Packaging with a packaging material having an oxygen scavenging function. [3-14] The package according to [3-13], in which a desiccant is enclosed. [3-15] A method for storing a biochip, comprising a step of storing the biochip according to any one of [3-5] to [3-8] in at least one of the following modes (a) to (d): (a) After being put into a package, evacuating the inside of the package and sealing it for storage; (b) Putting it into a package, replacing it with an inert gas, and sealing it for storage; (c) Enclosing it in a package together with an oxygen scavenger for storage; and ​(d) It is packaged and stored with a packaging material having an oxygen removal function. [3-16] The storage method according to [3-15], wherein the biochip is stored together with a desiccant.

Advantages of the Invention

[0029] The biochip of the present invention has one or more of the following effects. (1) Analysis is possible with a small amount of test sample. (2) The binding site of the immobilized substance can be controlled. (3) The uniformity of the distribution of the immobilized substance within the spot is high. (4) The S / N ratio is excellent. (5) The detection sensitivity is excellent. (6) High-speed analysis is possible. (7) It is possible to immobilize the immobilized substance without coating a coating layer such as a polymer. (8) It has excellent luminescence intensity. (9) Nonspecific adsorption of the substance to be detected can be suppressed. (10) It has high sensitivity. (11) It can be stored at room temperature. (12) The manufacturing process is simple. (13) The analysis cost is low. (14) It is easy to handle. (15) It can be applied to point-of-care test analysis.

Brief Description of the Drawings

[0030]

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[0031]

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Mode for Carrying Out the Invention

[0032] Hereinafter, embodiments of the present invention will be described with reference to the drawings. In each figure, components that exhibit the same or similar functions are denoted by the same reference numerals, and redundant explanations are omitted. Also, the materials, configurations, etc. described below do not limit the present invention and can be variously modified within the scope of the gist of the present invention. All documents, published gazettes, patent gazettes, and other patent documents cited in this specification are hereby incorporated herein by reference. Further, this specification includes the contents described in the specifications and drawings of two Japanese patent applications (Japanese Patent Application No. 2017-172453 and Japanese Patent Application No. 2017-172454) that form the basis of the priority claim of this application filed on September 7, 2017.

[0033] 1. Biochip The biochip of the present invention is composed of at least a substrate for a biochip and a substance to be immobilized, and has a reaction area on the upper surface of the substrate for biochemically reacting a test sample. One aspect of the biochip of the present invention is a biochip in which a substance to be immobilized containing a biological substance is immobilized in the reaction area of a substrate for a biochip having a surface including a hydrophilic reaction area, preferably a resin-made surface including a hydrophilic reaction area. Another aspect of the biochip of the present invention has a reaction area, preferably a substrate having a hydrophilic reaction area, and a spot of a substance to be immobilized containing a biological substance disposed in the reaction area, and the reaction area is surrounded by a boundary capable of holding a liquid inside. The test sample to which the biochip of the present invention can be applied is not particularly limited, and examples thereof include body fluids such as blood, plasma, serum, saliva, urine, lymph fluid, cerebrospinal fluid, synovial fluid, nasal discharge, ascites, aqueous humor, and tears, which are targets for allergy and tumor diagnosis, sputum, and biopsy samples.

[0034] As the substrate for the biochip of the present invention, there is no particular limitation as long as it does not have an excessive adverse effect on the test sample or biochemical reaction. For example, a resin material or the like can be used. As the resin material, without limitation, for example, a thermosetting resin or a thermoplastic resin can be used. In particular, by using a light-transmissive resin material such as polypropylene, polycarbonate, acrylic, polystyrene, polyethylene terephthalate, cycloolefin polymer, cycloolefin copolymer, etc., good visible light transmittance can be ensured. As polypropylene, without limitation, for example, homopolypropylene or a random copolymer of polypropylene and polyethylene can be used. Also, as acrylic, without limitation, for example, polymethyl methacrylate or a copolymer of methyl methacrylate and other monomers such as other methacrylic acid esters, acrylic acid esters, styrene, etc. can be used. In the present invention, "transparent" and "light-transmissive" mean that the average transmittance in the wavelength region of the detection light is 70% or more. If a material of a light-transmissive material is used in the visible light region (wavelength 350 to 780 nm), it is easy to visually recognize the sample state in the chip, but it is not limited to this. The thickness of the substrate is not particularly limited, but since it is desirable to have a certain degree of non-deformability in the manufacturing process, 0.3 mm to 3.0 mm is preferable, 0.5 mm to 1.5 mm is more preferable, and 0.7 mm to 1.0 mm is particularly preferable.

[0035] In some embodiments, the resin material used for the substrate of the biochip of the present invention is hydrophobic. In some embodiments, the resin material is formed from a water-insoluble polymer. In some embodiments, the resin material does not contain dextran, polyethylene glycol or its derivatives.

[0036] The substrate can be subjected to a hydrophilic treatment. By the hydrophilic treatment, non-specific adsorption to the substrate can be prevented, and the substrate and the substance to be immobilized can be firmly immobilized. As used herein, immobilization means that a strong chemical bond such as a covalent bond is formed between the substance to be immobilized and the substrate surface, and is distinguished from a weak chemical bond (also referred to as chemisorption) such as a hydrogen bond or physical adsorption due to van der Waals forces. Further, as used herein, non-specific adsorption means that the substance to be immobilized and / or a substance that specifically reacts with the substance to be immobilized adsorbs to a region on the substrate surface where the substance to be immobilized is not immobilized. Specific examples of the substance that specifically reacts with the substance to be immobilized include, for example, an antibody when the substance to be immobilized is an antigen, and an antigen when the substance to be immobilized is an antibody.

[0037] The hydrophilic treatment may be performed on one or a plurality of portions of the substrate surface, or on the entire substrate surface. When performing the hydrophilic treatment on a portion of the substrate surface, for example, it may be performed on the entire upper surface of the substrate or on one or a plurality of portions of the upper surface of the substrate. The area to be hydrophilized may be 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, etc. of the total surface area of the substrate or the total surface area of the upper surface of the substrate. Further, the portion to be subjected to the hydrophilic treatment may have various shapes. For example, the portion to be subjected to the hydrophilic treatment may have a shape such as a circle, an ellipse, a polygon, etc., whereby, for example, it is possible to form a plurality of circular hydrophilic portions on the substrate surface in a spot shape.

[0038] The hydrophilic treatment is not particularly limited, and examples thereof include coating the surface with an inorganic material such as silica having hydrophilicity or a surfactant, chemical hydrophilic treatments such as plasma treatment, UV-ozone treatment, corona treatment, and flame treatment, and imparting hydrophilicity by physically forming a fine nanostructure (WO2011 / 024947). When the substrate is made of resin, chemical hydrophilic treatments such as plasma treatment, UV-ozone treatment, corona treatment, and flame treatment that can break the chemical bonds of the molecules on the resin surface and generate polar functional groups according to the type of resin, such as OH (hydroxyl group), CO (carbonyl group), COOH (carboxyl group), methoxy group, peroxide group, polar ether group, etc., are more preferable, and UV-ozone treatment is particularly preferable. Further, in one aspect, the hydrophilic treatment in the present invention does not involve the formation of a coating layer with a polymer, particularly a water-soluble polymer. Therefore, the substrate in this aspect has a hydrophilic surface but does not have a coating layer. In the present invention, "hydrophilic" means that the contact angle (θ / 2 method) measured by the static contact angle measurement method (liquid application method) is 1° or more and less than 80°, preferably 5° or more and less than 75°, and more preferably 10° or more and less than 70°.

[0039] In one aspect, the substrate for the biochip of the present invention has a surface including a hydrophilic reaction area, preferably a resin surface including a hydrophilic reaction area. The reaction area means an area on the substrate where a substance to be immobilized is immobilized and reacted with a substance to be detected. The reaction area may be part or the whole of the substrate surface. When the reaction area is part of the substrate surface, the substrate surface other than the reaction area may be hydrophilic or hydrophobic. When the reaction area is part of the substrate surface, the reaction area may have various shapes, for example, circular, elliptical, polygonal or a combination thereof. The reaction area may be continuous or discontinuous. For example, the reaction area may form a plurality of spots that do not contact each other. The reaction area may be surrounded by a boundary capable of holding a liquid inside. Further, the reaction area may be covered with a polar functional group generated by breaking the carbon-related bond of the resin forming the substrate surface and bonding the broken portion to oxygen. Such functional groups include, but are not limited to, for example, hydroxyl group, carbonyl group, carboxyl group, methoxy group, peroxide group, polar ether group and the like.

[0040] A coating layer for preventing non-specific adsorption and immobilizing a substance to be immobilized can be provided on the substrate. By immobilizing a substance on the substrate through the coating layer, non-specific adsorption can be prevented and the detection sensitivity can be enhanced. Further, by the method of immobilizing a substance to be immobilized on the substrate through the coating layer, compared with the method of mixing a polymer, a substance to be immobilized and a photo-crosslinking agent and applying them on the substrate, since the amount of the substance to be immobilized exposed on the outermost layer is large, a substrate for a biochip with excellent S / N ratio and detection sensitivity can be obtained.

[0041] The coating layer is not particularly limited as long as it can promote the immobilization of the substance to be immobilized and / or suppress non-specific adsorption. For example, it can be composed of various polymers. Among the polymers, water-soluble polymers are preferred. By using a water-soluble polymer, it is possible to avoid the use of non-aqueous solvents other than water and alcohol that may denature the substance to be immobilized. Therefore, in the present invention, it is preferable to use a water-soluble polymer to prevent the denaturation of the substance to be immobilized. Furthermore, water-soluble polymers have the advantage of being excellent in the non-specific adsorption suppression effect. Here, "water-soluble" means, for example, that the solubility of the polymer in water (the number of grams dissolved in 100 g of water) is 5 or more.

[0042] The number average molecular weight of the polymer is not particularly limited and is usually about 350,000 to 5 million. By setting the molecular weight of the polymer to about 500,000 to several hundred thousand, the number of cross-links between the polymers can be appropriately maintained, and the reaction between the substance to be immobilized and the substance (such as a photo-crosslinking agent) used in the reaction with the substance to be immobilized can proceed.

[0043] Examples of the water-soluble polymer include amphiphilic polymers such as phosphorylcholine-containing polymers and nonionic polymers. Examples of the nonionic polymer include polyalkylene glycols such as polyethylene glycol (PEG) and polypropylene glycol; monomers such as vinyl alcohol, methyl vinyl ether, vinyl pyrrolidone, vinyl oxazolidone, vinyl methyl oxazolidone, 2-vinyl pyridine, 4-vinyl pyridine, N-vinyl succinimide, N-vinyl formamide, N-vinyl-N-methyl formamide, N-vinyl acetamide, N-vinyl-N-methyl acetamide, 2-hydroxyethyl methacrylate, polyethylene glycol methacrylate, polyethylene glycol acrylate, acrylamide, methacrylamide, N,N-dimethyl acrylamide, N-isopropyl acrylamide, diacetone acrylamide, methylol acrylamide, acryloyl morpholine, acryloyl pyrrolidine, acryloyl piperidine, styrene, chloromethyl styrene, bromomethyl styrene, vinyl acetate, methyl methacrylate, butyl acrylate, methyl cyanoacrylate, ethyl cyanoacrylate, n-propyl cyanoacrylate, isopropyl cyanoacrylate, n-butyl cyanoacrylate, isobutyl cyanoacrylate, tert-butyl cyanoacrylate, glycidyl methacrylate, ethyl vinyl ether, n-propyl vinyl ether, isopropyl vinyl ether, n-butyl vinyl ether, isobutyl vinyl ether, tert-butyl vinyl ether, etc., and nonionic vinyl polymers composed of a single monomer unit or a mixture thereof; natural polymers such as gelatin, casein, collagen, gum arabic, xanthan gum, tragacanth gum, guar gum, pullulan, pectin, sodium alginate, hyaluronic acid, chitosan, chitin derivatives, carrageenan, starches (carboxymethyl starch, aldehyde starch), dextrin, cyclodextrin, etc., and natural polymers such as water-soluble cellulose derivatives such as methyl cellulose, viscose, hydroxyethyl cellulose, hydroxyethyl methyl cellulose, carboxymethyl cellulose, hydroxypropyl cellulose, etc., but are not limited thereto.In addition, it is also possible to use commercially available photocrosslinkable water-soluble polymers based on these polymers, such as "BIOSURFINE-AWP" manufactured by Toyo Gosei Co., Ltd. based on polyvinyl alcohol. Among these, particularly preferred are polyethylene glycol-based polymers, and more preferably vinyl polymers of polyethylene glycol (meth)acrylate.

[0044] The substrate can be surface-treated to enhance the adhesion between the coating layer and the substrate. The surface treatment is not particularly limited. For example, it can be a treatment that cleaves the chemical bonds of the molecules on the resin surface of the substrate and generates hydrophilic functional groups such as OH (hydroxyl group), CO (carbonyl group), COOH (carboxyl group), methoxy group, peroxide group, polar ether group, etc. according to the type of resin. Examples include plasma treatment, UV-ozone treatment, corona treatment, and flame treatment.

[0045] The biochip of the present invention is composed of at least the above substrate and the immobilized substance, and has a reaction area on the upper surface of the substrate for subjecting the test sample to a biochemical reaction. The immobilized substance includes biological substances, and examples of the biological substances include peptides, nucleic acids, sugar chains, or a mixture of one or more selected from these. Here, the peptide is a general term for substances in which two or more amino acids are peptide-bonded. Peptides include oligopeptides and polypeptides, and polypeptides include proteins.

[0046] The biological substances are not particularly limited, and examples thereof include food allergens such as eggs, milk, meat, fish, crustaceans, mollusks, cereals, beans, nuts, fruits, vegetables, brewer's yeast, and gelatin, non-food allergens such as pollen, mites, and house dust, and tumor markers such as liver cancer and breast cancer markers. Among them, αs1-casein, αs2-casein, β-casein, κ-casein, α-lactalbumin, β-lactoglobulin as the main components of milk allergens, ovomucoid, ovalbumin, conalbumin as the main components of egg white allergens, gliadin as the main component of wheat allergens, proteins with molecular weights of 24 kDa and 76 kDa which are the main proteins of buckwheat, and Ara h1 which is the main protein of peanuts can be specifically exemplified. In particular, it is desirable to use at least one or more allergens selected from sodium caseinate, α-casein, β-casein, κ-casein, α-lactalbumin, β-lactoglobulin, ovomucoid, ovalbumin, and conalbumin. The biological substances also include antibodies against various allergens, biological substances (antigens, antibodies, aptamers, lectins, polynucleotides, enzymes, substrates, etc.) that react with various biomarkers (for example, tumor markers, infectious disease markers, genetic disease markers, endocrine disease markers, inflammatory markers, fatigue markers, stress markers, nutritional markers), antigens related to pathogens and autoimmune diseases, etc.

[0047] The immobilization carrier is not particularly limited, and examples thereof include particulate carriers such as organic fine particles and inorganic fine particles. The particulate carrier may be magnetic fine particles. Non-limiting examples of magnetic fine particles include beads with a uniform particle size covered with a polymer having a uniformly hydrophilic property (for example, a water-soluble polymer such as glycidyl methacrylate) of a magnetizable substance such as γFe2O3 and Fe3O4. Commercially available products include Dynabeads manufactured by Thermo Fisher Scientific, FG beads manufactured by Tama Seiki Co., Ltd., Sera-Mag magnetic beads manufactured by GE Healthcare, and Magnosphere manufactured by JSR Life Sciences.

[0048] (a) in Fig. 1 is a plan view showing one aspect of the biochip of the present invention (hereinafter sometimes referred to as biochip (A)). In the biochip (A) 10, a ring-shaped boundary 102 is formed on the upper surface 101A of the substrate 101. The boundary 102 is formed in an annular shape with a preset width and protrudes from the substrate 101 at a predetermined height, forming a reaction area 103 for holding a liquid in the space surrounded by the upper surface 101A of the substrate 101 and the boundary 102. Although a ring-shaped boundary is illustrated, it is not limited thereto, and it can have various shapes such as an ellipse or a polygon (for example, a quadrilateral, a pentagon, a hexagon, an octagon). In the reaction area 103, spots 104 to which components (substances to be immobilized) that react with the components of the test sample are immobilized are arranged. A plurality of the spots 104 can be arranged, and they are arranged as a plurality of independent spots at preset intervals at preset positions.

[0049] (b) in Fig. 1 is a cross-sectional view showing the cross-sectional structure of the biochip (A) 10 cut along the line A-A' (see (a) in Fig. 1). The lower limit of the height of the boundary 102 from the upper surface 101A of the substrate 101 is not particularly limited, but it is preferably 1 mm or more, and more preferably 2 mm or more. The upper limit of the height of the boundary 102 from the upper surface 101A of the substrate 101 is also not particularly limited, but considering the use as a chip, it is preferably 10 mm or less. The width of the boundary 102 is not particularly limited, but considering the structural stability and the size of the chip, it is preferably in the range of 0.5 mm to 5.0 mm, and more preferably in the range of 1.0 mm to 3.0 mm. The material of the boundary 102 is not particularly limited as long as it does not affect the test sample or biochemical reactions, and it can be appropriately selected from rubber compositions such as natural rubber, synthetic rubber, silicone rubber, fluororubber, urethane rubber, and resin materials that can be used as the substrate.

[0050] At spot 104, the substance to be immobilized or the immobilization carrier on which the substance to be immobilized is immobilized is fixed. The number and arrangement of spots 104 are not particularly limited, and any number and arrangement are possible. Furthermore, from the viewpoint of simultaneous multi-item inspection, the lower limit of the number of spots 104 is preferably 3 or more, more preferably 12 or more, and particularly preferably 18 or more. Also, due to limitations in the size of the reaction area, in order to prevent the signals of the spots from overlapping with those in the vicinity during detection, the upper limit of the number of spots 104 is preferably 168 or less, and more preferably 144 or less.

[0051] (a) in Fig. 2 is a plan view showing another mode of the biochip of the present invention (hereinafter sometimes referred to as biochip (B)). Biochip (B) 11 is different from biochip (A) in that there is no boundary and the entire upper surface 101A of the substrate 101 serves as the reaction area 103, but the other configurations are the same as those of biochip (A). Since biochip (B) does not require the installation of a boundary, it has the advantages of a simple manufacturing process and low manufacturing cost.

[0052] (a) in Fig. 3 is a plan view showing one mode of the biochip of the present invention (hereinafter sometimes referred to as biochip (C)). In biochip (C) 12, a ring-shaped boundary 102 is formed on the upper surface 101A of the substrate 101. The boundary 102 is formed in an annular shape with a preset width and protrudes from the substrate 101 at a predetermined height, forming a reaction area 103 for holding liquid within the space surrounded by the upper surface 101A of the substrate 101 and the boundary 102. Although the boundary is shown as ring-shaped, it is not limited thereto and can have various shapes such as elliptical or polygonal (e.g., quadrilateral, pentagon, hexagon, octagon). A coat layer 104 for preventing non-specific adsorption and immobilizing biological substances is formed in the reaction area 103, and a plurality of spots 105 on which components (substances to be immobilized) that react with the components of the test sample are immobilized are arranged via the coat layer 104. The spots 105 are arranged as a plurality of independent spots on the coat layer 104 at preset positions with a preset interval.

[0053] In Fig. 3, (b) is a cross-sectional view showing the cross-sectional structure obtained by cutting the biochip (C) 12 along the line A-A' (see (a) in Fig. 3). The lower limit of the height of the boundary 102 from the upper surface 101A of the substrate 101 is not particularly limited, but it is preferably 1 mm or more, and more preferably 2 mm or more. The upper limit of the height of the boundary 102 from the upper surface 101A of the substrate 101 is also not particularly limited, but considering the use as a chip, it is preferably 10 mm or less. The width of the boundary 102 is not particularly limited, but considering the structural stability and the size of the chip, it is preferably in the range of 0.5 mm to 5.0 mm, and more preferably in the range of 1.0 mm to 3.0 mm. The material of the boundary 102 is not particularly limited as long as it does not affect the test sample or biochemical reaction, and it can be appropriately selected from rubber compositions such as natural rubber, synthetic rubber, silicone rubber, fluororubber, urethane rubber, and resin materials that can be used as the substrate.

[0054] The thickness of the coating layer 104 is not particularly limited and can be formed to any thickness, but it is preferably 1 nm to 10 μm, more preferably 2 nm to 1 μm, and particularly preferably 10 nm to 100 nm.

[0055] The immobilized substance or the immobilization carrier on which the immobilized substance is immobilized is fixed to the spot 105. The number and arrangement of the spots 105 are not particularly limited, and any number and arrangement are possible. Then, from the point of simultaneous multi-item inspection, the lower limit of the number of the spots 105 is preferably 3 or more, more preferably 12 or more, and particularly preferably 18 or more. Also, due to the limitation of the size of the reaction area and to prevent the signals of the spots from overlapping with those in the vicinity during detection, the upper limit of the number of the spots 105 is preferably 168 or less, and more preferably 144 or less.

[0056] Fig. 4(a) is a plan view showing another aspect of the biochip of the present invention (hereinafter sometimes referred to as biochip (D)). The biochip (D) 13 is different from the biochip (C) in that a coating layer 104 for preventing non-specific adsorption and immobilizing biological substances is formed on the entire upper surface of the substrate 101. Therefore, the ring-shaped boundary 102 is formed on the coating layer 104. The boundary 102 is formed in an annular shape with a preset width and protrudes from the coating layer 104 at a predetermined height, forming a reaction area 103 for holding a liquid in the space surrounded by the coating layer 104 and the boundary 102. Although the cross-section of the boundary 102 in this aspect is kamaboko-shaped, the cross-sectional shape of the boundary 102 is not particularly limited as long as it can hold a liquid in the space surrounded by the boundary 102, and it can be various shapes such as a rectangle like biochip A, a triangle, a trapezoid, an L-shape, etc. On the coating layer 104 of the reaction area 103, a plurality of spots 105 on which components (substances to be immobilized) that react with the components of the test sample are immobilized are arranged. The spots 105 are arranged as a plurality of independent spots on the coating layer 104 at preset positions with a preset interval.

[0057] Fig. 4(b) is a cross-sectional view showing the cross-sectional structure of the biochip (D) 13 cut along the line A - A' (see Fig. 4(a)). The height range of the boundary 102 from the coating layer 104 is the same as the height range of the boundary 102 from the upper surface 101A of the substrate 101 in the biochip (C). Also, the width, material, thickness of the coating layer 104, and the spots 105 are the same as those in the biochip (C).

[0058] 2. Manufacturing method of biochip The manufacturing of the biochip of the present invention including a hydrophilic reaction area manufactures a substrate for the biochip by a step of hydrophilizing the substrate (hereinafter referred to as "step 1 - 1"), and then, A step of spotting at least a substance to be immobilized, a photocrosslinking agent having at least two photoreactive groups in one molecule, a thickening agent and / or a surfactant on the substrate for the biochip and irradiating with light (hereinafter referred to as "Step 1-2"). It may be included.

[0059] Hereinafter, each component of the production method of the present invention will be described in order. Step 1-1 In Step 1-1, it may include a step of hydrophilizing the substrate. The hydrophilization treatment of the substrate is not particularly limited as long as it is a treatment for hydrophilizing the surface of the substrate. For example, a method of coating silica, a surfactant, etc. dissolved or suspended in a liquid on the substrate by spin coating, coating, spraying, dipping, etc. and then drying, a method of chemically hydrophilizing the substrate by plasma treatment, UV-ozone treatment, corona treatment, frame treatment, etc., a method of transferring a fine nanostructure to the substrate or roughening the substrate with a chemical solution to physically form a nanostructure to impart hydrophilicity, a method of coating the substrate with a hydrophilic polymer, etc. can be mentioned. A method of chemically hydrophilizing by plasma treatment, UV-ozone treatment, corona treatment, frame treatment, etc. that can break the chemical bonds of the molecules on the resin surface and generate functional groups OH (hydroxyl group), CO (carbonyl group), COOH (carboxyl group), methoxy group, peroxide group, polar ether group, etc. having hydrophilic polarity according to the type of resin is more preferable, and that by UV-ozone treatment is particularly preferable.

[0060] A step of forming a boundary on the substrate to form a reaction area can be provided. The method of forming the reaction area is not particularly limited, but a method of adhering a ring previously formed by the rubber composition or the resin material to the substrate using an adhesive, etc., various resin molding methods such as injection molding and vacuum molding of the substrate composed of the resin material, or a method of molding a ring by machining cutting, etc. can be used. The adhesive is not particularly limited as long as it does not affect the test sample or biochemical reaction, and can be appropriately selected from commercially available adhesives.

[0061] Step 1-2 In Step 1-2, a step of forming lattice-shaped spots of at least a substance to be immobilized, a photo-crosslinking agent having at least two photoreactive groups in one molecule, a thickening agent and / or a surfactant on the substrate for the biochip, and a step of irradiating light may be included.

[0062] The production of the biochip of the present invention having a coating layer and a reaction area surrounded by a boundary capable of holding a liquid inside it includes a step of providing a coating layer for preventing non-specific adsorption and immobilizing a biological substance on the substrate, and a step of forming a boundary on the substrate to provide a reaction area. The substrate for the biochip is produced by a step selected therefrom (hereinafter referred to as "Step 2-1"), and then, a step of spotting at least a substance to be immobilized, a photo-crosslinking agent having at least two photoreactive groups in one molecule, a thickening agent and / or a surfactant on the substrate for the biochip and irradiating light (hereinafter referred to as "Step 2-2"), may be included.

[0063] Hereinafter, each component of the method for producing the biochip of the present invention having a coating layer and a reaction area surrounded by a boundary capable of holding a liquid inside it will be described in order. Step 2-1 In Step 2-1, a step selected from a step of providing a coating layer for preventing non-specific adsorption and immobilizing a biological substance on the substrate and a step of forming a boundary on the substrate to provide a reaction area may be included. If these steps selected from the above are included, the order of the steps can be arbitrarily changed. Further, a step of surface-treating the substrate may be included before the step of providing the coating layer.

[0064] The method of surface treatment in the step of surface-treating the substrate is not particularly limited as long as it improves the adhesion between the substrate and the coating layer. For example, known surface modification methods such as plasma treatment, UV ozone treatment, and corona treatment can be mentioned.

[0065] To prevent non-specific adsorption to the substrate and form a coating layer for immobilizing biological substances, it can be carried out by known methods such as spin coating, coating, spraying, dipping into the coating solution, etc. of a coating solution containing a water-soluble polymer. For example, the coating solution can be prepared by dissolving a water-soluble polymer in a solvent. As the solvent, water, lower alcohols that are miscible with water in any ratio, and mixtures thereof can be used. As the lower alcohol, methanol, ethanol, and isopropanol are preferred. Among them, it is preferable to use a mixed solvent of ethanol and water as the solvent.

[0066] The concentration of the polymer in the coating solution is not particularly limited, but the concentration of the polymer can be, for example, 0.0001 to 10 parts by mass, preferably 0.001 to 1 part by mass, and the concentration of the photo-crosslinking agent can be, for example, 1 to 20 parts by mass, preferably 2 to 10 parts by mass with respect to the polymer.

[0067] The coating solution containing the polymer preferably contains a photo-crosslinking agent having at least two photo-reactive groups in one molecule. In the present invention, the "photo-reactive group" means a group that generates radicals by irradiation with light. The photo-crosslinking agent can form a covalent bond with an amino group, a carboxyl group, a carbon atom constituting an organic compound, etc. by the photo-reactive group generating radicals upon light irradiation. Thereby, after applying the coating solution containing the photo-crosslinking agent onto the substrate and irradiating with light, the substrate and the polymer can be bonded via the photo-crosslinking agent, and a polymer layer having an effect of preventing non-specific adsorption can be formed on the substrate. In the present invention, without using the photo-crosslinking agent, or by introducing a photo-reactive group and / or a group capable of forming a covalent bond or a coordination bond with the substrate surface into the polymer together with the photo-crosslinking agent, it is also possible to bond the substrate and the polymer by utilizing the groups possessed by the polymer.

[0068] The water-soluble polymer and photo-crosslinking agent contained in the coating layer of the present invention are known per se, can be produced by known production methods, and are also commercially available. There is no particular limitation on the film thickness of the coating layer, but it is preferably 1 nm to 10 μm, more preferably 2 nm to 1 μm, and particularly preferably 10 nm to 100 nm.

[0069] In the present invention, after coating the coating layer as described above, it is preferable to stabilize the coating layer by aging under constant temperature and humidity conditions. The temperature is preferably 5°C to 40°C, more preferably 20°C to 30°C. The humidity is preferably 40% to 80%, more preferably 50% to 70%. The aging period is preferably 1 day to 1 month, more preferably 3 days to 2 weeks.

[0070] The method for forming the boundary on the substrate is not particularly limited, but a method of adhering a ring pre-formed from the rubber composition or the resin material to the substrate using an adhesive or the like, various resin molding methods such as injection molding and vacuum molding of a substrate composed of the resin material, or a method of molding a ring by mechanical cutting or the like can be used. The adhesive is not particularly limited as long as it does not affect the test sample or biochemical reaction, and can be appropriately selected from commercially available adhesives.

[0071] Step 2-2 In Step 2-2, it may include a step of spotting at least a substance to be immobilized, a photo-crosslinking agent having at least two photo-reactive groups in one molecule, a thickening agent and / or a surfactant in a lattice pattern on the substrate for the biochip, a step of irradiating light, and a step of removing unreacted components.

[0072] In Step 1-2 or 2-2, when the substance to be immobilized is immobilized on the immobilization carrier in advance, a known method can be used as the immobilization method. As the immobilization method, for example, a method can be mentioned in which the magnetic fine particles as the immobilization carrier and the substance to be immobilized prepared in advance are reacted in a solution such as a known buffer solution appropriately selected, and the magnetic fine particles on which the substance to be immobilized is immobilized are recovered, but it is not limited thereto. For example, the magnetic fine particles may be washed in advance using a known buffer solution, or the magnetic fine particles on which the substance to be immobilized is immobilized may be washed using a known buffer solution when recovered.

[0073] The photocrosslinking agent having at least two photoreactive groups in one molecule used in Step 1-2 or 2-2 is not particularly limited. Examples of the photoreactive group of the photocrosslinking agent include an azide group (-N3), an acetyl group, a benzoyl group, a diazirine group, etc. In particular, when the azide group is irradiated with light, nitrogen molecules are released and nitrogen radicals are generated, and this nitrogen radical can bond not only to functional groups such as amino groups and carboxyl groups but also to carbon atoms constituting organic compounds, and is preferable because it can form a covalent bond with most organic substances. Examples of the photocrosslinking agent having an azide group include diazidosulylbenzene. The photocrosslinking agent is preferably water-soluble. "Water-soluble" for the photocrosslinking agent means that an aqueous solution having a concentration of 0.5 mM or more, preferably 2 mM or more, can be provided.

[0074] It is preferable that the substance to be immobilized or the immobilization carrier on which the substance to be immobilized is immobilized and the photocrosslinking agent are dispersed or dissolved in a solution. The solution is not particularly limited, but a known buffer solution can be used. Examples of the buffer solution composition include PBS buffer solution, HEPES buffer solution, Tris buffer solution, MES buffer solution, etc. When using the substance to be immobilized as it is, phosphate buffered saline is preferable, and when using the substance to be immobilized after immobilizing it on the immobilization carrier, HEPES buffer solution is preferable in order to prevent aggregation of the immobilization carrier. The solution in which the substance to be immobilized and the photocrosslinking agent are dispersed or dissolved may be referred to as a stamp solution.

[0075] The concentration of the substance to be immobilized is not particularly limited. However, when the substance to be immobilized is not immobilized on the immobilization carrier, it is preferably 0.05 mg / mL to 2 mg / mL, more preferably 0.1 mg / mL to 1 mg / mL. When the substance to be immobilized is immobilized on the immobilization carrier, it is preferable to react the immobilization carrier with the substance to be immobilized at 0.5 mg / mL to 50 mg / mL, more preferably 1 mg / mL to 25 mg / mL. It is preferable to use the immobilization carrier on which the substance to be immobilized is immobilized at 1 mg / mL to 10 mg / mL, more preferably 2.5 mg / mL to 7.5 mg / mL. The concentration of the photocrosslinking agent is not particularly limited, but is preferably 0.01 mg / mL to 1 g / mL, more preferably 0.2 mg / mL to 0.2 g / mL.

[0076] The solution in which the substance to be immobilized or the immobilization carrier on which the substance to be immobilized is immobilized and the photocrosslinking agent are dispersed or dissolved in the solution preferably further contains a thickening agent and / or a surfactant. By including a thickening agent, when spotting the solution onto the substrate for the biochip, the size of the spot can be adjusted. More specifically, the higher the concentration of the thickening agent in the solution, the smaller the size of the spot (contact area with the substrate) tends to be when spotting the same volume of solution. In addition, by including a surfactant, it is possible to suppress the accumulation of the substance to be immobilized at the gas-liquid interface and the localization of the substance to be immobilized at the edge of the spot in the spot. The surfactant also contributes to improving the affinity with the substrate, and the higher the concentration of the surfactant in the solution, the larger the size of the spot (contact area with the substrate) tends to be when spotting the same volume of solution. Therefore, by adjusting the concentrations of the thickening agent and the surfactant, a stamp of any size can be formed.

[0077] The thickener is not particularly limited as long as it does not affect the test sample or biochemical reactions, and commercially available products can be used. For example, cellulose-based and its derivatives, polysaccharides, vinyl-based compounds, vinylidene-based compounds, polyglycol-based compounds, polyvinyl alcohol-based compounds, polyalkylene oxide-based compounds, etc. can be mentioned. Specifically, gellan gum, xanthan gum, curdlan, pullulan, guar gum derivatives, locust bean gum, carrageenan, pectin, β-glucan, tamarind gum, psyllium seed gum, dextran, glycerin, carboxymethyl cellulose, hydroxyethyl cellulose, hydroxymethylpropyl cellulose, lanolin, methyl cellulose, petrolatum, polyethylene glycol, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, polyvinyl pyrrolidone, polyvinyl alcohol, dextrin phosphate fatty ester, inulin phosphate fatty ester, etc. can be used. It is preferable to use one or more selected from hydroxyethyl cellulose, methyl cellulose, polyvinyl pyrrolidone, and polyvinyl alcohol, and it is more preferable to use polyvinyl alcohol.

[0078] The concentration of the thickener is not particularly limited. For example, when using polyvinyl alcohol, 0.01 to 1 part by weight, more preferably 0.02 to 0.5 part by weight, and particularly preferably 0.03 to 0.3 part by weight are preferred with respect to 100 parts by weight of the solution.

[0079] As for the surfactant, there is no particular limitation as long as it does not affect the test sample or biochemical reactions, and commercially available nonionic surfactants can be used. Polyoxyethylene (10) octyl phenyl ether [Triton X-100], polyoxyethylene (8) octyl phenyl ether [Triton X-114], polyoxyethylene sorbitan monolaurate [Tween20], polyoxyethylene sorbitan monooleate [Tween80], polyoxyethylene (23) lauryl ether [Brij35], polyoxyethylene (20) lauryl ether [Brij58], Pluronic F-68, polyethylene glycol, etc., or a mixture of two or more of these can be used. It is preferable to use one or more selected from Triton X-100, Tween20, and Tween80, and it is more preferable to use Tween20.

[0080] The concentration of the surfactant is not particularly limited. For example, when using Tween20, 0.001 part by weight to 1 part by weight is preferable, 0.005 part by weight to 0.5 part by weight is more preferable, and 0.01 part by weight to 0.3 part by weight is particularly preferable, based on 100 parts by weight of the solution.

[0081] The viscosity of the stamp solution is not particularly limited as long as spots of a desired shape can be formed. For example, at 25°C, it can be 0.4 mPa·s to 40 mPa·s, preferably 0.5 mPa / s to 10 mPa·s, more preferably 0.6 mPa·s to 4 mPa·s, particularly preferably 0.8 mPa·s to 2 mPa·s, and particularly about 1.3 mPa·s. Also, the viscosity of the stamp solution can be 0.6 mPa / s to 10 mPa·s, 0.8 mPa·s to 4 mPa·s, 1.0 mPa·s to 2 mPa·s, 1.12 mPa·s to 2 mPa·s, 1.13 mPa·s to 2 mPa·s, 1.14 mPa·s to 2 mPa·s, 1.15 mPa·s to 2 mPa·s, 1.16 mPa·s to 2 mPa·s, 1.17 mPa·s to 2 mPa·s, 1.18 mPa·s to 2 mPa·s, 1.19 mPa·s to 2 mPa·s, 1.2 mPa·s to 2 mPa·s, etc. at 25°C.

[0082] The method of spotting the substance to be immobilized or the immobilization carrier on which the substance to be immobilized is immobilized and the photo-crosslinking agent on the substrate for the biochip is not particularly limited. For example, methods such as spotting with a micropipette or the like, spotting by a pin method, or spotting by a piezoelectric method can be used. A method of spotting with a micropipette or the like that is not restricted by the size of the fine particles, or spotting by a pin method is preferable. The diameter of the spot can be, for example, 400 μm to 800 μm, preferably 500 μm to 700 μm.

[0083] The immobilization of the substance to be immobilized or the immobilization carrier on which the substance to be immobilized is immobilized can be carried out by irradiating light after coating, preferably after drying the coated solution. The light may be any light that can cause the photoreactive group used to generate radicals. In particular, when an azide group is used as the photoreactive group, ultraviolet light (for example, wavelength 10 to 400 nm) is preferable. The irradiation time can be, for example, 10 seconds to 120 minutes, preferably 30 seconds to 60 minutes, more preferably 1 minute to 30 minutes. Since the substance to be immobilized is rapidly immobilized by irradiation, the irradiation time is approximately equal to the time required for immobilization. The dose of the light beam to be irradiated is not particularly limited, but is usually about 1 mW to 100 mW per 1 cm 2 In this light irradiation, the photoreactive group contained in the photo-crosslinking agent (also, when the polymer has a photoreactive group, the photoreactive group) generates radicals. When the substance to be immobilized is not immobilized on the immobilization carrier, the polymer layer and the substance to be immobilized can be bonded via the photo-crosslinking agent. When the substance to be immobilized is immobilized on the immobilization carrier, the polymer layer and the substance to be immobilized and / or the immobilization carrier can be bonded.

[0084] In the present invention, after immobilizing a desired substance as described above, the substrate can be washed by a known method to remove unreacted components and the like, but such washing is not essential, and the product may be commercialized with the stamp solution left as it is. In this way, a biochip on which a desired immobilized substance is immobilized can be obtained. When washing is not performed, components of the stamp solution such as a thickening agent and / or a surfactant may adhere to the spots of the biochip in addition to the immobilized substance. Such residues of the stamp solution can be appropriately washed and removed before using the biochip.

[0085] 3. Method for storing the biochip In the present invention, for an immobilized substance susceptible to oxidation, particularly amino acids in proteins, etc., by taking deoxidation means, the effect of preventing oxidation of the immobilized substance and / or suppressing the growth of aerobic bacteria and fungi on the biochip can be obtained. The deoxidation means is not limited, and for example, the biochip can be packaged in at least one of the following modes (a) to (d): (a) vacuum packaging, (b) packaging with an inert gas, (c) packaging with a deoxidizer, (d) packaging with a packaging material having a deoxidation function. Therefore, the deoxidation means includes, in addition to each of the above packaging modes (a) to (d), for example, a combination of (a) and (c) (i.e., vacuum packaging with a deoxidizer), a combination of (b) and (c) (i.e., packaging with an inert gas and a deoxidizer), a combination of (b) to (d) (i.e., packaging with an inert gas and a deoxidizer using a packaging material having a deoxidation function), and the like.

[0086] Specific examples of such deoxidation means include a method of performing inert gas substitution such as nitrogen or argon when packaging the biochip as described above, a method of enclosing a deoxidizer in the package, a packaging material having a deoxidation function in the packaging material itself, for example, a method of using a packaging film as the packaging material, and the like.

[0087] Examples of the oxygen scavenger to be coexisted in the package include inorganic oxygen scavengers such as iron and cerium oxide, and organic oxygen scavengers. Such oxygen scavengers are not particularly limited, and commercially available oxygen scavengers that can be used for medical applications, for example, "Ageless" manufactured by Mitsubishi Gas Chemical Company, "Farmakeep" manufactured by Mitsubishi Gas Chemical Company, etc. can be mentioned.

[0088] The packaging material having an oxygen scavenging function is not particularly limited, but a packaging film containing an oxygen scavenger is preferable. Such a packaging film can be specifically classified into those containing an inorganic oxygen scavenger such as iron and cerium oxide, or an organic oxygen scavenger-containing film. These films are not particularly limited, and commercially available films that can be used for medical applications, for example, as a packaging material containing cerium oxide, "Oxycatch" manufactured by Kyodo Printing Co., Ltd., "Highster 02" manufactured by Star Plastic Industry Co., Ltd., etc., and as a packaging material containing iron, "Ageless O-mac" manufactured by Mitsubishi Gas Chemical Company, "Oxyguard" manufactured by Toyo Seikan Kaisha, Ltd., etc. can be used.

[0089] Furthermore, even when the biochip of the present invention cannot sufficiently stably maintain the immobilized substance on the biochip only by the oxygen scavenging means, there may be a case where the immobilized substance on the biochip can be stably maintained by using a desiccant in combination. In such a case, it is preferable to use a desiccant in combination. Of course, it is also possible to use a desiccant having only a moisture removal function and coexist it in the package, and use a packaging material having an oxygen scavenging function for packaging. As such a desiccant, those commonly used in the pharmaceutical field can be used.

[0090] 4. Package in which the biochip is packaged The present invention also relates to a package obtained by packaging a biochip according to the above storage method. The package of the present invention may be one obtained by packaging the biochip of the present invention in at least one of the following modes (a) to (d): (a) vacuum packaging, (b) packaging together with an inert gas, (c) packaging together with an oxygen scavenger, (d) packaging with a packaging material having an oxygen scavenging function. Further, a desiccant may be further enclosed in the package of the present invention. Each of the above configurations of the package of the present invention is as described above for the storage method of the present invention. The package of the present invention can be stored for a long time without impairing the quality of the biochip of the present invention and is excellent in practicality.

[0091] 5. Inspection of biological samples using biochips The present invention relates to a method for inspecting a biological sample using the above biochip, a method for inspecting the health state of a subject, a method for inspecting or diagnosing a disease in a subject, the above biochip for use in these inspection / diagnosis applications, and a kit for use in these inspection / diagnosis applications including the above biochip.

[0092] The method for inspecting a biological sample using the biochip of the present invention comprises (i) a step of providing the above biochip containing a substance that reacts with a target substance as a substance to be immobilized, (ii) a step of reacting the biochip with a biological sample, and (iii) a step of detecting the reaction between the immobilized substance and the target substance contained in the biological sample, wherein the detection of the reaction indicates the presence of the target substance in the biological sample, and non-detection indicates the absence of the target substance in the biological sample.

[0093] The target substance includes, without limitation, proteins such as antibodies, antigens, enzymes, hormones, cytokines, nucleic acid molecules such as RNA and DNA, sugar chains, and the like. The substances that react with the target substance include, without limitation, antigens, antibodies, aptamers, lectins, polynucleotides, enzymes, substrates, and the like. The detection of the reaction between the immobilized substance and the target substance can be carried out by various known methods. For example, when the target substance is a protein, it can be reacted with a labeled antibody against the protein, and the label of the labeled antibody bound to the protein can be detected by a method suitable for the detection of the label. Also, when the target substance is a nucleic acid molecule, it can be reacted with a labeled probe that hybridizes with the nucleic acid molecule, and the label of the labeled probe bound to the nucleic acid molecule can be detected by a method suitable for the detection of the label. The label is not limited, and examples include fluorescent substances, luminescent substances, enzymes, radioisotopes, etc. The methods for detecting the label are not limited, and examples include spectrophotometry, absorptiometry, fluorometry, colorimetry, autoradiography, etc. The detection can be qualitative or quantitative. When detecting quantitatively, information such as the concentration of the target substance in the biological sample can be obtained.

[0094] A method for examining the health status of a subject using the biochip of the present invention is (i) a step of providing the biochip containing a substance that reacts with the target substance as the immobilized substance, (ii) a step of reacting the biochip with a biological sample derived from the subject, and (iii) a step of detecting the reaction between the immobilized substance and the target substance contained in the biological sample, and the detection or non-detection of the reaction provides information regarding the health status of the subject. Examples of information regarding the health status include fatigue, stress, nutritional status, etc. Examples of the target substance include biological substances related to this information, such as proteins, nucleic acid molecules, sugar chains, etc. Examples of the immobilized substance include substances that react with these biological substances, such as antigens, antibodies, aptamers, lectins, polynucleotides, enzymes, substrates, etc. Biomarkers that serve as indicators for fatigue, stress, nutritional status, etc. are known.

[0095] A method for examining or diagnosing a disease in a subject using the biochip of the present invention is (i) Providing the above biochip containing a substance that reacts with the target substance as the substance to be immobilized; (ii) Reacting the biochip with a biological sample derived from the subject; and (iii) Detecting the reaction between the immobilized substance and the target substance contained in the biological sample. The detection or non-detection of the reaction indicates the presence or absence of a disease in the subject. Examples of the disease include diseases that can be examined and diagnosed by detecting substances in a biological sample, and are not limited to, for example, allergic diseases, endocrine diseases, infectious diseases, diseases associated with gene abnormalities, inflammatory diseases, autoimmune diseases, neoplastic diseases, etc. Examples of the target substance include biological substances related to these diseases, such as proteins, nucleic acid molecules, sugar chains, etc. Examples of the immobilized substance include substances that react with these biological substances, such as antigens, antibodies, aptamers, lectins, polynucleotides, enzymes, substrates, etc. Various disease markers such as tumor markers, infectious disease markers, genetic disease markers, endocrine disease markers, and inflammation markers are known.

[0096] The biochip of the present invention can be used for the above-mentioned various applications, that is, examination of biological samples, examination of the health status of the subject (such as fatigue, stress, nutritional status, etc.), examination or diagnosis of diseases in the subject (such as allergic diseases, endocrine diseases, infectious diseases, diseases associated with gene abnormalities, inflammatory diseases, autoimmune diseases, neoplastic diseases, etc.). The biochip of the present invention for use in the above-mentioned various applications preferably contains an immobilized substance suitable for each application.

[0097] The kit of the present invention can be used for each of the above applications, namely, the inspection of biological samples, the inspection of the health status of a subject (e.g., fatigue, stress, nutritional status, etc.), the inspection or diagnosis of diseases in a subject (e.g., allergic diseases, endocrine diseases, infectious diseases, diseases associated with gene abnormalities, inflammatory diseases, autoimmune diseases, neoplastic diseases, etc.). The kit of the present invention includes the biochip of the present invention, preferably the biochip of the present invention containing an immobilized substance suitable for each of the above applications. In addition to the biochip of the present invention, the kit of the present invention may include a reagent for detecting the reaction between the immobilized substance and the target substance contained in the biological sample, a standard sample, an instruction indicating the method of using the kit, such as an instruction manual, or a medium recording information on the method of use, such as a flexible disk, CD, DVD, Blu-ray disk, memory card, USB memory, etc.

Example

[0098] Hereinafter, the present invention will be described in detail with reference to examples, but the content of the present invention is not limited thereto. <Example 1: Production of Substrate for Biochip (1)> A polycarbonate sheet (manufactured by Mitsubishi Gas Chemical Company, MU58U, thickness 0.8 mm) was irradiated with a UV-ozone irradiation device (manufactured by Sen Special Light Source Co., Ltd., SSP16-110, UV lamp: SUV110GS-36L) at an irradiation distance of 50 mm for 2 minutes. Next, 0.5 part by weight of a polymer of polyethylene glycol monomethacrylate (manufactured by Sanyu Chemical Research Institute, polyethylene glycol molecular weight 350) and 0.025 part by weight of 4,4'-diazidostilbene-2,2'-disulfonic acid (manufactured by Tokyo Chemical Industry Co., Ltd., 98%, hereinafter referred to as bisazide) were dissolved in a 75% aqueous ethanol solution to obtain a coating solution for preventing non-specific adsorption. Using a spin coater (manufactured by MIKASA Co., Ltd., MS-A100), 15 μL of this coating solution was coated on the UV-irradiated polycarbonate under the conditions of 800 rpm for 5 seconds and 5000 rpm for 10 seconds. After coating, using a UV irradiation device (manufactured by UVP Co., Ltd., CL-1000), 120 mW / cm 2It was irradiated for 10 minutes. An O-ring (14φ) made of silicone rubber was adhered to the coated substrate with an adhesive to form a reaction area. Aging was performed at a temperature of 25°C and a humidity of 65% for 4 days to obtain a substrate 1 for biochip.

[0099] A substrate 2 for biochip was obtained in the same manner as the substrate 1 for biochip, except that a reaction area was not formed with silicone rubber.

[0100] <Example 2: Preparation of Stamp Solution> (1) (Stamp Solution 1-1) Sodium chloride was dissolved in ultrapure water to 0.8 parts by weight, potassium chloride was dissolved to 0.02 parts by weight, disodium hydrogen phosphate dodecahydrate was dissolved to 0.29 parts by weight, and potassium dihydrogen phosphate was dissolved to 0.02 parts by weight to obtain a PBS solution. 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid was dissolved in ultrapure water to 0.59 parts by weight to obtain a HEPES solution. Polyvinyl alcohol (manufactured by Wako Pure Chemical Industries, Ltd., 160-03055) and Tween 20 (manufactured by Sigma-Aldrich, P7949-100ML) were dissolved in the HEPES solution to 0.1 parts by weight and 0.05 parts by weight, respectively, to obtain a solvent for stamp solution. As the substance to be immobilized, a biotinylated peptide obtained by biotinylating the terminal of a partial peptide of αs1-casein (consisting of 15 amino acid residues) was reacted with Streptavidin beads (manufactured by Tamagawa Seiki Co., Ltd.) in a PBS solution at 4°C for 1.5 hours, and then purified and dispersed in the solvent for stamp solution to obtain stamp solution 1-1.

[0101] The viscosity of stamp solution 1-1 was measured. The measurement was performed using a B-type viscometer DV2T (manufactured by Brookfield) under the conditions of 20°C and 200 rpm. Before measuring the sample, calibration was performed with water and the viscosity meter calibration standard solution JS2.5 (manufactured by Nippon Grease Co., Ltd.). As a result of performing the measurement 3 times and obtaining the average value, the viscosity of stamp solution 1-1 was 1.31 mPa·s.

[0102] (Stamp Solution 1-2) Stamp solution 1-2 was obtained in the same manner as stamp solution 1-1, except that polyvinyl alcohol was not added to the solvent for the stamp solution. As a result of measuring the viscosity in the same manner as stamp solution 1-1, the viscosity of stamp solution 1-2 was 1.21 mPa·s. (Stamp solution 1-3) Stamp solution 1-3 was obtained in the same manner as stamp solution 1-1, except that Tween 20 was not added to the solvent for the stamp solution. As a result of measuring the viscosity in the same manner as stamp solution 1-1, the viscosity of stamp solution 1-3 was 1.22 mPa·s. (Stamp solution 1-4) Stamp solution 1-4 was obtained in the same manner as stamp solution 1-1, except that polyvinyl alcohol and Tween 20 were not added to the solvent for the stamp solution. As a result of measuring the viscosity in the same manner as stamp solution 1-1, the viscosity of stamp solution 1-4 was 1.11 mPa·s.

[0103] (Stamp solution 1-5) Morpholineethanesulfonic acid, monohydrate was dissolved in ultrapure water to a concentration of 0.53 parts by weight to obtain an MES solution. Polyvinyl alcohol (manufactured by Wako Pure Chemical Industries, Ltd., 160-03055) and Tween 20 (manufactured by Sigma-Aldrich, P7949-100ML) were dissolved in the HEPES solution to concentrations of 0.1 parts by weight and 0.05 parts by weight, respectively, to obtain a solvent for the stamp solution. As the substance to be immobilized, αs1-casein (manufactured by Sigma-Aldrich, C6780-250MG) was reacted with Linker beads (manufactured by Tamagawa Seiki Co., Ltd.) in an MES buffer solution at 40°C for 20 hours, and then purified and dispersed in the solvent for the stamp solution to obtain stamp solution 1-5.

[0104] (Stamp solution 1-6) Stamp solution 1-6 was obtained in the same manner as stamp solution 1-5, except that polyvinyl alcohol was not added to the solvent for the stamp solution. (Stamp solution 1-7) Stamp solution 1-7 was obtained in the same manner as stamp solution 1-5, except that Tween 20 was not added to the solvent for the stamp solution. (Stamp solution 1-8) Stamp solution 1-8 was obtained in the same manner as stamp solutions 1-5, except that polyvinyl alcohol and Tween 20 were not added to the solvent for the stamp solution.

[0105] (Stamp solution 1-9) In a PBS solution, 0.1 part by weight of polyvinyl alcohol (manufactured by Wako Pure Chemical Industries, Ltd., 160-03055) and 0.05 part by weight of Tween 20 (manufactured by Sigma-Aldrich, P7949-100ML) were each dissolved to obtain a solvent for the stamp solution. As the substance to be immobilized, αs1-casein (manufactured by Sigma-Aldrich, C6780-250MG) was dissolved in the solvent for the stamp solution to a concentration of 0.1 mg / mL to obtain stamp solution 1-9. (Stamp solution 1-10) Stamp solution 1-10 was obtained in the same manner as stamp solution 1-9, except that polyvinyl alcohol was not added to the solvent for the stamp solution. (Stamp solution 1-11) Stamp solution 1-11 was obtained in the same manner as stamp solution 1-9, except that Tween 20 was not added to the solvent for the stamp solution. (Stamp solution 1-12) Stamp solution 1-12 was obtained in the same manner as stamp solution 1-9, except that polyvinyl alcohol and Tween 20 were not added to the solvent for the stamp solution. The preparation conditions of each stamp solution are summarized in Table 1.

[0106]

Table 1

[0107] <Example 3: Manufacture of Biochip (1)> Bisazide was dissolved in ultrapure water to prepare a 10 mg / mL bisazide solution. The 10 mg / mL bisazide solution was diluted 5-fold (bisazide solution A) or 5000-fold (bisazide solution B). Bisazide solution B was dissolved in stamp solutions 1-1 to 1-8 to a concentration of 4.8 parts by weight. Bisazide solution A was dissolved in stamp solutions 1-9 to 1-12 to a concentration of 4.8 parts by weight. Using a stamper (Geneqs, Genex Arrayer), for each of the 12 types of stamp solutions, 3 spots were spotted onto the substrate 1 or 2 for the biochip prepared in Example 1, for a total of 36 spots, in a lattice-point type multi-spot dispensing method such that it was 6×6 (in the order from stamp solution 1-12 to stamp solution 1-1). The diameter of the spots was 0.36 to 0.54 μm, and the interval between spots was approximately 1.1 mm. After spotting, it was dried at 0.09 MPa for 10 minutes using a vacuum dryer. After drying, in order to immobilize the substance to be immobilized with a photo-crosslinking agent, it was irradiated with UV using a UV irradiation device (UVP, CL-1000) at 120 mW / cm 2 for 10 minutes to obtain the biochip 1 or 2.

[0108] <Example 4: Measurement Using the Chip> Using the biochip manufactured in Example 3, the measurement of IgE antibody in casein-positive human serum (PlasmaLab) was performed. The chip was washed three times with TBS-T solution (137 mM sodium chloride, 2.68 mM potassium chloride, 25 mM tris(hydroxymethyl)aminomethane, pH 7.4, 0.1 wt% Tween 20). 130 μL of the serum diluted 8-fold as a test sample was added to the reaction area of Biochip 1, and while shaking, it was reacted at room temperature for 8 minutes. The test sample was aspirated and removed, and washed with TBS-T solution. After washing, 130 μL of anti-human IgE antibody (manufactured by Seracare Life Sciences, 0751-1004) diluted 2000-fold with Can Get Signal Immunoreaction Enhancer Solution 2 (manufactured by Toyobo Co., Ltd., NKB-301) was added, and while shaking, it was reacted at room temperature for 4 minutes. The antibody was aspirated and removed, and washed with TBS-T. 130 μL of a luminescent reagent (Dynalight Substrate with Rapid Glow Enhancer, Molecular Probes, 4475406) was added, and after shaking for 1 minute, the luminescence intensity was measured by counting the number of pixels in the luminescent part using SpotSolver manufactured by Dynam Com and ImageJ manufactured by the National Institutes of Health of the United States with the part where no spot exists as the background. The measurement image by an optical microscope (manufactured by Olympus, IX71) is shown in Fig. 5, the image of the luminescence intensity is shown in Fig. 6, and the planar image, 3D image, and profile graph by a laser microscope (manufactured by Keyence, VK-X250) are shown in Figs. 7 to 14, respectively. Also, the luminescence signal intensity (number of pixels in the luminescent part) obtained from the image of Fig. 6 is shown in Table 2, and the average value (n = 3) of the volume of the immobilized solid substance (substance to be immobilized + immobilization carrier) obtained from the 3D images of Figs. 7 to 14 is shown in Table 3, respectively.

[0109]

Table 2

[0110]

Table 3

[0111] From the results of FIGS. 5 and 7 to 14, it can be seen that when neither the thickener nor the surfactant is included, the spot does not become round, and the shape of the spot can be stabilized by including the thickener and / or the surfactant. From the results of FIGS. 7 to 14 and Table 3, in the spots containing the thickener and / or the surfactant, the volume of the solid matter present on the chip is larger than that in the spots containing neither the thickener nor the surfactant, and it can be seen that more immobilized carriers to which the substance to be immobilized is bound are immobilized. Further, from the results of FIG. 6 and Table 2, the spots containing neither the thickener nor the surfactant have unstable luminescence intensity and reduced detection sensitivity, but the luminescence intensity of the spots can be stabilized and the detection sensitivity can be improved by including the thickener and / or the surfactant. Furthermore, the biochip of the present invention can be analyzed with a very small amount of test sample of 16 μL. Also, according to the method for manufacturing the biochip of the present invention, a biochip in which the binding site of the substance to be immobilized can be controlled and the shape of the spot is stable can be obtained.

[0112] <Example 5: Manufacture of Biochip (2)> Stamp solutions 2-1 to 2-8 were prepared in the same manner as in Example 2, except that Streptavidin Mag Sepharose (manufactured by GE Healthcare) was used instead of Streptavidin beads and Sera-Mag Carboxylate-Modified Magnetic Particles (Hydrophylic) (manufactured by GE Healthcare) was used instead of Linker beads. The preparation conditions for each stamp solution are summarized in Table 4. In the table, the immobilized carrier "A" indicates Streptavidin Mag Sepharose, and "B" indicates Sera-Mag Carboxylate-Modified Magnetic Particles.

[0113]

Table 4

[0114] Each of the stamp solutions 2-1 to 2-8 and 1-9 to 1-12 was spotted onto the substrate 1 or 2 for the biochip prepared in Example 1 in the same manner as in Example 3, and dried and UV-irradiated in the same manner to obtain biochips 3 or 4. Using these biochips, the same measurements as in Example 4 were performed. The image of the luminescence intensity is shown in Fig. 15, and the luminescence signal intensity obtained from the image of Fig. 15 is shown in Table 5, respectively. Observation with an optical microscope and a laser microscope was also performed, and the same results as in Example 4 were obtained. In Table 5, the numerical value of spot 4 is higher than those of spots 1 to 2, and spots 12 and spots 9 to 10 and spots 8 and spots 5 to 6, which have the same relationship, show different results. This is because, in the process of spotting the stamp solution in the order from stamp solution 1-12 to stamp solution 1-9, and then from stamp solution 2-8 to stamp solution 2-1, the stamp solution 2-5 remaining slightly on the stamper was mixed into the stamp solution 2-4 to be spotted next and was spotted onto spot 4 as it was.

[0115]

Table 5

[0116] <Example 6: Storage of Biochip (1)> (1) Preparation of buffer solution Sodium chloride was dissolved in ultrapure water to 0.8 parts by weight, potassium chloride to 0.02 parts by weight, disodium hydrogen phosphate dodecahydrate to 0.29 parts by weight, and potassium dihydrogen phosphate to 0.02 parts by weight to obtain a PBS solution. 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid was dissolved in ultrapure water to 0.59 parts by weight to obtain a HEPES solution. Morpholineethanesulfonic acid, monohydrate was dissolved in ultrapure water to 0.53 parts by weight to obtain a MES solution.

[0117] (2) Preparation of immobilization reaction solution t-Butyl alcohol (manufactured by Wako Pure Chemical Industries, Ltd., 028-03386) and dimethyl sulfoxide were mixed at a volume ratio of 4:1, and Tris「(1-benzyl-1H-1,2,3-triazol-4-yl)methyl」amine (manufactured by Sigma-Aldrich, 678937-50MG) was dissolved to a weight of 0.27 parts to obtain a TBTA solution.

[0118] (3) Preparation of solvent for stamp Polyvinyl alcohol (manufactured by Wako Pure Chemical Industries, Ltd., 160-03055) and Tween 20 (manufactured by Sigma-Aldrich, P7949-100ML) were dissolved in PBS solution to 0.1 part by weight and 0.05 part by weight, respectively, to obtain Solution A. Polyvinyl alcohol (manufactured by Wako Pure Chemical Industries, Ltd., 160-03055) and Tween 20 (manufactured by Sigma-Aldrich, P7949-100ML) were dissolved in 25 mM HEPES solution to 0.1 part by weight and 0.05 part by weight, respectively, to obtain Solution B.

[0119] As the substances to be immobilized, αs1-casein (manufactured by Sigma-Aldrich, C6780-250MG), β-casein (manufactured by Sigma-Aldrich, C6905-250MG) were each at 0.1 mg / mL, α-lactalbumin (manufactured by Sigma-Aldrich, L5385-25MG), β-lactoglobulin (manufactured by Sigma-Aldrich, L3908-250MG), ovalbumin (manufactured by Sigma-Aldrich, T2011-250MG), and ovalbumin (manufactured by Sigma-Aldrich, A2512-250MG) were used. Each of the substances to be immobilized was dissolved in Solution A to obtain Stamp Solutions 3-1 to 3-6. As the substances to be immobilized, αs1-casein, α-lactalbumin, and ovalbumin were each reacted with Linker beads in MES buffer at 40°C for 20 hours, then purified and dispersed in Solution B to obtain Stamp Solutions 3-7 to 3-9. Also, only Linker beads without the immobilized solidified substances were dispersed in Solution B to obtain Stamp Solution 3-10. As the substances to be immobilized, the terminals of different partial peptides A and B of αs1-casein (each consisting of 15 amino acid residues), biotinylated peptide A and biotinylated peptide B, were each reacted with Streptavidin beads in PBS solution at 4°C for 1.5 hours, then purified and dispersed in Solution B to obtain Stamp Solutions 3-11 to 3-12. Similarly, azidated peptide A and azidated peptide B with the terminals of peptides A and B azidated were each reacted with Alkyne beads (manufactured by Tamagawa Seiki Co., Ltd.) in HEPES solution at 25°C for 20 hours, then purified and dispersed in Solution B to obtain Stamp Solutions 3-13 to 3-14. The preparation conditions for each stamp solution are summarized in Table 6.

[0120]

Table 6

[0121] (4) Fabrication of the biochip The bisazide was dissolved in ultrapure water to prepare a 10 mg / mL bisazide solution. The 10 mg / mL bisazide solution was diluted 5-fold (bisazide solution A) and 5000-fold (bisazide solution B). The bisazide solution A was dissolved in the stamp solutions 3-1 to 3-6 to a concentration of 4.8 parts by weight. The stamp solutions 3-7 to 3-14 were dispersed with beads and the bisazide solution B was dissolved to a concentration of 4.8 parts by weight. Using a stamper (Geneqs, Genex Arrayer), 42 spots in total, 3 spots for each of the 14 stamp solutions, were spotted onto the biochip substrate 1 prepared in Example 1 in a grid pattern multi-spot dispensing method so as to form a 6×7 array. After spotting, drying was performed at 0.09 MPa for 10 minutes using a vacuum dryer. After drying, in order to immobilize the substance to be immobilized with a photo-crosslinking agent, irradiation was performed at 120 mW / cm 2 for 10 minutes using a UV irradiation device (UVP, CL-1000) to obtain the biochip 3.

[0122] (5) Storage of the biochip The prepared biochip 3 was stored under the following conditions. · Storage Example 1: The biochip was enclosed in an aluminum bag manufactured by Mitsubishi Gas Chemical Company together with an oxygen scavenger (Ageless manufactured by Mitsubishi Gas Chemical Company) and stored in a thermostat at 25°C for 2 weeks. · Storage Example 2: The biochip was stored in the same manner as Storage Example 1, except that after being put into the package, the inside of the package was evacuated and sealed without enclosing an oxygen scavenger. · Storage Example 3: The biochip was stored in the same manner as Storage Example 1, except that no oxygen scavenger was enclosed.

[0123] Using the biochips obtained in Storage Examples 1 to 3, the same measurements as in Example 4 were performed. The measurement results of the luminescence intensity are shown in Fig. 16, and the luminescence signal intensities obtained from the image of Fig. 16 are shown in Table 7, respectively.

Table 7

[0124] From the results of Storage Examples 1 to 2 in FIG. 16 and Table 7, it can be seen that the biochip of the present invention maintains the stability of the biochip even after long-term storage by storing it in an oxygen-free state or in a vacuum. On the other hand, from the results of Storage Example 3 in FIG. 16 and Table 7, the biochip that did not use the storage method of the present invention had the chip surface deteriorated and the luminescence intensity could not be read.

[0125] <Example 7: Manufacture of Substrate for Biochip (2)> <Substrate 1> A polycarbonate sheet (MR58U, manufactured by Mitsubishi Gas Chemical Company, thickness 0.8 mm) was irradiated with a UV-ozone irradiation device (SSP16-110, manufactured by Sen Special Light Source Co., Ltd., UV lamp: SUV110GS-36L) at an irradiation distance of 50 mm for 2 minutes to hydrophilize the entire upper surface of the sheet. An O-ring (14φ) made of silicone rubber was adhered to the hydrophilized sheet with an adhesive to form a reaction area, and Substrate 1 for biochip was obtained.

[0126] <Substrate 2> Substrate 2 for biochip was obtained in the same manner as Substrate 1 except that the treatment with the UV-ozone irradiation device was set to 1 minute.

[0127] <Substrate 3> Substrate 3 for biochip was obtained in the same manner as Substrate 1 except that the treatment with the UV-ozone irradiation device was set to 30 seconds.

[0128] <Substrate 4> Substrate 4 for biochip was obtained in the same manner as Substrate 1 except that the treatment with the UV-ozone irradiation device was set to 8 minutes.

[0129] <Substrate 5> Substrate 5 for biochip was obtained in the same manner as Substrate 1 except that a tabletop semi-automatic atmospheric pressure plasma surface modification device (MyPL-Auto100, manufactured by Well Co., Ltd.) was used for one treatment at 100 W and 50 mm / second instead of the treatment with the UV-ozone irradiation device.

[0130] <Substrate 6> A substrate 6 for a biochip was obtained in the same manner as in Example 1, except that UV-ozone treatment was not performed.

[0131] <Substrate 7> Instead of the treatment with a UV-ozone irradiation device, 15 μL of a coating solution in which 0.5 part by weight of polyethylene glycol monomethacrylate (manufactured by Sanyu Chemical Laboratory Co., Ltd., polyethylene glycol molecular weight 350) and 0.025 part by weight of 4,4'-diazidostilbene-2,2'-disulfonic acid (manufactured by Tokyo Chemical Industry Co., Ltd., 98%, hereinafter referred to as bisazide) were dissolved in a 75% ethanol aqueous solution was coated on a polycarbonate sheet using a spin coater (manufactured by MIKASA Co., Ltd., MS-A100) under the conditions of 800 rpm for 5 seconds and 5000 rpm for 10 seconds. Then, using a UV irradiation device (manufactured by UVP Co., Ltd., CL-1000), it was irradiated at 120 mW / cm 2 for 10 minutes to crosslink the bisazide and form a coating layer of a non-specific adsorption inhibitor. A substrate 7 for a biochip was obtained in the same manner as in Substrate 1, except for the above.

[0132] The functional groups on the resin surface were measured using an infrared spectrophotometer (manufactured by JASCO Corporation, FT / IR-4200). The state of the resin surface was determined by the absorption intensity (D -1 ) of the characteristic absorption of CH, which is the main structure of the substrate, and the absorption intensity (D CH ) of the characteristic absorption of -OH contained in the hydroxyl group and carboxyl group with respect to the absorption intensity (D -1 ) of the characteristic absorption of C=O contained in the carboxyl group and carbonyl group in the range of 3200 - 3600 cm OH ) and in the range of 1600 - 1950 cm -1 ) and the ratio of the absorption intensity (D CO ).

[0133] The D OH / D CH and D CO / D CH of Substrates 1 - 6 for a biochip are shown in Table 8.

Table 8

[0134] <Measurement of Contact Angle> The contact angle was measured using a contact angle measuring device (Drop Master 500, manufactured by Kyowa Interface Science Co., Ltd.), and the θ / 2 method (calculated by θ / 2 = arctan(h / r). Here, θ represents the contact angle, r represents the radius of the liquid droplet, and h represents the height of the liquid droplet) was used to measure the average value of the contact angles at three locations. Here, as the first measurement point, the central part of the sample was selected, and as the second and third measurement points, two points that were more than 20 mm away from the first measurement point and were point-symmetric to each other with respect to the first measurement point were selected.

[0135] Table 2 shows the contact angles of the substrates 1 to 7 for the biochip.

Table 9

[0136] From Tables 8 and 9, substrates 1 to 5 for the biochip have an increase in the hydroxyl groups and / or carboxyl groups and / or carbonyl groups on the surface and an increase in the contact angle compared to substrate 6 for the biochip.

[0137] <Example 8: Fabrication of Biochip (3)> Using a stamper (Genex Arrayer, manufactured by Geneqs), the stamp solutions 3-1 to 3-14 prepared in Example 6 were spotted on substrates 1 to 7 for the biochip at 42 spots in total, 3 spots for each of the 14 types of stamp solutions, in a grid-point type multi-spot dispensing method so as to be 6×7. After spotting, it was dried at 0.09 MPa for 10 minutes using a vacuum dryer. After drying, in order to immobilize the substance to be immobilized with a photo-crosslinking agent, it was irradiated with UV light at 120 mW / cm 2 for 10 minutes using a UV irradiation device (CL-1000, manufactured by UVP) to obtain a biochip.

[0138] Using the obtained biochip, the luminescence intensity was measured in the same manner as in Example 4. The measurement results of the luminescence intensity of the biochips fabricated with substrates 1 to 7 for the biochip are shown in Fig. 17, and the luminescence signal intensities obtained from the image of Fig. 17 are shown in Table 10, respectively. The luminescence signal intensity represents the number of pixels in the luminescent part.

Table 10

[0139] From FIG. 17 and Table 10, it can be seen that a biochip using the substrate 6 for biochips cannot obtain sufficient luminescence intensity. However, the substrates 1 to 5 for biochips having a resin surface containing a hydrophilic reaction area can immobilize the substance to be immobilized without using a coating layer, and show luminescence intensity equal to or higher than that of the substrate 7 for biochips coated with a water-soluble polymer. Therefore, by using a substrate for biochips having a resin surface containing a hydrophilic reaction area, it is possible to immobilize the substance to be immobilized using the same amount of photo-crosslinking agent as in the case of coating a non-specific adsorption prevention layer such as a water-soluble polymer without coating the non-specific adsorption prevention layer, and to provide a highly sensitive biochip with excellent luminescence intensity and suppression of non-specific adsorption of the substance to be detected.

[0140] <Example 9: Storage of Biochip (2)> Save Example 4 Using the substrate 1 for biochips and the stamp solution shown in Table 6, a biochip manufactured in the same manner as in Example 8 was sealed together with a deoxidizer (Ageless manufactured by Mitsubishi Gas Chemical Company) and placed in a thermostat at 25°C for 2 weeks of storage.

[0141] Storage Example 5 A biochip manufactured in the same manner as in Storage Example 4 was stored in the same manner as in Storage Example 4, except that the storage period was 8 weeks.

[0142] The luminescence intensities of the biochips obtained in Storage Examples 4 and 5 were measured in the same manner as in Example 4. The measurement results of the luminescence intensities are shown in FIG. 18, and the luminescence signal intensities obtained from the image of FIG. 18 are shown in Table 11, respectively.

Table 11

[0143] As can be seen from FIG. 18 and Table 11, the biochip of the present invention can maintain the stability of the biochip even after long-term storage by storing it in an oxygen-free state.

[0144] Although some embodiments of the present invention have been described, these embodiments are presented as examples and are not intended to limit the scope of the invention. These novel embodiments can be implemented in various other forms, and various omissions, replacements, and changes can be made without departing from the gist of the invention. These embodiments and their modifications are included in the scope and gist of the invention, and are included in the invention described in the claims and its equivalent scope.

Industrial Applicability

[0145] According to the present invention, for example, by using a biochip in which a plurality of different types of biomolecules are fixed as a plurality of spots, it is possible to simultaneously perform multi-item analysis on a small amount of test sample quickly, inexpensively, and easily, and it can be used for point-of-care test analysis for use at clinical sites and the like. Further, according to the present invention, it is possible to obtain a substrate for a biochip that immobilizes the immobilized substance without coating a coating layer such as a water-soluble polymer, has excellent luminescence intensity, and suppresses non-specific adsorption of the substance to be detected. This makes it possible to manufacture a biochip inexpensively with a simple manufacturing process.

Explanation of Signs

[0146] 10: Biochip (A) 11: Biochip (B) 12: Biochip (C) 13: Biochip (D) 101: Substrate 101A: Upper surface of the substrate 102: Boundary 103: Reaction area 104: Coating layer 105: Spot

Claims

1. On the reaction area of a substrate for a biochip having a resin surface including a hydrophilic reaction area, a substance to be immobilized containing a biological substance is immobilized. The substance to be immobilized is present in spots containing a thickener and a surfactant that are present in the reaction area. A coat layer for holding the substance to be immobilized is formed in the reaction area. The coat layer contains a water-soluble polymer selected from bipolar polymers and nonionic polymers. The thickener includes gellan gum, xanthan gum, curdlan, pullulan, guar gum derivative, locust bean gum, carrageenan, pectin, tamarind gum, psyllium seed gum, dextran, glycerin, carboxymethyl cellulose, hydroxyethyl cellulose, hydroxymethylpropyl cellulose, lanolin, methyl cellulose, petrolatum, polyethylene glycol, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, polyvinyl pyrrolidone, polyvinyl alcohol, fatty acid ester of dextrin phosphate, fatty acid ester of inulin phosphate, or a mixture of two or more selected from these. The substance to be immobilized, the thickener, and the surfactant are present in a plurality of spots on the coat layer. A biochip.

2. The biochip according to claim 1, wherein the reaction area is surrounded by a boundary capable of holding a liquid inside the reaction area, and a bond related to carbon of the resin is broken, and the broken portion is covered with a polar functional group formed by bonding with oxygen.

3. It has a substrate having a hydrophilic reaction area and a plurality of spots of a substance to be immobilized containing a biological substance disposed in the reaction area. The reaction area is surrounded by a boundary capable of holding a liquid inside thereof. The plurality of spots further contain a thickener and a surfactant, a coating layer for holding the substance to be immobilized is formed in the reaction area, the coating layer contains a water-soluble polymer selected from a bipolar polymer and a nonionic polymer, the thickener is gellan gum, xanthan gum, curdlan, pullulan, guar gum derivative, locust bean gum, carrageenan, pectin, tamarind gum, psyllium seed gum, dextran, glycerin, carboxymethyl cellulose, hydroxyethyl cellulose, hydroxymethylpropyl cellulose, lanolin, methyl cellulose, petrolatum, polyethylene glycol, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, polyvinyl pyrrolidone, polyvinyl alcohol, fatty acid ester of dextrin phosphate, fatty acid ester of inulin phosphate, or a mixture of two or more selected from these, and the substance to be immobilized, the thickener and the surfactant are present in the plurality of spots present on the coating layer, a biochip.

4. The biochip according to any one of claims 1 to 3, wherein the substance to be immobilized is immobilized on the substrate via a coating layer for holding the substance to be immobilized.

5. The biochip according to any one of claims 1 to 4, wherein the water-soluble polymer is polyethylene glycol methacrylate.

6. The biochip according to any one of claims 1 to 5, wherein the substance to be immobilized is immobilized in the reaction area by a photo-crosslinking agent having at least two photoreactive groups in one molecule.

7. The biochip according to any one of claims 1 to 6, wherein the substance to be immobilized is a peptide, a nucleic acid, a sugar chain, or a mixture of one or more selected from these.

8. The biochip according to any one of claims 1 to 7, wherein the substance to be immobilized is immobilized on an immobilization carrier, and the immobilization carrier on which the substance to be immobilized is immobilized is immobilized in the reaction area.

9. The biochip according to any one of claims 1 to 8, wherein the surfactant is Triton X-100, Tween 20, Tween 80, or a mixture of two or more selected from these.

10. The biochip according to any one of claims 1 to 9, wherein the substrate is a resin material of any one of polystyrene, polypropylene, polycarbonate, and acrylic resin.

11. A step of forming a coating layer for holding a substance to be immobilized on a reaction area of a substrate for a biochip having a resin surface including a hydrophilic reaction area; A step of adhering, as a plurality of spot-shaped lattices, a liquid containing a substance to be immobilized, a photo-crosslinking agent having at least two photo-reactive groups in one molecule, a thickening agent, and a surfactant onto the coating layer; A step of immobilizing the substance to be immobilized adhered onto the coating layer; The method for manufacturing a biochip according to any one of claims 1 to 2 and 4 to 10, wherein the coating layer contains a water-soluble polymer selected from a bipolar polymer and a nonionic polymer, and the thickening agent is gellan gum, xanthan gum, curdlan, pullulan, a guar gum derivative, locust bean gum, carrageenan, pectin, tamarind gum, psyllium seed gum, dextran, glycerin, carboxymethyl cellulose, hydroxyethyl cellulose, hydroxymethylpropyl cellulose, lanolin, methyl cellulose, petrolatum, polyethylene glycol, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, polyvinyl pyrrolidone, polyvinyl alcohol, a fatty acid ester of dextrin phosphate, a fatty acid ester of inulin phosphate, or a mixture of two or more selected therefrom.

12. A method for manufacturing a biochip according to any one of claims 3 to 10, comprising: A step of forming a hydrophilic reaction area on a substrate, on which a coating layer for holding a substance to be immobilized is formed; A step of forming a plurality of spots of the substance to be immobilized containing a biological substance on the coating layer; comprising, wherein the coating layer contains a water-soluble polymer selected from a bipolar polymer and a nonionic polymer, the spot contains a thickener and a surfactant, and the thickener is selected from gellan gum, xanthan gum, curdlan, pullulan, guar gum derivative, locust bean gum, carrageenan, pectin, tamarind gum, psyllium seed gum, dextran, glycerin, carboxymethyl cellulose, hydroxyethyl cellulose, hydroxymethylpropyl cellulose, lanolin, methyl cellulose, petrolatum, polyethylene glycol, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, polyvinyl pyrrolidone, polyvinyl alcohol, fatty acid ester of dextrin phosphate, fatty acid ester of inulin phosphate, or a mixture of two or more selected from these, a method.

13. The step of forming the plurality of spots is a step of adhering, as a plurality of lattice-shaped spots on the coating layer, a liquid containing a substance to be immobilized, a photocrosslinking agent having at least two photoreactive groups in one molecule, a thickener and a surfactant; a step of immobilizing the substance to be immobilized adhered on the coating layer; The production method according to claim 12, comprising.

14. A package obtained by packaging the biochip according to any one of claims 1 to 10 in at least one of the following modes (a) to (d): (a) Vacuum packaging; (b) Packaging together with an inert gas; (c) Packaging together with an oxygen scavenger; and (d) Packaging with a packaging material having an oxygen scavenging function.

15. The package according to claim 14, in which a desiccant is enclosed.

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