JP2007171144A - Microarray having cover - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a technique capable of constraining formation of air bubbles on and near the inner surface of a substrate. <P>SOLUTION: In a microarray, including a substrate and a cover that is attachable firmly to a part of the substrate, the microarray is characterized by making the inner surface of the cover to be hydrophilic property, making the cover to have at least one through hole, making the substrate to contain a plurality of relief structures, and making selective binding matter to be fixed on the upper surface of convex parts. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a microarray having a cover whose inner surface is hydrophilic.

  A microarray is a small device for simultaneously measuring thousands or more gene expressions, and is a molecule in which molecules such as DNA are arranged at high density on a base substrate such as glass or silicon. By using the microarray, systematic and comprehensive gene expression analysis in animal models of various diseases and cell biology can be performed. Specifically, gene functions can be analyzed. That is, it becomes possible to clarify the protein encoded by the gene and specify the time when the protein is expressed and the place where it acts. Analyzing gene expression fluctuations at the cell or tissue level of an organism by microarray and constructing a gene expression profile database in combination with physiological, cell biological, and biochemical event data, thereby enabling disease genes and treatment-related genes Searching for and searching for treatment methods will be possible.

  Currently, two basic methods are employed for microarrays, namely the GeneChip method and the cDNA microarray method.

  In the GeneChip method, an oligo DNA of about 25 mer is synthesized on a glass plate by a photolithographic method, and 25 mers of 16 to 20 sites are set for each gene from the base sequence data. This is a method developed by Affymetrix, which uses as a probe a set of oligomers with a single base mismatch that is intentionally different from the 13th base. This method is ideal for quantitative analysis of the expression level because the length of the probe DNA is constant and the sequence is known, so that the GC content that affects the strength of hybridization can be made constant. It is considered an array.

  The cDNA microarray method is a method developed by Stanford University, and is a method of fixing DNA to a glass plate by using a method such as spotting with a capillary pen or an inkjet method.

  In any of the above methods, after the sample to be measured is fluorescently labeled, it binds to the probe on the microarray by hybridization, and the fluorescence intensity is measured using a scanner to measure the expression of the target gene. .

  One analysis of microarray data is hierarchical clustering. This is a method by which genes with similar expression patterns can be collected to create a phylogenetic tree, and the expression levels of a large number of genes can be schematically displayed in color. By such clustering, genes related to a certain disease can be identified.

  Microarrays have come to be used not only for nucleic acids such as DNA, but also for testing and analyzing proteins and sugars. In particular, in protein microarrays, proteins such as antibodies, antigens, and enzyme substrates are immobilized on a substrate.

  The present inventors previously developed a microarray having a special shape using a polymer as a base material and having a concavo-convex structure instead of a general microarray based on glass or silicon (Patent Document 1). By this method, the variation of each spot of the substance immobilized on the array is reduced, and as a result, the S / N ratio and the detection sensitivity are greatly improved. The immobilizing substance is fixed on the upper surface of the convex portion. Furthermore, the presence of the fine particles inside the recesses makes it possible to increase the stirring efficiency of the reaction solution, and as a result, a reaction promoting effect is also achieved (Non-Patent Document 1).

JP 2004-264289 A Reiko Takizawa et al., 2005 7-8 Biotechnology Journal pages 418-420

  In the microarray having a concavo-convex structure developed by the present inventors, it is desirable to provide a cover on the top of the microarray in order to use the special structure and fine particles. However, when a cover made of a hydrophobic polymer of the same material as the substrate is used, if a liquid such as a test sample is injected into the inside of the microarray, bubbles are generated on or near the cover surface. Turned out to be. In particular, it has been found that when fine particles are encapsulated in a microarray having a concavo-convex structure, bubbles are likely to be generated on the surface of the cover or in the vicinity thereof.

  An object of the present invention is to provide a microarray capable of preventing the generation of bubbles in a microarray having a cover.

  Another object of the present invention is to provide a method of using such a microarray.

  The present inventors have surprisingly found that the above problem can be solved by making the inner surface of the cover of the microarray hydrophilic, and have completed the present invention.

Therefore, in summary, the present invention has the following features.
(1) A microarray including a substrate and a cover that can be in close contact with a part of the substrate, the inner surface of the cover being hydrophilic and the cover having at least one hole therethrough, A microarray, wherein the substrate includes a plurality of concavo-convex structures, and a selective binding substance is immobilized on an upper surface of the convex portion.
(2) The microarray according to (1), wherein the cover is made of a polymer.
(3) The microarray according to (1) or (2), wherein an inner surface of the cover includes a hydrophilic polymer.
(4) The microarray according to (3), wherein the hydrophilic polymer is formed by graft polymerization.
(5) The microarray according to (4), wherein a hydrophilic polymer is covalently bonded to the surface of the cover with high energy rays.
(6) The microarray according to any one of (3) to (5), wherein the hydrophilic polymer is polyalkylene glycol.
(7) The microarray according to (1), wherein an inner surface of the cover is hydrophilized by plasma treatment.
(8) The microarray according to (7), wherein the plasma treatment is an oxygen plasma treatment.
(9) The microarray according to (1), wherein the hole of the cover is for injecting a test sample onto the microarray.
(10) The microarray according to (1), wherein there are four holes in the cover.
(11) The microarray according to (1), wherein the cover is bonded to a part of the substrate.
(12) The microarray according to any one of (1) to (11), including a plurality of fine particles in a gap between the cover and the substrate.
(13) The microarray according to (12), wherein the fine particles have a diameter of 10 to 500 μm.
(14) The microarray according to (12) or (13), wherein the fine particles are for stirring the liquid.
(15) The microarray according to any one of (1) to (14), wherein the whole or a part of the substrate is black.
(16) The microarray according to any one of (1) to (15), wherein the whole or a part of the cover is transparent.

  According to the present invention, it is possible to prevent the generation of bubbles on or near the cover surface in a microarray including a substrate having a concavo-convex structure and a cover, and thus the S / N ratio inherent in such a microarray. And improve detection sensitivity.

  Hereinafter, the present invention will be described more specifically.

  The microarray of the present invention is a microarray including a substrate 1 and a cover 2 that can be in close contact with a part of the substrate 1, as shown in FIG. 1, wherein the inner surface of the cover is hydrophilic and the cover is And the substrate includes a plurality of concavo-convex structures, and a selective binding substance is immobilized on the upper surface of the convex portion. Accordingly, the gap 6 is a closed space that does not communicate with the outside except for the plurality of through holes 4.

The substrate usable in the present invention has a fine uneven structure.
The shape of the concave portion and the convex portion is not particularly limited. In particular, the convex portion is preferably a columnar structure such as a prism or a cylinder. Moreover, the shape of the upper surface of the convex portion is preferably a circle or a square such as a tri-octagon. The recesses or protrusions have completely or substantially the same structure, and are preferably alternately arranged regularly. In the case of such a regular arrangement, the shape of the concave portion is determined according to the shape of the convex portion.

  The size of the convex portion is, for example, about 10 to about 200 μm in height and about 50 to about 150 μm in width, but is not limited to such a range. The interval between the protrusions is, for example, about 50 to about 600 μm, and is preferably a space in which a plurality of fine particles can enter. Further, for the reason shown in Japanese Patent Application Laid-Open No. 2004-264289 (Patent Document 1), the height of the convex portion is preferably the same height as the surrounding flat portion. The relationship between the convex portion and the flat portion is shown in FIG.

  The selective binding substance 8 shown in FIG. 3 is immobilized on the upper surface of the convex portion. Therefore, it is necessary to provide a space between the upper surface of the convex portion and the cover so that the selective binding substance and the test substance can be combined. The size of such a space is, for example, about 1 to about 500 μm. A range smaller than this is not preferable because the signal after hybridization becomes extremely small. On the other hand, when the space size exceeds 500 μm, a large amount of liquid is required, and when a very small amount of sample is used, the concentration tends to be low and the signal after hybridization tends to be small.

  Examples of the material for the substrate include inorganic materials such as glass, ceramic, and silicon, and polymers such as polymethyl methacrylate (PMMA), polyethylene terephthalate, cellulose acetate, polycarbonate, polystyrene, polydimethylsiloxane, and silicone rubber. Preferably, it is a synthetic polymer that can be easily molded, such as PMMA.

  Examples of the molding of the substrate include a method such as an injection molding method or a hot embossing method in the case of a polymer, a method such as a sand blasting method in the case of glass or ceramic, and a method used in a known semiconductor process in the case of silicon.

  In the present invention, the selective binding substance is immobilized on the upper surface of the convex portion of the substrate. For this reason, the upper surface of the convex portion may include a functional group (for example, an amino group, a hydroxy group, a carboxyl group, an aldehyde group, or an epoxy group) that can bind to the selective binding substance. In order to introduce such a functional group, the polar group is introduced by, for example, subjecting the surface to plasma treatment or radiation treatment (for example, γ-ray, electron beam, etc.) or further graft polymerization treatment after these treatments. Or a polycation (for example, poly L-lysine, a silane coupling agent, etc.) can be coated.

  Examples of the silane coupling agent include 3-aminopropyltriethoxysilane, 3-aminopropyltrimethoxysilane, 3-aminopropyldiethoxymethylsilane, 3- (2-aminoethylaminopropyl) trimethoxysilane, and the like.

  The substrate can be entirely or partially black. Black means that in the visible light range (wavelength 400 to 800 nm), the spectral reflectance of the black portion of the substrate does not have a specific spectral pattern, is uniformly low, preferably 7% or less, and the black portion This means that the spectral transmittance of the light does not have a specific spectral pattern and is uniformly low, preferably 2% or less. By making at least a part of the substrate black, the S / N ratio can be improved. As a means for blackening, a black material such as carbon black, graphite, titanium black, aniline black, oxides of metals (Ru, Mn, Ni, Cr, Fe, Co, Cu, etc.), Si, This is achieved by mixing carbides of Ti, Ta, Zr, and Cr.

  A preferable substrate that can be used in the present invention and a method for producing the same are disclosed in, for example, Japanese Patent Application Laid-Open No. 2004-264289 (Patent Document 1), Takizawa Atsuko et al., 2005 7-8, Biotechnology Journal, pages 418 to 420 (Non-Patent Document 1). , Etc.

  Furthermore, fine particles 9 can be included in the gap 6 between the substrate and the cover (FIG. 3). The presence of the fine particles 9 enables efficient stirring of the liquid, resulting in a reaction promoting effect. The size of the fine particles is several tens to several hundreds of μm in diameter, but is a size such that a plurality of fine particles can freely move in the gap between the substrate and the cover. Examples of the material of the fine particles include glass, metal, or polymer, and more specifically, glass, ceramic (for example, yttria stabilized zirconia), stainless steel, nylon, and polystyrene. Among these, ceramic fine particles are preferable because they are chemically stable and have a large specific gravity. By adding such fine particles during hybridization and moving the beads, the liquid can be efficiently stirred. As means for moving the fine particles, there are a method of rotating the substrate and dropping the fine particles in the direction of gravity, and a method of setting the substrate containing the fine particles on a shaker and shaking the substrate. Moreover, the fine particles may be moved by magnetic force using magnetic fine particles. The size (diameter) of the fine particles is not particularly limited, but a range of 10 μm to 500 μm is preferable. If the area is smaller than this range, the effect of stirring may not be sufficiently obtained. If the area is larger than this range, a large space is required between the cover and the substrate described later in order to enclose the fine particles. As a result, the amount of liquid required increases. Further, for example, considering that the liquid is stirred by moving the fine particles by gravity, it is preferable to hold the cover in close contact with the substrate so that the liquid and beads do not spill from the chip. In consideration of operability, it is preferable to enclose fine particles between the substrate and the cover in advance. Further, considering the measurement of the signal (fluorescence) after hybridization, the cover is preferably removable after hybridization.

  The microarray of the present invention is characterized in that the inner surface includes a hydrophilic cover together with the substrate. If the cover inner surface is subjected to a hydrophilic treatment, it is possible to prevent bubbles from remaining in the liquid when the liquid is applied to the space between the substrate and the cover as described above. In particular, when the fine particles are encapsulated in the space between the cover and the substrate in advance, bubbles are likely to remain in the liquid unless the inside of the cover is subjected to a hydrophilic treatment. However, if the inside surface of the cover is subjected to a hydrophilic treatment, bubbles in the liquid can be effectively prevented. The reason for this is thought to be related to static electricity, but the details are unknown.

  The cover is in close contact with a flat portion provided around the uneven portion of the substrate, but can be detached therefrom. For example, if the cover has a gap structure or if the flat portion and the cover are attached via a spacer, a space is generated between the upper surface of the convex portion after the cover is in close contact. The size of this space is as described above. The adhesion can be performed by a method such as providing the adhesive layer 3 (FIGS. 1 and 3) on the flat portion of the substrate and bonding the cover through the adhesive layer. The adhesive layer is not particularly limited as long as it can bond materials such as synthetic resin, glass, silicone, polymer and glass, polymer and silicone, glass and silicone, but after hybridization. In view of the detachability, double-sided tape and PDMS (polydimethylsiloxane) can be preferably used.

  Alternatively, packing can be used instead of the adhesive layer. The packing can be arranged in a form such as bonding to the flat portion of the substrate, or embedding part of the packing uniformly in the flat portion. The shape of the packing includes, but is not limited to, a cutting section having a square shape or a circular shape. The material of the packing is not limited, but an elastomer such as rubber having an appropriate thickness (for example, silicone rubber) is preferable. The fixing of the cover and the substrate includes, but is not limited to, for example, fixing by a protrusion and a receiving hole. Specifically, a plurality of protrusions (for example, columnar shapes) are formed on the surface of the cover, while a receiving hole having an appropriate depth is formed on the flat portion of the substrate so that the protrusions can fit into the surface. The cover and the substrate can be fixed by the receiving hole. Due to such a fixing method, it is also possible to detach the substrate and the cover.

  The material of the cover may be the same as or different from the material of the substrate. For example, inorganic materials such as glass, ceramics, and silicon, polymethyl methacrylate (PMMA), polyethylene terephthalate, cellulose acetate, polycarbonate, polystyrene And polymers such as polydimethylsiloxane and silicone rubber. In particular, since a through-hole described later can be easily formed, a polymer such as PMMA, polycarbonate, polystyrene, or polyethylene terephthalate can be preferably used as the cover material.

  The cover as described above can be manufactured by, for example, an injection molding method or a cutting method, but the injection molding method is preferable in view of mass productivity.

  According to the present invention, the inner surface of the cover, that is, the surface facing the concavo-convex structure of the substrate is hydrophilic. By making it hydrophilic, generation | occurrence | production of the bubble adhering to the inner surface of a cover at the time of applying a liquid is prevented. The presence of bubbles causes a decrease in sensitivity, variation in measurement values, and the like, so that it is difficult to obtain accurate and reliable measurement results. The hydrophilic property means a property that is easy to adjust to water. Generally, as the hydrophilic property becomes higher, the contact angle formed between the water droplet and the cover surface becomes smaller. In order to obtain the effect of the present invention, the contact angle is usually 60 degrees or less, preferably 55 degrees or less, more preferably 50 degrees or less.

  In order to impart hydrophilicity to the cover, a hydrophilic polymer is coated on the inner surface of the cover, or a hydrophilic polymer is grafted on the surface by graft polymerization, or the cover material is, for example, polymethyl methacrylate (PMMA) In this case, it is possible to generate a hydrophilic group such as a carboxyl group in the side chain by immersing in a strong alkali.

  The hydrophilic polymer in the present invention means a water-soluble polymer or a polymer that is not readily water-soluble but has hydrophilicity. Specific examples include polyvinyl alcohol, carboxymethyl cellulose, ethylene-vinyl alcohol copolymer, polyhydroxyethyl methacrylate, poly α-hydroxy vinyl alcohol, polyacrylic acid, poly α-hydroxy acrylic acid, polyvinyl pyrrolidone, polyethylene glycol and polypropylene glycol. And polyalkylene glycols such as potato starch, corn starch, wheat starch and the like, glucomannan, silk fibroin, silk sericin, agar, gelatin, egg white protein, sodium alginate and the like. These sulfonated products can also be used.

  As the hydrophilic polymer of the present invention, polyalkylene glycol is preferred. The polyalkylene glycol is, for example, a chain polymer containing an oxygen atom in the main chain represented by polyethylene glycol or polypropylene glycol, but may be a polymer grafted with polyalkylene glycol. The molecular weight of the polyalkylene glycol is not particularly limited, and a number average molecular weight of 600 to 4000000 is preferable, and a molecular weight of about 10,000 to 1000000 is particularly preferably used.

  The coating of the hydrophilic polymer can be performed by uniformly applying the polymer to the inner surface of the cover. Briefly, in this method, for example, a solution in which a hydrophilic polymer is dissolved in an organic solvent is uniformly applied by a technique such as pouring or brushing on the surface of the cover, and then the solvent is evaporated and removed. Including.

  Alternatively, a hydrophilic polymer can be formed on the surface of the cover using graft polymerization. Briefly, reactive species (especially radicals) are formed on the surface of the cover by radiation (eg, γ-ray, electron beam, etc.) irradiation, light irradiation, plasma treatment, etc. The hydrophilic monomer is polymerized to graft the hydrophilic polymer onto the surface of the cover.

  In particular, in the present invention, it is preferable to covalently bond a hydrophilic polymer to the surface of the cover with high energy rays. For example, the cover is first immersed or brought into contact with a solution of a hydrophilic polymer, preferably polyalkylene glycol, and then irradiated with a high energy beam such as a gamma ray or an electron beam. When a polyalkylene glycol solution is used as the hydrophilic polymer, the temperature of the polyalkylene glycol solution is not particularly limited, but is preferably 0 ° C. or higher and 30 ° C. or lower, more preferably 10 ° C. or higher and 25 ° C. or lower. The solvent for preparing the polyalkylene glycol solution is not particularly limited, and water, methanol, ethanol, acetone, or the like is used as a good solvent, but water is preferably used from the viewpoint of cost and safety.

High energy rays in the present invention are energy rays having a certain amount of energy, and microwaves, infrared rays, visible rays, ultraviolet rays, X-rays, gamma rays, electron rays, proton rays, and neutron rays are also included in the high energy rays. . Among them, the gamma ray is an electromagnetic wave having a wavelength of 10 ⁻¹² to 10 ⁻¹⁵ m, and is excellent in transparency, and therefore, it is preferable because a large amount of cover can be grafted at a time.

  The irradiation amount of the high energy ray is not particularly limited, and it is sufficient that the irradiation amount is sufficient to immobilize the polyalkylene glycol chain on the cover to be hydrophilic. When using gamma rays, the absorbed energy is usually 100 kGy or less. Preferably, 40 kGy or less is used, and more preferably 10 kGy or less is used because the resin substrate is less affected by yellowing of the test sample.

  Alternatively, the surface of the cover may be hydrophilized by plasma treatment. Plasma treatment is generally used as a method for making the surface of a material such as a hydrophobic polymer or glass hydrophilic. This method involves placing the material between two electrodes and causing a discharge in an atmosphere of oxygen, nitrogen, air, etc., if necessary under reduced pressure, thereby forming polar groups on the surface of the material and making it hydrophilic. To be improved. Since various types of plasma generators are commercially available, they can be used as appropriate. Furthermore, in order to enhance the hydrophilicity retention effect, the material is immersed in a polar organic solvent such as methanol or acetone (Mitsuhide Yoshikawa and Tsutomu Shinoda, Tokyo Metropolitan Industrial Technology Research Institute Report No. 3, pages 165-166 (2000) )) Alternatively, it is desirable to graft the hydrophilic polymer by graft polymerization as described above.

  Among the above hydrophilization means, the method of irradiating the cover with a polyalkylene glycol solution or irradiating a high energy beam such as a gamma ray or an electron beam is compared with a coating method or a plasma treatment to measure fluorescence. In this case, since there is almost no eluate that causes noise, it can be particularly preferably used.

  The cover can be transparent in whole or in part. By making it transparent, it is possible to visually capture the injection amount of the test sample and to confirm the state of stirring by the fine particles. As long as it can be so, only a part of the cover can be transparent, not the whole. Moreover, as long as it can be transparent and can confirm the state inside the substrate, it may be colored and transparent, but is preferably colorless and transparent.

  The cover may cover at least part of the surface of the selective binding substance-immobilized carrier, and may be bonded so as to have a gap between the carrier and the cover. The carrier preferably has a selective binding substance immobilized on a region of the carrier located on the surface of the carrier. That is, preferably, the cover is bonded to a part of the carrier so that the region where the selective binding substance is immobilized exists in the gap. The cover may be bonded in any manner as long as the gap is formed, but is preferably bonded through an adhesive layer 3 such as a double-sided tape or a resin composition.

  The cover may have one or more through holes communicating with the gap, and preferably has a plurality of through holes. This hole is for injecting a liquid such as a test sample and a binding buffer, and at the same time, for maintaining the pressure inside the substrate at atmospheric pressure. It is preferable that there are a plurality of through-holes with respect to one gap, and among these, it is particularly preferable that the number of through-holes is 3 to 6 because the sample solution can be easily filled. As will be described later, when the gap is divided into a plurality of spaces that do not communicate with each other, it is preferable that each space has a plurality, more preferably 3 to 6, through holes. When the cover has a plurality of through holes, the diameters thereof may be the same or different, but one of the plurality of through holes serves as an apply port, and the other through hole functions as an air vent. In this case, from the viewpoint of easy application of the sample solution and hermetic retention of the sample solution, it is preferable that only the apply port has a wide hole diameter necessary for the application and other through holes have a narrow hole diameter. Specifically, the through-hole size of the apply port is preferably in the range of 0.01 to 2.0 mm in diameter, and the diameters of the other through-holes are preferably 0.01 to 1.0 mm.

  At least one of the through-holes 4 may be provided with a portion having a large diameter at the upper end, that is, a so-called liquid level holding chamber 5 by changing the diameter thereof (FIG. 4). By providing the liquid level holding chamber, the rise of the liquid level of the sample solution applied from the through hole 4 and filled in the gap 6 is suppressed, and sealing when the through hole is sealed with the sealing member 7 is easy. This is preferable because it can be performed reliably and a large number of bubbles can enter the sample solution or the sample solution can be prevented from flowing out. The shape of the liquid level holding chamber is not particularly limited, and may be a cylindrical shape, a prismatic shape, a conical shape, a pyramid shape, a hemispherical shape, or a shape similar thereto. Among these, the columnar shape is particularly preferable from the viewpoints of ease of manufacture and the high effect of suppressing the rise in the sample solution.

  The hole diameter size of the through hole is not particularly limited. For example, in the case of a combination of the cylindrical through hole 4 having a longitudinal cross-sectional shape and the liquid level holding chamber 5 shown in FIG. The hole diameter size (diameter) of the hole 4 is preferably 0.01 to 2.0 mm, and more preferably 0.3 to 1.0 mm. By setting the pore diameter to 0.01 mm or more, it is possible to easily apply the sample solution. On the other hand, by setting the diameter of the through hole 4 to 1.5 mm or less, it is possible to more effectively suppress the evaporation of the sample solution before sealing after the application. About the hole diameter size (diameter) of the chamber 5 for liquid level parking, 1.0 mm or more is preferable. By setting it to 1.0 mm or more, a sufficient difference in size from the through hole 4 can be obtained, and a sufficient liquid level retaining effect can be obtained, which is preferable. The upper limit of the diameter of the liquid level chamber 5 is not particularly limited, but can be 10 mm or less. Further, the depth of the liquid level holding chamber 5 is not particularly limited, but can be within a range of 0.1 to 5 mm.

  Such a cover is preferably detachably bonded to the above-described selective binding substance-immobilized carrier. When the analysis chip of the present invention is used as a DNA chip, it is usually necessary to read the DNA chip with a dedicated scanner. However, when the cover is adhered, it is difficult to set in the dedicated scanner. However, if the scanning operation is performed, the cover and the optical parts of the scanner may come into contact with each other, which may cause a failure. Even if reading through the cover is possible, the reading value may be inaccurate. Therefore, it is preferable that the cover is removable so that the cover can be removed in the reading step.

  A mode in which the cover is detachably bonded to the selective binding substance-immobilized carrier is not particularly limited, but a mode in which the cover and the carrier can be detached without being damaged is preferable. For example, it can adhere | attach via the adhesive layers 3, such as a double-sided tape and a resin composition.

  When using a double-sided tape as the adhesive layer, it is preferable to use a double-sided tape with different adhesive strengths on both sides. Specifically, the surface with low adhesive strength of the double-sided tape is bonded to the carrier side, It is preferable to adhere to the cover side. By adopting such an embodiment, when the cover is peeled off, the double-sided tape is easily detached from the carrier in a state where the double-sided tape is adhered to the cover, thereby inconvenience in the reading process due to the remaining adhesive layer on the carrier. It can be avoided. As such a double-sided tape, Nitto Denko Corporation product number No. 535A, product numbers 9415PC and 4591HL manufactured by Sumitomo 3M Limited, and product numbers No. 7691 etc. are mentioned.

  When using a resin composition as an adhesive layer, as the resin composition, a resin composition containing a polymer selected from the group consisting of acrylic polymers, silicone polymers, and mixtures thereof can be used. By using these resin compositions, it becomes possible to improve the sealing property as compared with the double-sided tape, and it is more stable for long-term incubation than the double-sided tape. It is particularly preferred in analytical systems that require a period of incubation. In particular, when a silicone-based elastomer is used as the adhesive layer, the sealing property is good and the cover can be bonded in a state where it can be easily detached. Specific examples of such an elastomer include Sylgard (Silgard is a registered trademark of Dow Corning) and two-component RTV rubber (for mold making) manufactured by Shin-Etsu Chemical Co., Ltd.

  The shape of the cover is not particularly limited as long as it covers at least part of the surface of the selective binding substance-immobilized carrier and can be bonded so as to have a gap between the carrier and the cover. In the outer peripheral portion, a structure having a protruding portion at a portion far from the carrier than a portion near the carrier, that is, an overhang structure may be provided. Providing an overhang structure is preferable because it is easy to remove the cover without damaging the carrier. In the microarray of the present invention, the selective binding substance is immobilized on the upper surface of the convex portion of the substrate.

  The selective binding substance refers to a substance that can selectively bind to a test substance directly or indirectly. Examples include nucleic acids, proteins, saccharides, or other antigenic compounds. Nucleic acids include DNA, RNA, PNA, cDNA, cRNA and the like. Proteins include antibodies, fragments thereof, antigens, and enzyme substrates. The saccharide includes an oligosaccharide and a polysaccharide. Other antigenic compounds include peptides and small molecules. Preferred selective compounds are nucleic acids and proteins (especially antibodies, antigens, etc.). In this respect, preferred examples of the microarray of the present invention are a DNA microarray (also referred to as a DNA chip) or a protein microarray. Such a selective binding substance may be commercially available, or may be synthesized or prepared from a natural source such as a living tissue or a cell.

  For DNA such as a gene whose characteristics and primary structure (base sequence) are clear, a probe or primer is prepared based on the sequence, and the target DNA can be selected from, for example, a cDNA library prepared from a biological tissue. Alternatively, total RNA can be extracted from a biological tissue, poly A (+) RNA (ie, mRNA) can be prepared using an oligo dT column, and cDNA and further cRNA can be produced by cDNA cloning. The obtained nucleic acid can be detected by a known method such as Southern or Northern blotting method, Southern or Northern hybridization method, cleavage and mapping with a restriction enzyme, sequencing or the like. In addition, amplification of the obtained nucleic acid can be performed by polymerase chain reaction (PCR), gene recombination technology (for example, use of a vector) or the like. Alternatively, DNA of 100 mer or less can be synthesized using a DNA synthesizer. The above series of techniques is described in, for example, Ausbel et al., Current Protocols in Molecular Biology, John Willy & Sons, US (1993); Sambrook et al., Molecular Cloning A Laboratory, Laboratories. be able to.

  Proteins can be purified from nature according to literature methods or synthesized by genetic recombination techniques (vector / host systems). By immunizing non-human mammals such as rabbits, mice and goats using the protein as an antigen, an antibody against the protein can be produced. Further, in mice such as mice, monoclonal antibodies can be produced by a method including fusion of spleen cells and myeloma cells that have been immunostimulated with the target antigen. Antibodies include polyclonal antibodies, monoclonal antibodies, anti-peptide antibodies and the like. These antibodies can be prepared using a known method.

  For monoclonal antibodies, see, for example, Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyzes, Plenum Press, US (1980); Ikuzaki Ikuo et al., Monoclonal Antibody Hybridoma and ELISA, For example, reference can be made to Naohisa Matsuhashi et al., Introduction to Immunology Experiments (Biochemical Experiment Method 15), and Academic Publishing Center (1982).

  About anti-peptide antibody, since Takara Bio (Kyoto) etc. has commissioned synthesis, it is also possible to obtain it using it. Briefly, the primary sequence of a protein is predicted to be mobile, hydrophilic / hydrophobic, secondary structure prediction, polarity prediction, etc. to predict the site located on the protein surface, Predict that there is a homology search (DNASIS software) and determine the peptide sequence; then, based on that sequence, synthesize the peptide by peptide synthesis; immunize animals such as rabbits; isolate anti-peptide antibodies from blood And purified using an affinity column or the like.

  Saccharides can be obtained by chemically synthesizing or by cleaving from glycoproteins with glycosidases.

  In order to immobilize the selective binding substance on the substrate, for example, a known method such as a spotting method using a capillary pen or an ink jet method can be used.

  The spotting method is a method of spotting a selective binding substance using a high-density dispenser called a spotter or an arrayer. Specifically, for example, a different solution is put in each well of a plate having a large number of wells, and this solution is picked up by a pin (needle) and sequentially spotted on the substrate.

  The ink jet method is a method in which a minute droplet is ejected from a nozzle by a piezoelectric element or the like and a selective binding substance is sprayed on a substrate. Specifically, genes are ejected from a nozzle, and selective binding substances are arranged and arranged at high speed on a substrate.

  Alternatively, when the selective binding substance is a nucleic acid, nucleotide synthesis can be sequentially performed on the substrate by a photolithographic method.

  The present invention further provides a method of using the above-described microarray of the present invention for measuring the presence or amount of a substance in a test sample capable of directly or indirectly binding to a selective binding substance immobilized thereon. I will provide a.

The selective binding substance is the nucleic acid, protein, saccharide or other antigenic compound described above.
The test sample is a biological sample. The biological sample includes, for example, biological samples derived from plants and animals, biological samples such as tissues, cells and body fluids derived from mammals including humans. Specific examples include human disease-related genes and their expression products (proteins). Furthermore, biological samples include prokaryotic organisms such as bacteria, biological samples derived from other eukaryotic organisms such as yeast, basidiomycetes, algae and insects, samples derived from viruses, and the like.

  The measurement includes detecting the binding between the selective binding substance immobilized on the substrate and the substance in the test sample. When the selective binding substance is a nucleic acid, the measurement is based on DNA / DNA hybridization, DNA / RNA hybridization or RNA / RNA hybridization. When the selective binding substance is a protein, the measurement is based on an antigen-antibody reaction, that is, an immunological reaction. Conditions such as reaction temperature, time, and buffer are appropriately selected according to the type and chain length of the nucleic acid to be hybridized, the type of antigen and / or antibody involved in the immune reaction, and the like.

  Hybridization is generally performed under stringent conditions. Such conditions include, but are not limited to, for example, hybridization at 30-50 ° C., 3-4 × SSC, 0.1-0.5% SDS for 1-24 hours, followed by 2 × SSC and 0. Washing with a solution containing 1% SDS can be included. Here, 1 × SSC is a solution (pH 7.2) containing 150 mM sodium chloride and 15 mM sodium citrate.

  In a DNA microarray, cDNA is synthesized from mRNA extracted from a cell or tissue of a living organism, labeled with a Cy dye, and then used as a sample for hybridization with DNA on a substrate.

  The immunological reaction is, for example, a reaction between an antibody immobilized on a substrate and an antigen in a test sample. Detection is performed by measuring an immune complex of an antibody and an antigen, for example, using a spectroscopic method. As the measurement, for example, an enzyme immunoassay method (EIA, ELISA), a radioimmunoassay method, a fluorescent antibody method, or the like, a method using a label with an enzyme, a radioisotope or a fluorophore may be desirable. For example, after binding an antibody on a substrate and an antigen in a sample, a labeled antibody that can bind to an antigen of the formed immune complex (ie, a secondary antibody) is allowed to act on the basis of the intensity of the label. The amount or presence of the target antigen can be measured. It is also possible to immobilize the antigen in advance on the substrate instead of the antibody.

  After the above hybridization or immune reaction, the microarray is scanned using a scanner, and the intensity or presence of a signal such as fluorescence emitted from the label is measured. If necessary, analyze the data with a computer.

  Since the microarray of the present invention is substantially free of bubbles on or near the cover surface, the microarray characterized by a substrate having a concavo-convex structure inherently has a good S / N ratio, high detection sensitivity, etc. Maintains characteristics, improves measurement variability, and enables accurate and reliable measurements.

  The invention is illustrated in more detail by the following examples. However, the present invention is not limited to the following examples.

Example 1
(Preparation of DNA immobilization carrier)
A mold for injection molding was prepared by using a known method, LIGA (Lithographie Galvanforming Abforming), and a carrier made of PMMA having a shape as described later was obtained by the injection molding method. The average molecular weight of PMMA used in this example is 50,000, carbon black (Mitsubishi Chemical # 3050B) is contained in PMMA at a ratio of 1% by weight, and the carrier is black. When the spectral reflectance and spectral transmittance of this black carrier were measured, the spectral reflectance was 5% or less at any wavelength in the visible light region (wavelength of 400 nm to 800 nm), and in the same range of wavelengths, The transmittance was 0.5% or less. Neither spectral reflectance nor spectral transmittance had a specific spectral pattern (such as a peak) in the visible light region, and the spectrum was uniformly flat. Note that the spectral reflectance is the spectrum when specularly reflected light from a carrier is captured using an apparatus (manufactured by Minolta Camera, CM-2002) equipped with an illumination / light-receiving optical system conforming to condition C of JIS Z 8722. The reflectance was measured.

  The shape of the carrier was 76 mm in length, 26 mm in width, and 1 mm in thickness, and the surface was flat except for the central part of the carrier. In the center of the carrier, a concave part with a length of 22 mm and a depth of 0.15 mm is provided. In this recess, a convex part with a diameter of 0.15 mm and a height of 0.15 mm is 1296 (36 × 36). A place was provided. When the height difference between the height of the upper surface of the convex portion of the concave and convex portion (the average value of the height of 1296 convex portions) and the flat portion was measured, it was 3 μm or less. In addition, variations in the heights of the top surfaces of 1296 convex portions (the difference between the height of the top surface of the highest convex portion and the height of the top surface of the lowest convex portion), and the average value of the heights of the top surfaces of the convex portions and the flat portions The difference in height of the upper surface was measured and found to be 3 μm or less. Furthermore, the pitch (the distance from the central part of the convex part to the central part of the adjacent convex part) of the convex part of the uneven part was 0.5 mm.

  The PMMA carrier was immersed in a 10N aqueous sodium hydroxide solution at 70 ° C. for 12 hours. This was washed with pure water, 0.1N HCl aqueous solution, and pure water in this order to generate carboxyl groups on the surface of the carrier.

(Immobilization of probe DNA)
DNA having a base sequence represented by SEQ ID NO: 1 (60 bases, 5 ′ terminal amination) was synthesized. This DNA is aminated at the 5 ′ end.

This DNA was dissolved in pure water at a concentration of 0.3 nmol / μl to obtain a stock solution. When spotting on a carrier, PBS (NaCl 8 g, Na ₂ HPO ₄ · 12H ₂ O 2.9 g, KCl 0.2 g, KH ₂ PO ₄ 0.2 g in pure water was made up to 1 l. To adjust the final concentration of probe DNA to 0.03 nmol / μl by adding hydrochloric acid for adjusting pH to pH 5.5) and condensing the carboxylic acid on the carrier surface with the amino group at the end of the probe DNA 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) was added to give a final concentration of 50 mg / ml. Then, these mixed solutions were spotted on the entire upper surface of the carrier convex portion by an arrayer (manufactured by Nippon Laser Electronics; Gene Stamp-II). Next, the carrier was put in a sealed plastic container, incubated at 37 ° C. and 100% humidity for about 20 hours, and washed with pure water.

(Preparation of sample DNA)
As the sample DNA, DNA having a base sequence represented by SEQ ID NO: 4 (968 bases, hereinafter also referred to as DNA of SEQ ID NO: 4) capable of hybridizing with the probe DNA immobilized on the DNA immobilization carrier was used. . The preparation method is shown below.

  DNA having the base sequence represented by SEQ ID NO: 2 (hereinafter also referred to as DNA of SEQ ID NO: 2) and DNA having the base sequence represented by SEQ ID NO: 3 (hereinafter also referred to as DNA of SEQ ID NO: 3) were synthesized. . This was dissolved in pure water to make the concentration 100 μM. Next, pKF3 plasmid DNA (Takara Bio Inc. product number: 3100) (DNA having the base sequence represented by SEQ ID NO: 5: 2264 bases) is prepared, and this is used as a template. SEQ ID NO: 2 and SEQ ID NO: 3 Amplification was carried out by PCR reaction (Polymerase Chain Reaction) using the DNA of.

  The conditions for PCR are as follows. That is, ExTaq 2 μl, 10 × ExBuffer 40 μl, dNTP Mix 32 μl (the above is attached to product number RR001A manufactured by Takara Bio Inc.), DNA solution of SEQ ID NO: 2 is 2 μl, DNA solution of SEQ ID NO: 3 is 2 μl, template (SEQ ID NO: 0.2 μl of DNA having the base sequence represented by 5) was added, and the volume was made up to 400 μl with pure water. These mixed solutions were divided into four microtubes, and PCR reaction was performed using a thermal cycler. This was purified by ethanol precipitation and dissolved in 40 μl of pure water. When a part of the solution after the PCR reaction was taken and confirmed by electrophoresis, it was confirmed that the base length of the amplified DNA was about 960 bases and that the DNA of SEQ ID NO: 4 (968 bases) was amplified.

  Subsequently, a 9-base random primer (manufactured by Takara Bio Inc .; product number 3802) was dissolved at a concentration of 6 mg / ml, and 2 μl was added to the DNA solution purified after the PCR reaction. This solution was heated to 100 ° C. and then rapidly cooled on ice. To this, 5 μl of the buffer attached to Klenow Fragment (Takara Bio Inc .; product number 2140AK) and 2.5 μl of dNTP mixture (dATP, dTTP, and dGTP concentrations of 2.5 mM and dCTP concentration of 400 μM, respectively) were added. . Furthermore, 2 μl of Cy3-dCTP (manufactured by Amersham Pharmacia Biotech; product number PA53021) was added. To this solution, 10 U of Klenow Fragment was added and incubated at 37 ° C. for 20 hours to obtain sample DNA labeled with Cy3. Since the random primer is used for labeling, the length of the sample DNA varies. The longest sample DNA is the DNA of SEQ ID NO: 4 (968 bases). When the sample DNA solution was taken out and confirmed by electrophoresis, the strongest band appeared in the vicinity corresponding to 960 bases, and the region corresponding to a shorter base length was thinly smeared. This was purified by ethanol precipitation and dried.

  This labeled sample DNA is obtained by diluting 1% by weight BSA (bovine serum albumin), 5 × SSC (5 × SSC is 20 × SSC (manufactured by Sigma) 4 times with pure water. 20 × SSC diluted twice with pure water is expressed as 10 × SSC, 20 × SSC 2 × diluted solution is expressed as 10 × SSC, and 100 × diluted solution is expressed as 0.2 × SSC). A 0.1 wt% SDS (sodium dodecyl sulfate), 0.01 wt% salmon sperm DNA solution (each concentration is a final concentration) and 400 μl were dissolved to obtain a stock solution for hybridization.

  In the following examples and comparative examples, the sample solutions used for hybridization were 1 wt% BSA, 5 × SSC, 0.01 wt% salmon sperm DNA, unless otherwise specified. What was diluted 200 times with a 0.1 wt% SDS solution (each concentration is a final concentration) was used. The sample DNA concentration in this solution was measured and found to be 1.5 ng / μL.

(Production of cover)
A cover having four through holes shown in FIG. 1 (with an overhang structure on the outer periphery) was produced by an injection molding method.
This was immersed in a polyethylene glycol aqueous solution having a molecular weight of 50,000,1000 ppm, sealed, and irradiated with gamma rays. The cover irradiated with gamma rays was dried and adhered to the carrier so as to cover the probe DNA immobilization region using a double-sided tape (manufactured by Nitto Denko Corporation, No. 535A). The contact angle between the cover and water at this time was 50 degrees.

(Hybridization)
The sample DNA was hybridized to the carrier on which the probe DNA obtained above was immobilized. Specifically, 50 mg of 180 μm diameter zirconia beads (manufactured by Tosoh Corporation; TZB180) was loaded in advance from the through-hole of the cover. Next, 100 μl of hybridization solution is injected from the through hole using a micropipette. Then, the through hole was closed with Kapton tape (As One 5-5018-01), and this was fixed in a plastic container provided on the rotating surface of a microtube rotator (manufactured by As One, product number: 1-4096-01). Incubated at 0 ° C. for 16 hours. At that time, the rotation speed of the rotator was 3 rpm, and the rotation surface of the rotator was set to be perpendicular to the horizontal plane. Furthermore, the probe DNA immobilization surface of the carrier was set to be perpendicular to the rotation surface of the rotator. By doing so, the glass beads continue to move in the direction of gravity, and hybridization can be performed while stirring the sample solution. After incubation, the carrier and the double-sided tape were removed from the carrier, and the carrier was washed and dried.

(Measurement)
The carrier after the above treatment was set in a scanner for DNA chip (GenePix 4000B manufactured by Axon Instruments), and measurement was performed with the laser output set to 33% and the photomultiplier voltage set to 500. Here, the fluorescence intensity is an average value of the fluorescence intensity in the spot, and the background noise is the fluorescence intensity of the convex portion where the probe DNA is not immobilized. The results are shown in Table 1. Sufficient fluorescence signal was obtained and background noise was kept low. Although 20 DNA chips were used, the operation was simple and no bubbles remained.

Comparative Example 1
An experiment was conducted in the case where a cover that was not graft-polymerized was adhered to a carrier. Experiments were conducted in the same manner as in Example 1 except that the cover shown in FIG. 2 manufactured by cutting was bonded to a single body without any treatment. The contact angle of the cover with water at this time was 66 degrees. The results are shown in Table 1.

  When injecting the sample solution, bubbles remained in 13 of the 20 probe DNA immobilization regions, and the carrier in which bubbles were generated was uneven in the signal after hybridization.

Example 2
Experiments were performed using a cover that had been subjected to oxygen plasma treatment. The cover surface was washed with ethanol and dried, and then set in a vacuum chamber of a reactive ion etching apparatus (Samco Co., Ltd .: RIE-10NR). Next, after evacuating to a high vacuum (3 × 10 ⁻³ Pa), oxygen plasma treatment was performed for 20 seconds under the conditions of an oxygen gas flow rate of 100 sccm, an input power of 100 W, and a chamber pressure of 10 Pa. The experimental procedure was the same as in Example 1 except for the cover processing method. The contact angle of the cover with water at this time was 52 degrees. The results are shown in Table 1. Sufficient fluorescence signal was obtained, but background noise increased. The reason why the background noise increased is thought to be that the resin that reacted with oxygen on the cover surface eluted into the liquid. Although 20 DNA chips were used as in Example 1, the operation was simple and no bubbles remained.

Comparative Example 2
The same experiment as in Example 1 was performed, except that the untreated cover was adhered to the carrier and the beads were not filled. The results are shown in Table 1. When the sample solution was injected, air bubbles remained in the probe DNA immobilization region in 2 out of 20 samples. However, even with a carrier that did not generate bubbles, the signal after hybridization was uneven. On the other hand, the fluorescence signal after hybridization was significantly lower than when beads were loaded.

  In the present invention, stirring with beads during the hybridization reaction is indispensable in order to obtain a sufficient signal intensity after hybridization and to make the signal uniform.

In Table 1, the CV value is an index indicating the degree of variation in signal intensity, and the following formula:
CV value (%) = (standard deviation of signal intensity / average of signal intensity) × 100
It is represented by It can be said that the smaller the CV value, the higher the quality of the DNA chip. In the comparative example, the CV value was increased due to the generation of bubbles.

  According to the present invention, since the microarray of the present invention is substantially free of bubbles on or near the cover surface, the microarray characterized by a substrate having a concavo-convex structure inherently has a good S / N ratio, high Maintain characteristics such as detection sensitivity, improve measurement value variation, and enable accurate and reliable measurement. For this reason, it is possible to improve the precision of microarrays that are frequently used in fields such as biology, medicine, and microbiology, which is very useful industrially.

The schematic and sectional drawing of the microarray of this invention containing a board | substrate and a cover. Schematic and cross-sectional views of a substrate constituting the microarray of the present invention. The longitudinal cross-sectional view which shows an example of the microarray of this invention roughly. The fragmentary sectional view which shows an example of the through-hole in the cover which comprises the microarray of this invention, and a liquid level parking chamber.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Substrate 2 Cover 3 Adhesive layer 4 Through-hole 5 Liquid level holding chamber 6 Void 7 Sealing member 8 Selective binding substance 9 immobilized on substrate 9 Fine particles (beads)

SEQ ID NO: 1: Plasmid pKF3 DNA
SEQ ID NO: 2: Primer for amplification of plasmid pKF3 DNA SEQ ID NO: 3: Primer for amplification of plasmid pKF3 DNA SEQ ID NO: 4: Plasmid pKF3 DNA
SEQ ID NO: 5: plasmid pKF3 DNA

Claims (16)

  A microarray comprising a substrate and a cover that can be in close contact with a portion of the substrate, wherein the inner surface of the cover is hydrophilic and the cover has at least one hole therethrough, The microarray is characterized in that a selective binding substance is fixed on the upper surface of the convex part.   The microarray of claim 1, wherein the cover is made of a polymer.   The microarray according to claim 1 or 2, wherein an inner surface of the cover includes a hydrophilic polymer.   The microarray according to claim 3, wherein the hydrophilic polymer is formed by graft polymerization.   The microarray according to claim 4, wherein a hydrophilic polymer is covalently bonded to the surface of the cover with a high energy beam.   The microarray according to claim 3, wherein the hydrophilic polymer is polyalkylene glycol.   The microarray according to claim 1, wherein an inner surface of the cover is hydrophilized by plasma treatment.   The microarray according to claim 7, wherein the plasma treatment is an oxygen plasma treatment.   The microarray according to claim 1, wherein the hole of the cover is for injecting a test sample onto the microarray.   The microarray according to claim 1, wherein there are four holes in the cover.   The microarray according to claim 1, wherein the cover is bonded to a part of the substrate.   The microarray according to any one of claims 1 to 11, comprising a plurality of fine particles in a gap between the cover and the substrate.   The microarray according to claim 12, wherein the fine particles have a size of 10 to 500 μm in diameter.   The microarray according to claim 12 or 13, wherein the microparticles are for stirring the liquid.   The microarray according to any one of claims 1 to 14, wherein the whole or a part of the substrate is black. The microarray according to any one of claims 1 to 15, wherein all or part of the cover is transparent.

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