JP2008089414A - Analysis chip - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To solve a problem in an analysis chip formed of a substrate and a cover member stuck to the substrate wherein, when particulates are filled into a clearance surrounded by the substrate and cover, the generation of static electricity makes the filling of the particulates into the clearance difficult, and the reaction with specimen solution becomes insufficient due to fixation of the filled particulates, mixing of air bubbles into the clearance, or the like. <P>SOLUTION: The analysis chip is formed of the substrate and the cover member stuck to the substrate and has the clearance between the substrate and cover member. The particulates are filled into the clearance, and the center line average roughness (Ra value) of the surfaces of the particulates is set ≥40 nm and ≤300 nm. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to an analysis chip that can be used when performing an analysis based on a reaction between a test substance and a selective binding substance, in which fine particles are enclosed in a space holding a solution of the test substance.

  Research on genetic information analysis of various organisms has begun. Information on human genes and many other genes and their base sequences, proteins encoded by the gene sequences, and sugar chains that are secondarily produced from these proteins are rapidly being clarified. The functions of macromolecules such as genes, proteins, and sugar chains whose sequences have been clarified can be examined by various methods. Mainly, nucleic acids can be examined for the relationship between various genes and their biological function expression by methods utilizing complementarity between various nucleic acids / nucleic acids, such as Northern blotting or Southern blotting. On the other hand, the protein can be examined for the function and expression of the protein using a protein / protein reaction typified by Western blotting.

  In recent years, a new analysis method called a DNA chip method (DNA microarray method) has been developed and attracts attention as a method for analyzing many gene expressions at once. A DNA chip is a small device for simultaneously measuring hundreds to tens of thousands of gene expressions, and is a molecule in which molecules such as DNA are arranged at high density on a substrate such as glass or silicon. By using a DNA chip, systematic and comprehensive gene expression analysis in animal models of various diseases and cell biology phenomena can be performed. Specifically, it becomes possible to clarify the function of the gene, that is, the protein encoded by the gene, and to 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 using a DNA chip and constructing a gene expression profile database in combination with physiological, cell biology, and biochemical event data, thereby making disease genes and treatment-related It will be possible to search for genes and search for treatment methods.

  Currently, two basic methods, namely, the GeneChip method and the cDNA microarray method are adopted for the production of DNA chips.

  The GeneChip method is a method developed by Affymetrix, which synthesizes about 25-mer oligo DNA on a glass plate by photolithographic method, and from 16 to 20 25-mer from one nucleotide sequence data per gene. Is set as a probe DNA by combining a 25-mer perfect match and a one-base mismatch oligomer set intentionally different in 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. On the other hand, the cDNA microarray method is a method developed by Stanford University, in which DNA is fixed to a glass plate by a spotting method using a capillary pen or an ink jet method. In both methods, a sample (gene) to be measured, which is fluorescently labeled in advance, is combined with a probe on a DNA chip by hybridization, and the fluorescence intensity is measured using a scanner to measure gene expression. is there.

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

  An analysis chip in which not only DNA but also proteins and saccharides are arranged on a substrate has come to be used as an inspection and analysis means. In particular, in protein chips on which proteins are arranged, proteins such as antibodies, antigens and enzyme substrates are immobilized on the substrate.

A specially-shaped DNA chip having a concavo-convex structure using a polymer as a base material instead of general glass or silicon has been developed (Patent Document 1). By this method, variation in the amount of substance spotted on the upper surface of the convex portion of the substrate is reduced, and as a result, the S / N ratio and detection sensitivity are greatly improved. Furthermore, the presence of the fine particles in 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., Biotechnology Journal: July-August 2005, pages 418-420

  In the analysis chip that includes the substrate and the cover member bonded to the substrate as described above, and has a gap between the substrate and the cover member, and the fine particles are sealed in the gap, When enclosing the fine particles, for example, generation of static electricity may make it difficult to enclose the fine particles from the through hole provided in the cover into the gap. In addition, when a sufficient amount of fine particles cannot be sealed due to generation of static electricity, the stirring efficiency of the solution may not be sufficiently obtained. Furthermore, the encapsulated microparticles may become solidified and may not move. When the sample solution is injected into the space surrounded by the cover and the substrate in such a state, the sample solution does not penetrate into the area where the microparticles are solidified, and the void As a result of bubbles mixed in, reaction unevenness may occur.

  An object of the present invention is to provide an analysis chip that suppresses static electricity generated when enclosing fine particles in voids and facilitates the operation of enclosing fine particles.

  In view of the above problems, the present inventors have intensively studied. As a result, in the analysis chip having a substrate and a cover member bonded to the substrate, and having a gap between the substrate and the cover member, the substrate and the cover The inventors have found that the above problems can be solved by controlling the surface roughness of the fine particles when the fine particles are enclosed in the enclosed voids, and the present invention has been completed.

  That is, the present invention is an analysis chip comprising a substrate and a cover member bonded to the substrate, and having a gap between the substrate and the cover member, in which fine particles are enclosed in the gap, The analysis chip has a surface centerline average roughness, that is, an Ra value of 40 nm to 300 nm.

  A preferred embodiment of the present invention is an analysis chip in which the center line average roughness (Ra value) of the surface of the fine particles is 40 nm or more and 200 nm or less.

  A preferred embodiment of the present invention is an analysis chip in which the material of the fine particles is ceramic or glass.

  A preferred embodiment of the present invention is an analysis chip in which the maximum diameter of fine particles is 10 μm or more and 500 μm or less.

  In the DNA chip of the present invention, when fine particles having a surface centerline average roughness (Ra value) of 40 nm or more and 300 nm or less are used, generation of static electricity is suppressed when the fine particles are enclosed in a space surrounded by the substrate and the cover. Thus, the working efficiency when enclosing fine particles is remarkably improved. Furthermore, since a sufficient amount of fine particles can be easily enclosed in the space surrounded by the substrate and the cover, the solution stirring efficiency during hybridization is remarkably improved, and the S / N ratio and detection sensitivity are improved compared to conventional DNA chips. It can be greatly improved.

  Hereinafter, the present invention will be described more specifically.

  The analysis chip of the present invention is an analysis chip comprising a substrate and a cover member bonded to a part of the substrate, wherein there is a void formed by the substrate and the cover member, and fine particles are enclosed in the void. .

  FIG. 1 shows an example of the analysis chip of the present invention. The analysis chip of FIG. 1 is an analysis chip including a substrate 1 and a cover member 2 bonded to a part of the substrate 1 with an adhesive layer 3, and a fine particle 8 in a gap 6 formed by the substrate 1 and the cover 2. Is enclosed.

  The analysis chip is a chip used to apply a solution containing a test substance to the chip and measure the presence / absence of the test substance, the amount of the test substance, the property of the test substance, and the like. Specifically, a biochip that examines the amount of the specimen and the presence or absence of the specimen by a reaction between the selective binding substance immobilized on the substrate surface and the specimen. More specifically, a DNA chip having a nucleic acid immobilized on the substrate surface, a protein chip having a protein typified by an antibody immobilized on the substrate surface, a sugar chain chip having a sugar chain immobilized on the substrate surface, and a substrate surface Examples thereof include a cell chip on which cells are immobilized. The selective binding substance and its immobilization mode will be described later.

  The substrate of the present invention preferably has a fine uneven structure. The shape of the concave portion and the convex portion is not particularly limited, but the convex portion is preferably a columnar structure such as a prism, a cylinder, or a truncated cone. 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.

Examples of the material of the substrate include inorganic materials such as glass, ceramics, and silicon, and polymers such as polymethyl methacrylate (PMMA), polyethylene terephthalate, cellulose acetate, polycarbonate, polystyrene, polydimethylsiloxane, and silicon 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.

  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 Ti, Ta, Zr, and Cr carbides.

  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 convex portions is, for example, about 50 to about 600 μm, but preferably has a size that allows a plurality of fine particles to enter. In addition, 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 part and the flat part is shown in FIG. 2 and FIG.

  As shown in FIG. 3, the selective binding substance can be immobilized on the upper surface of the convex portion. In this case, 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, 1 to 500 μm in the height direction. If the range is smaller than this, the chance of the test substance coming into contact with the immobilized selective binding substance becomes extremely small, and the signal after hybridization becomes extremely small. On the other hand, if the spatial size exceeds 500 μm, When a very small amount of sample is used, the concentration of the sample solution decreases, the hybridization reactivity decreases, and the signal at the time of detection becomes weak.

  In order to immobilize the selective binding substance on the upper surface of the convex portion of the substrate, the upper surface of the convex portion has 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 material). Etc.). In order to introduce such a functional group, for example, plasma treatment or radiation treatment (for example, γ-ray, electron beam, etc.) is applied to the surface, and then a polar group is introduced by further graft polymerization treatment, or polycation (For example, poly-L-lysine, a silane coupling agent, etc.) can be coated.

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

  A preferable substrate that can be used in the present invention and a method for producing the same are described in, for example, Japanese Patent Application Laid-Open No. 2004-264289 (Patent Document 1), Biotechnology Journal: July-August 2005, pages 418-420 (Non-Patent Document 1). ), Etc.

  The shape of the cover may cover at least a part of the surface of the selective binding substance-immobilized substrate, and may be bonded so as to have a gap between the substrate and the cover member. The substrate preferably has a selective binding substance immobilized on a region of the substrate located in the void. That is, preferably, the cover member is bonded to the substrate so that the region where the selective binding substance is immobilized exists in the gap. The cover member may be bonded in any manner as long as the gap is formed, but is preferably bonded via an adhesive member 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 liquid such as fine particles, test sample, reaction buffer, etc., and at the same time, for maintaining the pressure inside the chip at atmospheric pressure. It is preferable that there are a plurality of through holes with respect to one gap, and among these, 3 to 6 is particularly preferable 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 inlet and the other through hole functions as an air outlet. In this case, from the viewpoint of easy application of the sample solution and hermetic retention of the sample solution, it is preferable that the inlet has a wide hole diameter necessary for the injection of the sample solution and the other through holes have a narrower hole diameter. Specifically, the through hole size of the injection port is preferably in the range of 0.01 to 2.0 mm, and the diameter of the other through holes is preferably in the range of 0.01 to 1.0 mm.

  At least one of the through holes may be provided with a portion having a large diameter at the upper end, that is, a so-called liquid level holding chamber, by changing the diameter thereof. (Herein, the term “parking” means that it is limited to the necessary part.) By providing a liquid level parking chamber, the liquid level of the sample solution applied from the through hole and filled in the gap is suppressed and penetrated. This is preferable because the hole can be easily and surely sealed with the sealing member, 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 with a cylindrical through hole having a longitudinal cross-sectional shape shown in FIG. The pore size (diameter) 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 to 1.5 mm or less, it is possible to more effectively suppress the evaporation of the sample solution before sealing after the application. The pore size (diameter) of the liquid level holding chamber is preferably 1.0 mm or more. By setting the thickness to 1.0 mm or more, a sufficient difference in size from the through hole can be obtained, and as a result, a sufficient liquid level retaining effect is obtained, which is preferable. The upper limit of the diameter of the liquid level chamber is not particularly limited, but can be 10 mm or less. Further, the depth of the liquid level holding chamber is not particularly limited, but can be within a range of 0.1 to 5 mm.

  Such a cover is preferably bonded to the above-described selective binding substance-immobilized substrate in such a manner that it can be removed and attached. 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. If the 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 can be detached so that the cover can be removed in the reading step.

  The analysis chip of the present invention includes fine particles in a space surrounded by the substrate and the cover (FIG. 3). The presence of the microparticles makes it possible to efficiently stir the injected sample solution, and as a result, the effect of promoting the hybridization reaction is brought about.

  The material of the fine particles is not particularly limited, and examples thereof include metals such as glass, ceramic and stainless steel, and polymers such as nylon and polystyrene. Among these, ceramic fine particles are preferably used because they are physically and chemically stable and have a large specific gravity. The ceramic includes fine ceramics such as alumina, zirconia, aluminum nitride, silicon nitride, silicon carbide, sialon, titania, and ferrite. Of these, yttria partially stabilized zirconia is more preferably used.

  As the fine particles used in the analysis chip of the present invention, known or commercially available fine particles can be used as they are. Fine particles comprising yttria partially stabilized zirconia can be produced as follows. The starting powder is produced by a known powder synthesis method such as a chemical synthesis method such as a hydrolysis method, neutralization coprecipitation method, thermal decomposition method, hydrothermal synthesis method, alkoxide method, or oxide mixing method. Powder can be used. Examples of means for forming such powder into fine particles include, for example, a known rolling granulation molding method, press molding method, spray granulation molding method, stirring granulation molding method, CIP molding method, cast molding method, extrusion molding method, and the like. Can be adopted. After forming into a desired size by the forming method, the obtained formed body is sintered in an oxidizing atmosphere or air. The sintering temperature is 1350 to 1500 ° C., and the sintering time is preferably 2 to 3 hours. Further, HIP (Hot Isostatic Pressing) treatment may be performed after the sintering. What is necessary is just to classify to a predetermined particle size with a vibration sieve etc. when adjusting a particle size as needed. When the surface roughness Ra of the fine particles is adjusted after sintering, it may be adjusted within the range of the present invention by, for example, polishing, heat treatment or chemical treatment, and the surface roughness after sintering is within the range of the present invention. It is also possible to use it as it is.

  By moving the microparticles during hybridization, the liquid is efficiently stirred. As a means for moving the fine particles, preferably the method of rotating the chip to drop the fine particles in the direction of gravity, the method of setting the chip containing the fine particles in a shaker and shaking the substrate, the magnetic force using magnetic fine particles The method of moving fine particles is used, but the method of setting the chip on a shaker and swirling and rotating in a horizontal plane has a large movement range of fine particles and moves evenly, so that the liquid can be stirred efficiently as a result. Therefore, it is preferable. At this time, the rotational speed of the turning rotation is preferably 10 to 1000 rotations / minute, more preferably 100 to 500 rotations / minute.

  The fine particles used in the analysis chip of the present invention have a center line average roughness (Ra value) of 40 nm or more and 300 nm or less. By enclosing fine particles having a surface roughness in this range, static electricity is less likely to be generated, and it is possible to efficiently perform the operation of enclosing fine particles. In the case of ceramic fine particles, considering the strength of the material, the Ra value is preferably 40 nm or more and 200 nm or less. If fine particles having a surface centerline average roughness (Ra value) of less than 40 nm are used, static electricity is generated, and the encapsulation efficiency is greatly reduced. Further, when the center line average roughness (Ra value) of the surface of the fine particle is larger than 300 nm, the sample solution becomes turbid after hybridization, and the detection result becomes uneven.

  The center line average roughness representing the surface roughness of the fine particles, that is, the Ra value is the arithmetic average roughness of the roughness curve defined in JIS B0601: 2001. The Ra value is measured by the stylus method in the JIS standard as follows. For the measurement, a stylus electric surface roughness measuring machine made in accordance with JIS B0651 2001 is used. This apparatus is composed of mechanical elements (positioning device, fixture for the object, measuring stand, feeding device, probe) related to the contact between the measurement target surface and the tip of the stylus. In measurement, the target surface is traced with the stylus at the tip of the probe, and the stylus moves up and down with respect to the curve according to the roughness of the symmetry plane. Begins. At this time, the measurement distance (the length of the curve) is defined by JIS B0651 2001, which is referred to as a reference length l (el). The arithmetic average roughness Ra value of the roughness curve is an average of absolute values of the height Z (x) of the roughness curve at an arbitrary position x within the reference length l.

  Further, the center line average roughness (Ra value) of the surface of the fine particles is determined by measuring the surface profile image of the fine particles using a scanning electron microscope when the fine particles of the present invention are to be measured. It can also be measured by calculating the value. This method is preferably used because a more accurate Ra value can be obtained.

  The size (diameter) of the fine particles is not particularly limited, but is preferably 10 μm to 500 μm, and more preferably 50 μm to 300 μm. If the range is smaller than this, the effect of stirring may not be sufficiently obtained. Conversely, if the range is larger than this, it is necessary to increase the gap between the cover and the substrate in order to enclose the fine particles. As a result, the necessary amount of liquid increases, which is not preferable. In addition, considering that the sample and other solutions are placed in the space surrounded by the substrate and the cover and the enclosed fine particles are moved and stirred, the solution and fine particles will not spill from the through-holes in the cover. It is preferable to close the through hole.

  The mode of adhering the cover to the substrate on which the selective binding substance is immobilized is not particularly limited, but as described above, the cover and the substrate can be detached without being damaged. An aspect is preferable, for example, it can adhere | attach through adhesive layers, such as a double-sided tape and a resin composition.

  When double-sided tape is used as the adhesive layer, it is preferable to use double-sided tapes with different adhesive strengths on both sides. Specifically, the surface with weak adhesive strength of the double-sided tape is bonded to the substrate side to cover the surface with strong adhesive strength. Adhering to the side is preferred. By adopting such an embodiment, when the cover is peeled off, the double-sided tape is easily detached from the substrate 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 substrate. 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 Dow Corning Sylgard (registered trademark) and Shin-Etsu Chemical Co., Ltd., two-component RTV rubber for molding.

  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 deoxyribonucleic acid (DNA), ribonucleic acid (RNA), peptide nucleic acid (PNA), complementary DNA (cDNA), complementary RNA (cRNA) and the like. Proteins include antibodies and fragments thereof, antigens, 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 analysis chip of the present invention are a DNA chip (also referred to as a DNA microarray) or a protein chip. 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, polyA RNA (ie, mRNA) can be purified using an oligo dT column, and cDNA or even 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. 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, Naoto Matsuhashi, et al., Introduction to Immunology Experiments (Biochemical Experiment Method 15), and Academic Publishing Center (1982) can be referred to.

  About anti-peptide antibody, since Takara Bio Co., Ltd. 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 a substrate by a photolithographic method.

  The present invention further uses the analysis chip of the present invention described above 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. Provide a method. The selective binding substance here is the nucleic acid, protein, saccharide or other antigenic compound.

  The test sample here 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 are not particularly limited. For example, hybridization at 30 to 50 ° C. in 3 to 4 × SSC, 0.1 to 0.5% SDS for 1 to 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 chip, 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 performing the above-described hybridization or immune reaction, the DNA chip 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 measured data with a computer.

  Since the analysis chip of the present invention is substantially free of bubbles on or near the cover surface, the analysis chip characterized by a substrate having a concavo-convex structure inherently has a good S / N ratio and high detection sensitivity. Maintain the characteristics, improve the dispersion of measured values, and enable accurate and reliable measurement.

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

Example 1
(Production of DNA-immobilized substrate)
A mold for injection molding was produced by using a known method of LIGA (Lithographie Galvanforming Abforming), and a substrate made of PMMA having a shape as described later was obtained by an 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 substrate is black. When the spectral reflectance and spectral transmittance of this black substrate were measured, the spectral reflectance was 5% or less at any wavelength in the visible light region (wavelength 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 a spectrum obtained when specularly reflected light from a substrate is captured using an apparatus (manufactured by Minolta Camera, CM-2002) equipped with an illumination / light-receiving optical system conforming to the condition C of JIS Z 8722. The reflectance was measured.

  The shape of the substrate was 76 mm in length, 26 mm in width, and 1 mm in thickness, and the surface was flat except for the central portion of the substrate. In the center of the substrate, there is a concave part with a length of 22mm and a depth of 0.15mm. In this recess, a convex part with a diameter of 0.15mm and a height of 0.15mm is 1296 (36 x 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 substrate was immersed in a 10N aqueous sodium hydroxide solution at 70 ° C. for 12 hours. This was washed in the order of pure water, 0.1N HCl aqueous solution, and pure water to generate carboxyl groups on the substrate surface.

(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 to a concentration of 0.3 nmol / μL to obtain a stock solution. When spotting on the substrate, PBS (NaCl 8g, Na2HPO4 · 12H2O 2.9g, KCl 0.2g, KH2PO4 0.2g dissolved in pure water and made up to 1L with hydrochloric acid for pH adjustment) In order to condense the carboxylic acid on the substrate surface with the amino group at the end of the probe DNA, the final concentration of the probe DNA is 0.03 nmol / μL by adding 10 times with the added one, pH 5.5) -Ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) was added to bring the final concentration to 50 mg / mL. These mixed solutions were spotted on the entire upper surface of the convex portion of the substrate by an arrayer (manufactured by Nippon Laser Electronics; Gene Stamp-II). The substrate was then placed in a sealed plastic container and incubated for about 20 hours at 37 ° C. and 100% humidity. Finally, it was washed with pure water, dried by centrifugation with a spin dryer. This reaction scheme is shown in FIG.

(Adhesion of cover)
A cover having four through holes shown in FIG. 6 (with an overhang structure on the outer peripheral portion) was produced by an injection molding method.

  Immerse the cover in a cleaning agent (Clean Ace (ASONE catalog, product number: 4-078-01) 25-fold diluted solution) and ultrasonically wash it for 5 minutes, then rinse thoroughly with reverse osmosis water (RO water), and air blow. After drying, the washed cover was adhered to the substrate on which the probe DNA obtained above was fixed with PDMS polymer (Toray Dow Corning Silicone). The bonding condition is 42 ° C. for 2 hours.

(Preparation and encapsulation of fine particles)
A commercially available zirconia fine particle (Toray Industries, Inc.) having a surface centerline average roughness (Ra value) of 20 (nm) and an average particle diameter of 197 μm is centrifuged using a silicon carbide abrasive (particle size # 20). Polishing was performed in water with a polishing machine for 1 hour, washed with water and dried. The surface roughness of the fine particles after polishing was Ra = 165 nm. The measurement of the center line average roughness of the surface of the fine particles is performed by vacuum-depositing the surface with Au, and then the surface center line average roughness (Ra value) using a scanning electron microscope (manufactured by Elionix Co., Ltd., model ESA-2000). ) (Nm) was measured. The centerline average roughness was measured for 10 arbitrary samples with an observation magnification of 10,000 times and a cutoff value of 0, and the average value was obtained. The particle size of such fine particles is obtained by taking an image of an arbitrary 100 or more fine particles with a stereomicroscope at a magnification of 50 to 150 times, and then obtaining an equivalent circle diameter by image processing analysis software (Mitani Corporation, Win Roof). The average value was calculated as the average particle size. Thereafter, it was immersed in an ethanol solution and subjected to ultrasonic cleaning for 5 minutes. Further, the same washing was repeated twice. 120 mg of the fine particles were sealed in the gap between the substrate and the cover from the through hole of the cover.

(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 substrate 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 a concentration of 100 μM. Next, pKF3 plasmid DNA (Takara Bio Inc.) (DNA having the base sequence represented by SEQ ID NO: 5: 2264 bases) is prepared, using this as a template, and the DNAs of SEQ ID NO: 2 and SEQ ID NO: 3 as primers As a result, amplification was carried out by PCR reaction (Polymerase Chain Reaction).

  The conditions for PCR are as follows. Specifically, ExTaq 2 μl, 10 × ExBuffer 40 μl, dNTP Mix 32 μl (manufactured by Takara Bio Inc.), SEQ ID NO: 2 DNA solution 2 μl, SEQ ID NO 3 DNA solution 2 μl, template (SEQ ID NO: 5) 0.2 μl of DNA having a base sequence) was added to make 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.) 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 a buffer attached to Klenow Fragment (manufactured by Takara Bio Inc.) and 2.5 μl of a dNTP mixture (dATP, dTTP, dGTP concentrations were 2.5 mM and dCTP concentrations were 400 μM, respectively) were added. Furthermore, 2 μl of Cy3-dCTP (manufactured by GE Healthcare Bioscience) 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 was 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 a 1% by weight BSA (bovine serum albumin), 5 × SSC (5 × SSC) refers to a solution obtained by diluting 20 × SSC (manufactured by Sigma) 4 times with pure water. Similarly, a solution obtained by diluting 20 × SSC twice with pure water is expressed as 10 × SSC, and a solution diluted 100 × is expressed as 0.2 × SSC), 0.1 wt% SDS (sodium dodecyl sulfate), A solution of 0.01% by weight salmon sperm DNA (each concentration is a final concentration) dissolved in 400 μl was used as a stock solution for hybridization.

  In the following examples and comparative examples, the sample DNA solution for hybridization is 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 of this solution was measured and found to be 1.5 ng / μL.

(Hybridization)
Using a micropipette, 165 μL of the hybridization sample solution was injected into the gap between the substrate and the cover (reaction vessel) from the through hole. At this time, the solution could be easily injected, and bubbles were not mixed. Silicon tape (As One) was used as a sealing material, and four through holes were closed. The hybridization chamber (Takara Hybridization chamber (Takara Bio Inc.)) was fixed in close contact with a sheet shaking table (MMS FIT-S manufactured by Tokyo Rika Kikai Co., Ltd.), and the substrate was set in the hybridization chamber. Then, 15 μL of ultrapure water was dropped into the dents at both ends of the position where the substrate was set, the hybridization chamber was closed, the six fixing screws were tightened, and fixed, and then a constant temperature chamber set at 42 ° C. (Tokyo Rika) Mounted on a shaker (MMS-310, Tokyo Rika Instruments Co., Ltd.) installed in FMS-1000 (manufactured by Instrument Co., Ltd.) The front surface of the thermostatic chamber was shielded with aluminum foil and 250 rpm / Incubate for 16 hours at 42 ° C. with swirling shaking in minutes. After chromatography DOO, hybridization chamber from the substrate is taken out, after elimination of the cover and the PDMS polymer adhered to the substrate, washed and dried.

(Measurement)
The substrate after the above treatment was set on a DNA chip scanner (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 intensity was obtained and background noise was also kept low. Further, the same experiment was performed using 10 substrates. However, in all the chips, the fine particles could be easily encapsulated, and it could be easily performed without mixing bubbles during solution application.

  Here, in Table 1, “O” in operability (at the time of enclosing fine particles) indicates that 120 mg of fine particles could be easily enclosed in the void, and “X” indicates that it was difficult. Show. In addition, “O” in operability (at the time of solution application) indicates that it was easy to inject without leaving bubbles in the void, and “X” indicates that it was difficult. .

Example 2
The same experiment as in Example 1 was performed except that zirconia fine particles polished for 2 hours with an alumina abrasive (particle size # 220) were encapsulated. The center line average roughness of the surface of the fine particles was Ra = 43 nm. The results are shown in Table 1. Sufficient fluorescence intensity was obtained and background noise was also kept low. Further, the same experiment was performed using 10 substrates. However, in all the chips, the fine particles could be easily encapsulated, and it could be easily performed without mixing bubbles during solution application.

Example 3
The same experiment as in Example 1 was conducted except that the commercially available zirconia fine particles described in Example 1 were immersed in 55% hydrofluoric acid (manufactured by Stella Chemifa Corporation) at 20 ° C. for 2 hours, washed with water and dried. It was. The center line average roughness of the surface of the fine particles was Ra = 110 nm. The results are shown in Table 1. Sufficient fluorescence intensity was obtained and background noise was also kept low. Further, the same experiment was performed using 10 substrates. However, in all the chips, the fine particles could be easily encapsulated, and it could be easily performed without mixing bubbles during solution application.

Comparative Example 1
An experiment similar to that of Example 1 was performed except that zirconia fine particles having a surface centerline average roughness Ra = 20 nm were encapsulated without polishing. The results are shown in Table 1. Although 10 substrates were used, static electricity was generated when fine particles were encapsulated in all the substrates, and only about 30 mg could be encapsulated. In addition, even in solution application, bubbles that cannot be removed remain between fine particles that have stopped moving due to static electricity, resulting in hybridization reaction unevenness. Since there were few encapsulated microparticles | fine-particles, the stirring effect of the solution was also inadequate and the fluorescence intensity was low compared with Example 1,2.

Comparative Example 2
The same experiment as in Example 1 was performed except that zirconia fine particles polished for 1 hour with an alumina abrasive (particle size # 220) were encapsulated. The center line average roughness of the surface of the fine particles was Ra = 32 nm. The results are shown in Table 1. Experiments were conducted using 10 substrates, and static electricity was generated when encapsulating fine particles in all substrates, and only about 40 to 60 mg could be encapsulated. In addition, even in solution application, bubbles that cannot be removed remain between fine particles that have stopped moving due to static electricity, resulting in hybridization reaction unevenness. Since there were few encapsulated fine particles, the stirring effect of the solution was insufficient, and the fluorescence intensity was low as compared with Example 1.

Example 4
The hydrofluoric acid-treated microparticles of Example 3 were polished in water for 30 minutes with a centrifugal barrel polisher using a silicon carbide abrasive (particle size # 20), washed with water and dried. The same experiment as in Example 1 was performed. The center line average roughness of the surface of the fine particles was Ra = 254 nm. The results are shown in Table 1. Although 10 substrates were used, the fine particle encapsulation and solution application operations were easily performed as in Example 1, and the background noise was larger than that in Example 1, but the fluorescence intensity was implemented. Similar to Example 1.

  The analysis chip of the present invention can easily encapsulate microparticles in the gap between the substrate and the cover, and therefore can easily encapsulate the amount of microparticles necessary for the hybridization reaction. Further, the analysis chip of the present invention can substantially inject bubbles on or near the cover surface because the specimen solution can be injected after the fine particles are moved and dispersed in the gaps. Therefore, the analysis chip characterized by a substrate with a concavo-convex structure maintains the characteristics such as the good S / N ratio and high detection sensitivity inherently, improves the dispersion of measured values, and provides accurate and reliable measurement. Enable. For this reason, it is possible to improve the accuracy of analysis chips that are frequently used in fields such as biology, medicine, and microbiology, which is very useful industrially.

It is the perspective view which shows an example of the analysis chip of this invention roughly, and the fragmentary sectional view cut | disconnected by the surface along A1. It is the schematic and longitudinal cross-sectional view of the board | substrate which comprise the analysis chip of this invention. It is a longitudinal section showing an example of an analysis chip of the present invention roughly. It is a fragmentary sectional view which shows an example of the through-hole in the cover which comprises the analysis chip of this invention, and a liquid level parking chamber. It is the schematic which shows fixation | immobilization of probe DNA in an Example of this application and a comparative example. It is the perspective view which shows an example of the analysis chip of this invention roughly, and the fragmentary sectional view cut | disconnected by the surface along A2.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Substrate 2 Cover member 3 Adhesive layer 4 Liquid level holding chamber 5 Through hole 6 Void 7 Selective binding substance 8 immobilized on substrate 8 Fine particles (beads)
9 Sealing material 10 Sample solution 11 Probe DNA

Claims (7)

  An analysis chip comprising a substrate and a cover member bonded to the substrate, and having an air gap between the substrate and the cover member, wherein fine particles are enclosed in the air gap, and the center line average of the surface of the fine particles An analysis chip having a roughness (Ra value) of 40 nm or more and 300 nm or less.   The analysis chip according to claim 1, wherein a material of the fine particles is ceramic or glass.   The analysis chip according to claim 1 or 2, wherein the center line average roughness (Ra value) of the surface of the fine particles is 40 nm or more and 200 nm or less.   The analysis chip according to claim 1, wherein the cover member has a through hole.   The analysis chip according to claim 1, wherein the substrate includes a plurality of uneven structures.   The analysis chip according to claim 1, wherein a selective binding substance is immobilized on the upper surface of the convex portion of the substrate.   The analysis chip according to claim 1, wherein the maximum diameter of the fine particles is 10 μm or more and 500 μm or less.

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