JP2010014669A - Analysis chip - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an analysis method equivalent to an analysis using two analysis chips without further thinning an analyte. <P>SOLUTION: In this analysis chip, selectively bonding substance immobilizing regions of a substrate to which selectively bonding substances are immobilized are arranged so as to face each other, and an analyte solution is held in space sandwiched between the selectively bonding substance immobilizing regions. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to an analysis chip including a substrate on which a substance that selectively binds (herein, “selective binding substance”) is immobilized.

  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 a method 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 instead of general glass or silicon has been developed (Patent Document 1). By this method, variation in the amount of material 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 an analysis using an analysis chip such as a DNA chip, it is necessary to prepare a sample at a high concentration in order to prioritize the sensitivity. If the sample solution can be prepared at a high concentration, highly sensitive detection is possible and it can be expected to detect minute changes. However, in the analysis using an analysis chip, an analysis using two or more analysis chips may be performed in order to increase the accuracy of the experiment. In this case, the sample solution has a volume compared to the analysis using one analysis chip. Is used after being diluted twice, and the analysis is performed in a state where the concentration is halved, the original sensitivity is lost, and important results may be missed. An object of the present invention is to enable an analysis equivalent to an analysis using two analysis chips without further diluting a specimen.

  In view of the above problems, the present inventors have intensively studied and found that the above problems can be solved by an analysis chip arranged so that substrates on which a selective binding substance is immobilized face each other, thereby completing the present invention. It was.

That is, this invention is comprised by the following (1)-(6).
(1) An analysis chip in which a selective binding substance-immobilized region of a substrate on which a selective binding substance is immobilized is arranged so as to face each other, and a sample solution in a space sandwiched between the selective binding substance-immobilized regions An analysis chip, characterized by holding.
(2) The analysis chip according to (1), wherein the two substrates are disposed so as to face each other.
(3) The analysis chip according to (1) or (2), wherein the substrate is provided with a through hole. (4) At least one of the selective binding substance-immobilized regions has an uneven structure. The analysis chip according to any one of (1) to (3).
(5) The analysis chip according to any one of (1) to (4), wherein fine particles are contained in a space sandwiched between the selective binding substance immobilization regions.
(6) The analysis chip according to any one of (1) to (5), wherein the selective binding substance is a nucleic acid.

  In the analysis chip of the present invention, the analysis of two conventional analysis chips can be performed at one time without diluting the prepared sample solution. Furthermore, in the analysis chip of the present invention, since the sample solution and the selective binding substance can be brought into contact with each other in the same environment in the two selective binding substance immobilization regions, more accurate analysis data can be obtained without losing sensitivity. Obtainable.

  Hereinafter, the present invention will be described more specifically.

  The analysis chip of the present invention is an analysis chip arranged on a substrate on which a selective binding substance is immobilized so that the selective binding substance immobilization regions face each other. The analysis chip here refers to a chip used to apply a solution containing a sample to the chip and measure the presence / absence of the sample, the amount of the sample, the properties of the sample, 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 analysis chip of the present invention is characterized in that the selective binding substance immobilization regions are arranged so as to face each other. As a specific example, the embodiment disclosed in FIG. 1 can be mentioned, but preferably the selective binding substance is immobilized. This is a mode in which two formed substrates are arranged to face each other (see the upper part of FIG. 1). The analysis chip in which the two substrates are arranged to face each other can be obtained by bonding the substrates so as to provide a space for holding the sample solution. The two substrates may be bonded in any manner as long as the space is formed, but an embodiment in which the substrate can be detached without being damaged is preferable. Double-sided tape, resin It is preferably bonded via an adhesive member such as a composition.

  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 surface of the selective binding substance immobilization region of the substrate preferably has a fine concavo-convex structure as seen in FIGS. 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.

  The selective binding substance is preferably bonded to the convex portion of the concavo-convex structure. In that case, the size of the space between the upper surfaces of the convex portions facing each other is preferably 1 to 500 μm in the height direction. If the area is smaller than this range, the chance that the specimen will come into contact with the immobilized selective binding substance is extremely reduced, and the signal after hybridization becomes extremely small. On the other hand, if the spatial size exceeds 500 μm, many liquids When a small amount of sample is used, the concentration of the sample solution is reduced, the hybridization reactivity is lowered, and the signal at the time of detection is weakened.

  The size of the projection is preferably 10 to 200 μm in height and 50 to 150 μm in width, but is not limited to such a range. The interval between the protrusions is, for example, 50 to 600 μm, and preferably a size that allows a plurality of fine particles to enter. Further, for the reason described in JP-A-2004-264289, the height of the convex portion is preferably the same height as the surrounding flat portion.

  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.

  Examples of the material of the substrate include glass, ceramics, inorganic materials such as silicon, 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, it is possible to improve the S / N ratio when detecting the specimen. 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.

  Preferred substrates that can be used in the present invention and methods for producing the same are described in, for example, Japanese Patent Application Laid-Open No. 2004-264289, Biotechnology Journal: July-August 2005, pages 418-420 by the present applicant.

  In the analysis chip of the present invention, it is preferable that a plane including one selectively binding substance immobilization region has a through hole communicating with the space. The through hole is used for injecting a liquid such as a test sample and a reaction buffer, and at the same time, used for maintaining the pressure inside the space at atmospheric pressure. It is preferable that there are a plurality of through-holes for one space, and it is more preferable that the number of through-holes is 3 to 6 because the specimen solution can be easily filled. In addition, as described later, when the space is divided into a plurality of spaces that do not communicate with each other, it is preferable that there are a plurality of spaces per space, and it is more preferable that each space has 3 to 6 through holes. .

  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 reliably sealed with the sealing member, and many bubbles can be prevented from entering the sample solution and 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.

  It is preferable that such a substrate is bonded to a selective binding substance-immobilized substrate having no detachable strength and mode. 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, in the state where two substrates are bonded, it is difficult to set the dedicated chip on the dedicated scanner. Impossible. Therefore, it is preferable that the two substrates can be detached so that the two substrates can be removed in the reading step.

  In the analysis chip of the present invention, it is preferable that fine particles are contained in the space sandwiched between the selective binding substance immobilization regions. The presence of the fine particles makes it possible to efficiently stir the injected specimen solution, and as a result, the effect of promoting the reaction between the specimen and the selective binding substance is brought about.

  As a stirring method using fine particles, a method in which the fine particles are dropped in the direction of gravity by rotating the chip, a method in which the chip containing fine particles is set on a shaker and the substrate is shaken, or the fine particles are removed by magnetic force using magnetic fine particles. The method of moving is used, but the method in which the chip is set on a shaker and swirled and rotated in a horizontal plane is preferable because the movement range of the fine particles is large and the particles can move evenly. As a result, the liquid can be efficiently stirred. 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 size (diameter) of the fine particles is not particularly limited, but is preferably 10 μm or more, and more preferably 50 μm or more. This is because the effect of stirring may not be sufficiently obtained in a range smaller than this range. On the other hand, if the range is too large, it is necessary to enlarge the gap sandwiched between the selective binding substance immobilization regions in order to enclose the fine particles, resulting in an increase in the amount of liquid required, which is not preferable. In addition, considering that a sample or other solution is put in a gap between two substrates and the encapsulated particles are moved and stirred, the solution and particles will not spill from the through-holes of the substrate. Furthermore, it is preferable to close the through hole.

  By controlling the surface roughness of the fine particles, it is possible to suppress the generation of static electricity when enclosing the fine particles. Here, as a preferable range of the surface roughness of the fine particles, the surface roughness (Ra) is 40 to 300 nm. 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, the thickness of 40 to 200 nm is particularly preferable in consideration of the strength of the material. If fine particles having a surface roughness (Ra) of less than 40 nm are used, static electricity is generated and the encapsulation efficiency is greatly reduced. Further, when the surface roughness (Ra) of the fine particles is larger than 300 nm, the sample solution becomes turbid after hybridization, and the detection result becomes uneven.

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

  When producing fine particles of yttria partially stabilized zirconia, the starting powder is, for example, a chemical synthesis method such as a hydrolysis method, a neutralization coprecipitation method, a thermal decomposition method, a hydrothermal synthesis method, an alkoxide method, or an oxide. A powder produced by a known powder synthesis method such as a mixing method 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. In the present invention, in consideration of the convenience of the user, it is preferable that fine particles are sealed in advance in a gap sandwiched between two substrates.

  The selective binding substance in the present invention refers to a substance that can selectively bind to a specimen 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 Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyzes, Plenum Press, US (1980); Iwasaki Ikuo et al. 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 provides a method of using the above-described analysis chip for measuring the presence or amount of a substance in an analyte that can bind directly or indirectly to a selective binding substance immobilized thereon.

  As the specimen, a biological sample is preferably used. The biological sample includes, for example, biological samples derived from plants and animals, biological samples such as tissues derived from mammals including humans, cells, and body fluids. 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 specimen. 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 an 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 an enzyme, a radioisotope or a fluorophore as a label is preferable. 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 hybridization or immune reaction, the analysis 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 method of the present invention is substantially free of bubbles on or near the substrate surface, the DNA chip characterized by the substrate having a concavo-convex structure has a good S / N ratio and high detection sensitivity inherently. 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. If necessary, four through-holes were made in the area without the pillars of the substrate by drilling or the like.

  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 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. The sample is diluted with hydrochloric acid for pH adjustment, pH 5.5), diluted 10-fold to make the final concentration of probe DNA 0.03 nmol / μL, and the carboxylic acid on the substrate surface and amino at the end of probe DNA In order to condense with the group, 1-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.

(Board bonding)
The substrate provided with the through hole to which the probe DNA obtained above was fixed and the substrate not provided were bonded with PDMS polymer (Toray Dow Corning Silicone). The bonding condition is 42 ° C. for 2 hours.

(Preparation and encapsulation of fine particles)
Commercially available zirconia fine particles (manufactured by Toray Industries, Inc.) having a surface roughness of 20 (nm) and an average particle diameter of 197 μm are submerged in water for 1 hour using a silicon carbide abrasive (particle size # 20) with a centrifugal barrel grinder. Polished, washed with water and dried. The surface roughness of the fine particles was Ra = 165 nm. The surface roughness of such fine particles was measured by vacuum-depositing the surface with Au and then measuring the surface roughness Ra (nm) with a scanning electron microscope (manufactured by Elionix Co., Ltd., model ESA-2000). The surface roughness was measured for any 10 samples with an observation magnification of 10,000 times and a cut-off 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 space between the two substrates from the through hole of the substrate.

(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. That is, ExTaq 2 μl, 10 × ExBuffer 40 μl, dNTP Mix 32 μl (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: 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, and dGTP concentrations were 2.5 mM and dCTP were each 400 μM) 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 3 ng / μL.

(Hybridization)
Using a micropipette, 165 μL of the hybridization sample solution prepared above was injected from the through hole into a space (reaction tank) sandwiched between two substrates through the through hole of the substrate. 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, peeling the two substrates, after it eliminated the PDMS polymer, respectively washed and dried.

(Measurement)
Set the two substrates (a, b) after the above treatment on the DNA chip scanner (Axon Instruments GenePix 4000B), and set the laser output to 33% and the photomultiplier voltage to 450. went. 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. The numerical values of the two substrates were almost equal and stable, and sufficient fluorescence intensity and low background noise were obtained.

Comparative Example 1
A cover having four through-holes shown in FIG. 6 was prepared by an injection molding method, and this was adhered to the DNA-immobilized region of the DNA-immobilized substrate produced in Example 1 by the following method to produce an analysis chip. . 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 (no through hole) on which the probe DNA obtained above was fixed with PDMS polymer (Toray Dow Corning Silicone). Adhesion conditions are 42 ° C. and 2 hours.

  Example 1 except that two chips (c, d) were prepared, the hybridization solution (3 ng / μL) was diluted 2-fold (1.5 ng / μL), and applied to each of the two chips. The same experiment was conducted. The results are shown in Table 1.

  Compared to Example 1, the background noise was kept low, but the fluorescence intensity was about half.

Comparative Example 2
Two chips (e, f) similar to Comparative Example 1 were prepared, and an experiment similar to Comparative Example 1 was performed, except that the hybridization solution was applied without dilution. The results are shown in Table 1. The results of fluorescence intensity and background noise were the same as in Example 1. However, the difference in fluorescence intensity between the two sheets was 300, which was 10 times that of Example 1 (Table 2). It is conceivable that the two chips show the difficulty in aligning the hybridization environment.

  The DNA chip of the present invention can use two chips to align the sample solution and the hybridization environment, and enables stable and highly sensitive analysis. In particular, analysis data using two chips can be obtained without diluting a very small amount of sample twice, thereby enabling highly reliable measurement. 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 board | substrate 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 schematically an example of the chip | tip in this-application comparative example, and the fragmentary sectional view cut | disconnected by the surface along A2.

Explanation of symbols

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

Claims (6)

  An analysis chip arranged such that a selective binding substance-immobilized region of a substrate on which a selective binding substance is immobilized faces each other, and holds a sample solution in a space sandwiched between the selective binding substance-immobilized regions An analysis chip characterized by that.   The analysis chip according to claim 1, wherein the two substrates are arranged so as to face each other.   The analysis chip according to claim 1, wherein a through hole is provided in the substrate.   The analysis chip according to claim 1, wherein at least one of the selective binding substance-immobilized regions has an uneven structure.   The analysis chip according to any one of claims 1 to 4, wherein fine particles are contained in a space sandwiched between the selective binding substance immobilization regions.   The analysis chip according to claim 1, wherein the selective binding substance is a nucleic acid.

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