JP2005030927A - Organism-related molecule microarray - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a microarray which is manufactured and carries out detection at low cost, operates simply, has a high S/N ratio, and further carries out highly accurate detection. <P>SOLUTION: This microarray has a constitution wherein organism-related molecules are carried between the first member 1 and the second member 2, and, on either of the first member and the second member, a plurality of grooves are formed in parallel on the contact surface to the other member, to thereby provide a plurality of spaces working as a reaction region 3. Hereby, highly accurate detection wherein interference between the samples is prevented is performed, and data is read simply and highly accurately by an inexpensive line sensor. In addition, furthermore cost reduction, and simple and highly accurate detection are realized by paying attention to polydimethyl siloxane as a member to be subjected to micro-fabrication. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a biorelevant molecular microarray, and more particularly to a biorelevant molecular microarray in which reaction regions formed by microfabrication of constituent members are arranged in a barcode.

  In the past, there has been a strong demand in the environmental business market to accurately grasp the dynamics of microorganisms that are problematic in the areas of soil purification and water safety. In other words, there is a demand for continuous or real-time monitoring of the presence of pathogenic microorganisms in upper and lower waters, and for quick and accurate quantification and identification of coliforms and enterobacteria that are water safety standards. is there. In the field of soil remediation business, there is also a demand to propose an accurate remediation method by accurately investigating what microorganisms are present in the contaminated soil before in-situ remediation. Furthermore, it is required to accurately grasp the dynamics of microorganisms in various environmental business fields such as the investigation of the dynamics of indicator microorganisms used for bioaugmentation for introducing useful microorganisms and the soil diagnosis after the ex-post facto.

  However, under the present circumstances, samples are brought back to the laboratory and inspected by culture and microscopic observation by skilled inspectors, and it has been difficult to perform real-time monitoring on a continuous or on-site side. In recent years, detection methods at the gene level such as identification by base sequence analysis of ribosomal RNA genes of microorganisms have been reported, but all remain at the laboratory level and are rarely used in the field. The current situation is that the following species are hardly identified. In view of these current conditions, for identification of species below the genus of microorganisms, identification methods at the species level focusing on genetic polymorphism between species have been attempted.

  In addition, with the progress of gene analysis research represented by recent genome projects, genetic research is entering a new stage, and in the future, functional analysis research of genes existing on the genome is becoming very important. . Genes in living organisms are temporally and spatially regulated, and gene expression monitoring studies are underway to comprehensively elucidate this regulatory mechanism. In addition, the nucleotide sequence of human genomic DNA varies from person to person. In particular, a state in which one base is different is called a single nucleotide polymorphism (SNP), and such differences are related to differences in individual drug susceptibility and susceptibility. Research on genetic polymorphisms for the realization of disease-related gene mapping, genomic new drugs, and tailor-made medicine is underway.

  The microarray technology that can comprehensively analyze many kinds of genes is an important fundamental technology in the post-genome era for comprehensive gene expression monitoring, genome mutation, and polymorphism detection. It is attracting attention from the medical field to the field of basic biology. Particularly in recent years, DNA microarray technology has made tremendous technological progress. A DNA microarray (DNA chip) uses a micro technique to hybridize a target nucleic acid labeled with a fluorescent molecule to gene DNA that is aligned and fixed at high density on a substrate such as glass. The label bound on the substrate is imaged by a fluorescent scanner or the like and analyzed.

  DNA microarrays are roughly classified into an optical lithography type and a spotting type depending on the manufacturing method. The optical lithography type is a method of synthesizing oligonucleotides on a substrate by fusing optical lithography and combinatorial chemistry. Specifically, the base bonded to the substrate is protected with a functional group that can be removed by light, and light is applied only to a specific region of the base by using an optical lithographic mask that can irradiate only the reaction site. After removing the functional group in this way, by repeating the process of reacting with a base whose hydroxyl group is protected and coupling with the base from which the protecting group on the substrate is eliminated, hundreds of thousands of oligonucleotides can be obtained. It is synthesized on a glass substrate (see, for example, Non-Patent Document 1 and Patent Document 1). On the other hand, the spotting type is a method in which a high-density high-speed robot spotter is used to prepare a high-density DNA microarray by arranging (spotting) DNA on a substrate by using a robot arm. Are densely spotted on a substrate coated with a solid phase agent (see, for example, Non-Patent Document 2 and Patent Document 2). By using probes that have already been prepared, it is possible to create a DNA microarray desired by researchers.

  However, the DNA microarray technology is in the stage of development and has technical problems to be solved. Since the optical lithography type is time-consuming to design and create and is expensive, the use is limited to some research institutions. On the other hand, the spotting type immobilizes the DNA probe on the substrate by dropping a small amount of the droplet containing the DNA probe on the substrate, so that the dropped droplet diffuses on the substrate surface and interferes with adjacent spots. In addition, there is a problem of causing contamination, and furthermore, since the density and uniformity of each spot cannot be secured, the accuracy and reproducibility are not satisfactory. In addition, there is a problem that the target DNA is non-specifically adsorbed on the substrate due to the presence of the solid-phase agent adhering to the periphery of the spot portion, and noise is increased to reduce the S / N ratio.

The fluorescence generated when the microarray is irradiated with light includes the fluorescence generated from the substrate itself in addition to the fluorescence generated from the fluorescent label, which becomes the background and reduces the S / N ratio. There was a problem. For detection, data is usually obtained by detecting the fluorescence of each spot using a commercially available microarray scanner. However, laser oscillators and optical systems are very expensive, and high-precision detection is possible. For this purpose, it is necessary to accurately position the spot on the micro scanner. Therefore, it is difficult to say that the conventional method is sufficiently satisfactory from the viewpoints of accuracy, cost, and ease of operation.
Fedder SP, Rava RP, Huang XC, Peace AC, Holmes CP, Adams CL, “Multiplexed biochemical assays with biologics”, Vol. 556 US Pat. No. 5,744,305 Shena, M .; Shalon, D .; Davis, R .; W. , And Brown, P.M. O. "Quantitative monitoring of gene expression patterns with a complementary DNA microarray", 1995, Science, 270, p. 467-470 US Pat. No. 5,807,522

  Therefore, the present invention has been made in view of the actual situation relating to the microarray technology as described above, and can be manufactured and detected at low cost, and is easy to operate, has a high S / N ratio, and realizes accurate detection. An object of the present invention is to provide a microarray that can be used.

  As a result of diligent investigations to achieve the above object, the present inventors have arranged interference regions between samples by arranging a plurality of reaction regions as sample-shaped spaces in parallel using microfabrication technology. It has been found that high-accuracy detection can be realized, and data can be read easily and with high accuracy by an inexpensive line sensor. Furthermore, it has been found that further cost reduction and simple and highly accurate detection can be realized by paying attention to polydimethylsiloxane as a member to be finely processed. Based on these findings, the present invention has been completed.

  The present invention relates to a biorelevant molecule microarray provided with a plurality of parallel, linear reaction regions, and a target substance that is selectively captured by the biorelevant molecule by supporting the biorelevant molecule in the space. Is detected. Hereinafter, the barcode array may be abbreviated from the arrangement shape of the space that becomes the reaction region.

That is, when the present invention is outlined, the first invention of the present invention is a biologically relevant molecular microarray carrying biologically relevant molecules between the first member and the second member,
In either one of the first member and the second member, a plurality of grooves are formed in parallel on the contact surface with respect to the other member, and a plurality of spaces serving as reaction regions are provided.

Furthermore, the second invention of the present invention is a bio-related molecule microarray carrying a bio-related molecule between the first member and the second member,
A plurality of grooves are formed in parallel on the contact surfaces of the first member and the second member, and a plurality of spaces serving as reaction regions are provided.

A third invention of the present invention is a bio-related molecule microarray carrying a bio-related molecule between the first member and the second member,
In the first member, a plurality of linear protrusions are formed in parallel on the contact surface with respect to the second member,
In the second member, a plurality of linear grooves that can be fitted in one-to-one with the linear protrusions on the contact surface with respect to the first member are formed in parallel.
When the first member and the second member are brought into contact with each other while fitting the linear convex portion and the linear groove portion, a space serving as a reaction region is formed between the linear convex portion and the linear groove portion. It is configured to be formed.

  A fourth invention of the present invention is the bio-related molecular microarray of the first to third inventions of the present invention, wherein the reaction region and the outside can communicate with each other, and a sample supply port for supplying a sample to the reaction region and reaction A sample collection port for collecting a sample from the region is provided.

  A fifth invention of the present invention is the biologically relevant molecular microarray of the first to fourth inventions of the present invention, wherein the base material constituting at least one of the first member and the second member is a poly It is characterized by being a transparent silicon rubber or plastic resin such as dimethylsiloxane or polymethyl methacrylate.

  According to a sixth aspect of the present invention, there is provided a biorelevant molecular microarray carrying biorelevant molecules, comprising a microarray body in which a plurality of linear reaction regions are arranged in parallel.

  The seventh invention of the present invention is characterized in that the substrate constituting the microarray body is a transparent silicon rubber or plastic resin such as polydimethylsiloxane or polymethyl methacrylate.

  Here, the term “biologically related molecule microarray” in the present specification means a microarray on which a biologically relevant molecule is supported or actually supported, and includes a concept that does not actually carry. . The term “biologically relevant molecule” means a probe molecule capable of recognizing a test object, that is, a molecule capable of selectively detecting one of substances having affinity for each other. Therefore, as long as it has molecular recognition ability that can selectively detect one of the substances immobilized on the substrate and having affinity for each other, it is a concept including any substance, such as DNA fragments such as cDNA and PCR products, oligos Nucleic acids such as nucleotides, polynucleotides, peptide nucleic acids, proteins, peptides, sugars, cells, microorganisms and the like are preferably exemplified, but not limited thereto. A commercially available product can be used as the biological substance, and those obtained from cells, tissues and the like using appropriate extraction and purification means, and those synthesized artificially can also be used. When nucleic acids are used as biologically relevant substances, DNA and RNA extracted from cells or tissues using known methods can be used. Furthermore, linear or circular plasmid DNA or chromosomal DNA can be used. It is also possible to use a DNA fragment that has been cleaved by a restriction enzyme or chemically, DNA that has been synthesized by an enzyme or the like in a test tube, or a chemically synthesized oligonucleotide. In the case of proteins and peptides, proteins extracted and purified from cells or tissues, peptide fragments obtained by enzymatic or chemical cleavage of proteins with proteases, proteins synthesized using recombinant gene technology, antibodies, An enzyme or the like can be used. Here, the substance discriminating ability means that substances having affinity to each other can be selectively identified. For example, hybridization between DNA-DNA, DNA-RNA, and RNA-RNA bases (complementary) Binding) ability, antibody, antibody fragment-antigen, or regulator, receptor-hormone, cytokine, neurotransmitter, lectin, etc., or biological response ability between enzyme-substrate, coenzyme, etc. Is done. Therefore, the microarray of the present invention can be configured as a DNA chip, protein chip, sugar chain chip, microbial chip, cell chip, etc., and determination of nucleic acid sequence information, gene expression, mutation, diversity, etc. And analysis of the target protein and functional analysis such as protein purification, identification, protein expression, interaction, post-translational modification and the like.

  The target substance is a substance that is captured by the biological substance, that is, a substance that interacts with the biological substance carried on the member with a selectively distinguishable affinity as described above. is there. For example, DNA fragments such as cDNA and PCR products, nucleic acids such as oligonucleotides, polynucleotides and peptide nucleic acids, proteins, peptides, sugars, cells, microorganisms and the like are preferably exemplified, but not limited thereto.

  The test sample provided to the microarray is a concept including any sample in which the presence of the target substance existing in the environment or the living body is suspected. Examples include environmental samples such as soil and water, biological samples, food samples and the like, and these are preferably subjected to an appropriate pretreatment, and preferably labeled with an appropriate label. For example, when the target substance is a nucleic acid, it is preferable to extract, purify, or isolate a microorganism, animal or plant cell or tissue from the sample and prepare a nucleic acid sample from the obtained cell or tissue by an appropriate means. . Preparation from cells and tissues is performed based on a normal method for preparing nucleic acids. In the case of mRNA, it is preferable to use labeled cDNA by reverse transcription reaction in the presence of labeled dNTP. In prokaryotic cells, total RNA is used, and when examining gene mutations and polymorphisms, labeled cDNA is used. It is preferable to amplify the target region by PCR in the presence of a primer or labeled dNTP. Furthermore, when the DNA fragment on the substrate is an oligonucleotide, it is preferable to reduce the molecular weight.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. However, the present invention is not limited to the embodiments exemplified below, and appropriate modifications are also included in the present invention. Can understand.
(First embodiment)
FIG. 1 is a top view of the barcode array of the present invention and is a schematic diagram showing the basic configuration of the present invention. FIG. 2 is a cross-sectional view taken along the line AA in FIG. 1, and schematically illustrates the barcode array of the present invention when configured as the first embodiment. Hereinafter, the upper side in FIG. 2 is referred to as “upper”, and the lower side is referred to as “lower”.

  As shown in FIGS. 1 and 2, the barcode array of the present invention is composed of a first member 1 and a second member 2 fixedly disposed on the upper surface of the first member 1. A space serving as a reaction region 3 arranged in a barcode shape is provided between the member 2 and the member 2.

  The first member 1 is configured as a flat plate-like body. In the second member 2, a surface treatment is performed on a flat plate-like base material, and a plurality of grooves 6 extending in one direction are formed in parallel in a barcode shape on the contact surface with the first member 1. That is, the second member 2 has a spatial structure in which a plurality of linear grooves 6 and partition walls 7 partitioning the grooves 6 are alternately arranged on the contact surface side with the first member. The grooves 6 are preferably arranged substantially parallel to each other. In FIG. 2, the cross-sectional shape of the groove 6 is a quadrangular shape with the bottom of the groove 6 being flat. However, the shape is not limited to this, and an arbitrary shape such as an inverted triangle or a trapezoid narrowing toward the bottom. You may form in.

  The lower end surface of each partition wall 7 formed on the second member 2 is closely bonded to the first member 1. With this configuration, the opening of each groove 6 formed in the second member 2 is sealed, and a space is formed that forms a linear reaction region 3 that is completely separated by the partition wall 7 and the first member 1. Is done. Accordingly, it is possible to reliably prevent the fluid from escaping from the reaction region 3 to the outside, that is, the adjacent reaction region 3, thereby reliably preventing sample contamination between the adjacent reaction regions 3. Is possible.

  As a base material which comprises the 1st member 1 and the 2nd member 2, a glass plate, a quartz plate, a silicon wafer etc. are illustrated preferably, for example, However, Any well-known material used conventionally can be used. In particular, from the viewpoint of moldability and optical properties, from the optical viewpoint, polydimethylsiloxane (hereinafter abbreviated as PDMS), acrylic resin polymethyl methacrylate (PMMA, polymethyl methacrylate). ) Is preferably exemplified. Moreover, the 1st member 1 and the 2nd member 2 can be formed using the same base material, and can also be formed using a different base material. There are no particular restrictions on the shape, size, thickness, etc. of the first member 1 and the second member 2, but the shape is particularly preferably a plate-like body. Further, the size of the member is appropriately determined according to the number, size, shape, and the like of the reaction region 3, and an example is 26 × 76 mm, but is not limited thereto. The thickness of the first member 1 and the second member 2 depends on the type of base material constituting the member, the stability of the shape required for the member, the number, size, shape, etc. of the reaction regions 3 provided. It is determined in consideration of the suitability of the first member 1 and the second member 2.

  PDMS is a kind of silicon elastomer, has very high transparency, excellent optical properties, low absorption in a wide wavelength region, especially extremely low absorption in the visible light region, and almost no fluorescence detection. Since there is no influence, the S / N value can be lowered by using PDMS as a microarray substrate. In addition, the microarray is optimally suited for various optical devices because it has the following characteristics of molds, easy microfabrication from nano to micron order, and easy to mold into any microstructure. There is an advantage that it can be finely processed. In addition, PDMS itself has a property of good adhesion to glass, acrylic resin, and the like. By bringing flat glass or acrylic resin member into contact with a finely processed PDMS member, adhesive or the like It is possible to form a flow path and a chamber without using an adhesion means such as adhesion.

  The shape of the reaction region 3 is a linear space, and a plurality of parallel regions are formed between the first member 1 and the second member 2 in a barcode shape. The reaction region 3 is a solid-phased region for biologically relevant molecules, a biologically related molecule to be immobilized on a substrate, a sample, a reaction solution injection, and a biopolymer as a flow path for diffusion due to capillary phenomenon. It is configured to be used for reactions that bind to substrates such as glass PDMS.

  In addition, it is configured to have a role as a chamber when storing in a state where biologically relevant molecules or the like are solid-phased as an implementation area for hybridization, antigen-antibody response reaction, and the like.

  In addition to this, hybridization, antigen-antibody response reaction, etc. are carried out by immobilizing a bio-related molecule in the reaction region 3 and then either one of the two members constituting the array (for example, the component member is made of glass. In the case of PDMS processed into a linear shape, the PDMS is peeled off and covered with another member (for example, a PDMS chamber) that can cover the entire area where the bio-related molecules are immobilized, hybridization, antigen-antibody response reaction It is also possible to perform the same in the chamber. With this configuration, the entire array can be reacted in a single reaction tank, so that each reaction region 3 formed in a linear shape can be made of the same sample without complicated processing. It becomes possible to contact evenly.

  There are no particular restrictions on the length, width and depth of the reaction region 3 and the spacing between the reaction regions 3, and the reaction can be performed satisfactorily by developing the bio-related molecules, reaction liquid, sample, etc. sufficiently and evenly. If the space can be maintained and a desired reaction can be performed, it is designed as appropriate. The width of the reaction region 3 is preferably exemplified as 10 to 200 μm, but is not limited thereto. It is also possible to change the width and interval of the reaction area 3 like a barcode so that the determination information can be read.

  And as shown in FIG. 1, it can be set as the structure which provided the sample supply port 4 and the sample collection | recovery port 5 which can communicate the reaction region 3 and the exterior. The sample supply port 4 is an opening that is connected to an appropriate sample supply unit and supplies a sample into the reaction region 3, and the sample recovery port 5 is connected to an appropriate sample recovery unit and collects the sample outside the reaction region 3, It is an opening to discharge. As long as the sample supply port 4 and the sample recovery port 5 have such functions and do not interfere with a desired reaction in the reaction region 3, there are no particular restrictions on the shape, size, setting position, etc. Can be done. Therefore, FIG. 1 discloses a configuration in which the sample supply port 4 is provided at one end in the longitudinal direction of the reaction region 3 of the second member 2 and the sample recovery port 5 is provided at the other end. It is not limited, and the sample supply port 4 and the sample recovery port 5 can be configured so as to share one opening, and the second member 2 is directly above or on the side of the reaction region 3. It is also possible to form the first member 1 in the direction.

  Appropriate sample supply means and sample collection means include glass syringes, plastic syringes, micropipetters, fluoroethylene resin tubes / silicon tubes, PVC tubes, peristaltic pumps, plunger pumps, syringe pumps, etc., or combinations thereof Is preferably exemplified. These materials are preferably those which do not adsorb nonspecifically substances in the sample such as nucleic acids and proteins.

    Next, the manufacturing method of the barcode array according to the first embodiment of the present invention will be described. First, the member surface is finely processed, that is, the groove 6 structure is formed on the contact surface of the second member 2 with the first member 1. As a method for forming the groove on the substrate surface, any of known microfabrication techniques can be used. As a direct forming method, a method for removing a groove portion by a mechanical cutting method using a fine processing drill, broad, etc., ionizing radiation lithography such as photolithography, electron beam lithography or AFM lithography, physical, chemical Examples include a method of removing a groove portion by etching, or a combination of these methods. Furthermore, it is also possible to form grooves indirectly by transferring the spatial structure using a mold having a convex structure that reverses to the desired groove structure, produced according to the method described above. For example, injection molding using a mold, press molding at a high temperature, or the like can be used.

  When PDMS is used as a base material constituting a member to be subjected to a fine structure, PDMS has a high followability with respect to a mold, and therefore a method of forming using a mold can be particularly preferably used. By using a mold, once the mold is produced, a large amount of chips can be produced easily and inexpensively, so that the manufacturing cost can be reduced.

  Next, the opening of the groove 6 is sealed by adhering the first member 1 to the upper end surface of the partition wall 7 (convex portion) of the second member 2 that has been finely processed. The first member 1 and the second member 2 are preferably bonded tightly, so that it is possible to reliably prevent sample contamination between reaction regions. The adhesion method can be adhered by applying an adhesive, but PDMS itself has the property of being in close contact with a flat surface such as glass or acrylic resin. Therefore, when PDMS is used for one member, For example, when a flat glass is used as the other member, it is bonded without an adhesive by removing organic substances and dust with a glass detergent (Semico Clean (manufactured by Furuuchi Chemical Co., Ltd.) or 0.3N sodium hydroxide solution). It becomes possible. Further, when the surface is treated with oxygen using a plasma etching apparatus, stronger adhesion is possible.

  Next, as a particularly preferable embodiment, a specific procedure for producing a PDMS chip using a mold method will be briefly described. For the formation of the mold, any known fine processing technique can be used, and it is preferable to use a combination of a photolithography technique and an etching technique. In photolithographic processing, a photoresist (photosensitive resin) is applied on a substrate to be processed such as silicon or glass, and after heat treatment, the resist is exposed through a photomask in which a desired flow path pattern is formed, and the resist layer is heat-treated. Then, it develops by developing with a predetermined developing solution. As the photoresist, both a positive type in which an exposed portion is developed and a negative type in which an unexposed portion is developed can be used. A known method such as spin coating, roll coating, or dip coating can be used on a substrate. Evenly applied. The developer is suitably selected according to the type of resist, and a tetramethylammonium hydroxide aqueous solution is preferably used for the positive resist, and a xylene / acetone mixed solution is preferably used for the negative resist.

  Next, the substrate subjected to the photolithography process is preferably etched. Etching is a wet etching performed in a chemical solution such as sulfuric acid, nitric acid, phosphoric acid, and hydrofluoric acid, and a reactive gas containing trifluoromethane, tetrafluoromethane, or hexafluoroethane is discharged into a plasma state. Any of the dry etching represented by plasma etching that forms fine patterns can be used by removing the radicals (reactive species) and ions generated from the reaction with the solid material to remove them as reaction products. From the viewpoint of facilitating the release of the member for transferring the material from the mold, on the other hand, the plasma etching that forms a protective film that facilitates the release by attaching CF, CF2 radicals, etc. to the substrate is performed. In particular, it is preferably exemplified.

  Subsequently, the mold having the concavo-convex space structure produced as described above is fixed in a mold, and uncrosslinked PDMS is mixed with an appropriate polymerizing agent, poured into a mold, and cured. The structure is transferred to PDMS. After curing, the PDMS substrate having a shape pattern with a desired structure can be produced by peeling from the mold. The curing temperature is preferably 23 ° C. to 150 ° C., and the curing time is set according to the curing temperature. The curing time is 24 hours at 23 ° C., 4 hours at 65 ° C., and 2 hours 30 at 75 ° C. For example, 1 minute at 100 ° C. and 15 minutes at 150 ° C. are exemplified. Preferably, the reaction is performed at 75 ° C. for 2 hours and 30 minutes.

  Next, a target substance detection method using the barcode array according to the first embodiment of the present invention will be exemplarily described. First, in the reaction region 3, a bio-related molecule having substance discrimination ability with respect to the target substance is carried. The bio-related molecules are solid-phased on the surfaces of the first member 1 and the second member 2 that surround the space serving as the reaction region 3. Both the first member 1 surface and the second member 2 surface can be solid-phased, or both can be solid-phased. Further, the solid phase can be performed after the first member 1 and the second member 2 are bonded, but can also be performed before bonding them, and is appropriately selected. When solidifying a biological substance after adhesion of a member, it is performed by injecting a sample containing the biological substance from a sample injection port.

  Any known method can be used to immobilize the bio-related molecule on the surface of the member, and the optimum method is appropriately selected according to the type of substance to be immobilized and the type of substrate constituting the member. A method is selected. For example, when the biologically relevant molecule to be immobilized is a nucleic acid, electrostatic binding or covalent binding, and when it is a protein, covalent binding, ionic binding, physical adsorption, or biological specificity A carrier bonding method such as bonding, a crosslinking method, and a comprehensive method (lattice type or microcapsule type) encapsulating with a polymer substance are preferably used. Regarding nucleic acids, specific methods include the method of electrostatically binding to the carrier surface with polycations such as polylysine and polyethyleneimine using the charge of DNA, various silane couplings having amino groups, aldehyde groups, epoxy groups, etc. Preferred examples include a method in which a carrier surface-treated with an agent is covalently bonded to a DNA having an amino group, aldehyde group, SH group, biotin or the like introduced as a functional group at the terminal. Thereafter, treatments such as ultraviolet irradiation, crosslink formation with a chemical crosslinking agent, surface blocking, and washing are performed as necessary. Here, the solid phase means that the biologically relevant molecule does not substantially desorb from the substrate surface without impairing the original activity.

  Next, a reaction for capturing the target substance is performed. A sample solution that has been subjected to appropriate pretreatment, a reaction solution such as a hybridization solution, a washing solution, and the like are supplied from the sample supply port 4 into the reaction region 3, and after a predetermined reaction, the sample solution is supplied through the sample recovery port 5. From the reaction zone 3, it is collected and discharged.

  In addition, hybridization, antigen-antibody response reaction, and the like are performed by immobilizing a bio-related molecule in the reaction region 3 and then either one of the two members constituting the array (for example, the component is made of glass and wire). In the case of PDMS processed into a shape, the PDMS) is peeled off and covered with another member (for example, a PDMS chamber) that can cover the entire area where the bio-related molecules are immobilized, and hybridization, antigen-antibody response reaction, etc. It is also possible to configure to perform simultaneously in this chamber. With this configuration, the entire array can be reacted in a single reaction tank, so that each reaction region 3 formed in a linear shape can be made of the same sample without complicated processing. It becomes possible to contact evenly.

  After the reaction, the target substance in the test sample that interacts with the bio-related molecule on the microarray is detected using, for example, a labeling substance as an index. The detection means is preferably a line sensor such as a barcode reader. Since the data can be read by an inexpensive line sensor, the detection cost can be reduced. When reading with a line sensor, as long as the reaction area is scanned, it can be read from any direction, such as the direction orthogonal to the reaction area or the oblique direction, so use a microscanner. Since strict positioning as in the case is unnecessary, the operation can be simplified.

Here, in the first embodiment of the present invention, the form in which the groove 6 is formed in the second member 2 has been exemplarily described, but the reverse form in which the groove 6 is formed in the first member 1 is also preferably present. It is illustrated as an embodiment.
(Second Embodiment)
Next, a barcode array according to the second embodiment of the present invention will be described with reference to FIG. FIG. 3 is a cross-sectional view taken along the line AA of FIG. 1 and illustrates the barcode array of the present invention when configured as the second embodiment. About the same structure as the barcode array of 1st Embodiment, description is abbreviate | omitted with the same code | symbol in a figure.

  Both the first member 1 and the second member 2 are subjected to surface treatment on a flat plate-like base material, and a plurality of grooves 6 extending in one direction are formed in a barcode shape on the contact surface with the other member. They are formed in parallel. That is, in both the first member 1 and the second member 2, a plurality of linear grooves 6 and partition walls 7 partitioning the grooves 6 are alternately arranged in parallel on the contact surface side with the first member 1. It has a spatial structure. And when the 1st member 1 and the 2nd member 2 are made to contact | abut, the groove | channel 6 formed in the 1st member 1 and the groove | channel 6 formed in the 2nd member 2 each face one-to-one. Is configured to do.

The upper end surface of each partition wall 7 formed on the first member 1 and the lower end surface of each partition wall 7 formed on the second member 2 are closely bonded. With this configuration, the opening of each groove 6 formed in the second member in the first member forms one space, and the linear shape is completely separated by the partition wall 7, the first member, and the second member. A space that becomes the reaction region 3 is formed.
(Third embodiment)
Next, a bar code array according to the third embodiment of the present invention will be described with reference to FIG. FIG. 4 is a cross-sectional view taken along the line AA of FIG. 1, and schematically illustrates the barcode array of the present invention when configured as the third embodiment. About the same structure as the barcode array of 1st Embodiment, description is abbreviate | omitted with the same code | symbol in a figure.

  The first member 1 has a surface treatment applied to a flat plate-like substrate, and a plurality of linear protrusions 8 extending in one direction are formed in parallel in a barcode shape on the contact surface with the second member 2. Yes. The second member 2 is also subjected to a surface treatment on a flat plate-like base material, and a plurality of linear groove portions 10 that can be fitted to the linear protrusions 8 on a one-to-one basis on the contact surface with the first member 1. They are formed in parallel. That is, the first member 1 has a spatial structure in which a plurality of linear convex portions 8 and concave portions 9 that partition the linear convex portions 8 are alternately arranged on the contact surface side with the second member 2. The second member 2 has a spatial structure in which a plurality of linear groove portions 10 and partition walls 7 partitioning the groove portions 10 are alternately arranged in parallel on the contact surface side with the first member 1. And when the said 1st member 1 and the said 2nd member 2 are made to contact | abut, making the said linear convex part 8 and the said linear groove part 10 fit, between the said linear convex part 8 and the said linear groove part 8 A space serving as a reaction region 3 is formed between them.

  And each recessed part 9 formed in the 1st member 1 and the partition 7 formed in the 2nd member 2 are closely fittingly adhere | attached. With this configuration, a linear space that becomes the reaction region 3 is formed between the linear convex portion 8 and the linear groove portion 10, and the space includes the partition wall 7, the first member 1, and the second member. 2 completely separated from the outside. In this way, the amount of noise detected from the background can be reduced by providing a difference in height between the reaction region and the region other than the reaction region on the substrate surface and focusing the detection on the reaction region surface. Thus, the S / N value can be improved.

  The height and shape of the linear protrusions 8 formed on the first member 1 and the height and shape of the linear grooves 10 formed on the second member 2 are the same as those of the linear protrusions 8 and the linear grooves 10. When the first member 1 and the second member 2 are brought into contact with each other, the space that becomes the reaction region 3 is formed between the linear convex portion 8 and the linear groove portion 10. As long as it is configured, the height of the linear protrusion 8 is preferably 1 μm to 1 cm, and particularly preferably 10 μm to 1 mm, although it is appropriately set.

Here, as a third embodiment of the present invention, the form in which the linear protrusions 8 are formed in the first member 1 and the linear grooves 10 are formed in the second member 2 has been exemplarily described. The reverse form which forms the linear convex part 8 in the 2nd member 2 for the linear groove 10 is also illustrated as this preferable embodiment.
(Other embodiments)
Hereinafter, other embodiments of the present invention will be described.
(1) The biorelevant molecule microarray according to the present invention has been described as a structure in which two or more microfabricated members are bonded to each other and a biorelevant molecule is supported in a reaction region formed between them. Alternatively, it may be composed of only one member subjected to fine processing. That is, one member may be grooved to form a plurality of linear grooves in parallel, and the reaction may be performed by supporting biological molecules with the linear grooves as reaction regions. As the member to be processed, transparent silicon rubber or plastic resin such as polydimethylsiloxane or polymethyl methacrylate is exemplified from the viewpoint of easy molding and optical characteristics, and from an optical viewpoint. By configuring in this way, reactions such as hybridization can be carried out not only in a closed system region but also in an open system region, and it is easy to mold and has excellent optical properties. Molecular microarrays can be provided.
(2) Next, one member may be drilled to form a plurality of linear holes in parallel, and the reaction may be carried out by supporting biological molecules with the linear holes as reaction regions. Examples of the member include transparent silicone rubber or plastic resin such as polydimethylsiloxane or polymethyl methacrylate from the viewpoint of easy molding and optical characteristics.

EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited to these Examples.
[Example 1] Production of PDMS chip (1) Production of mold The surface of a silicon wafer (diameter 4 inches, manufactured by Shin-Etsu Chemical Co., Ltd.) was formed with hydrogen peroxide / sulfuric acid (hydrogen peroxide: sulfuric acid = 1: 2), followed by buffered foot. Washed with acid, and after washing, the wafer was rinsed with distilled water and dried. Next, negative-type epoxy-based film resist SU-8 (manufactured by Micro Chem.) Was spin-coated on the wafer using a spin coater. The conditions for spin coating are shown below.

Spin coating conditions (1 case, flow path height 100μm)
Slope 5 seconds-> 500 rpm 5 seconds-> slope 10 seconds-> 2800 rpm 30 seconds-> slope 5 seconds After spin coating, let stand for at least 1 hour and then leave the wafer at 65 ° C for 10 minutes and then to 95 ° C For 30 minutes.

  Next, the photomask from which the convex pattern reversed to the desired flow path pattern was obtained was placed on the wafer, and exposure was performed by irradiating with ultraviolet rays on the exposure apparatus for 45 seconds (when the flow path height was 100 μm). Here, the exposure time is adjusted to obtain a desired flow path height. After exposure, the wafer was heat treated at 65 ° C. for 3 minutes and subsequently at 95 ° C. for 10 minutes. The unpolymerized SU-8 was removed by placing the wafer in a SU-8 developer (Merck) and developing the resist layer while shaking for 15 minutes.

Next, the wafer was washed while shaking with 2-propanol for 10 minutes. Subsequently, the wafer was dried after washing with distilled water. After drying, the wafer surface was treated with trifluoromethane (CHF ₃ ) in a plasma etching apparatus. A mold in which convex portions inverted to a desired channel structure were constructed was used as a mold for PDMS microfabrication shown below.

(2) Transfer from Template Sylgard (registered trademark) 184 (manufactured by Dow Corning) and a curing agent (manufactured by Dow Corning) were measured at a weight ratio of 10: 1 and mixed according to the manufacturer's instructions. About 5g is required to make a 24mm x 76mm x 2mm size, and an appropriate amount is measured according to the size of the substrate to be formed.

Next, this mixed solution was deaerated in a desiccator, and after deaeration, degassed PDMS was poured into a mold on which a wafer was set. Then, it was cured by heating in an oven at 75 ° C. for 2.5 hours. The cured PDMS was peeled from the mold to produce a PDMS chip having a groove in which the convex portion of the mold was transferred.
(The invention's effect)

  According to the barcode array of the present invention, since the sample is poured into the reaction region partitioned into the linear space, interference by the adjacent reaction region can be prevented and the target DNA can be applied to the non-specific substrate. Since adsorption can be prevented and noise can be reduced, highly accurate detection is possible.

  Furthermore, by providing a difference in height between the reaction region and the region other than the reaction region on the substrate surface, it becomes possible to reduce background fluorescence and the like, and to improve the S / N value. Become.

In addition, it is possible to detect with a general-purpose line sensor without using an expensive microarray scanner, and it is possible to reduce costs, and it is possible to detect without accurate positioning like a microarray scanner. Simplification can be achieved.
By using PDMS with excellent optical properties as the base material that constitutes the substrate, low background can be achieved, detection is possible with a high S / N ratio, and it is also excellent in moldability. , Nano- and micron-order processing becomes easy, and it is possible to manufacture in an optimal shape for various optical devices. By adopting a molding method using a mold, mass production is possible and further cost reduction effect is achieved. I can expect.
In addition, this device can be applied to any sample by using a method suitable for each sample only for pretreatment. Therefore, microorganisms contained in environmental samples, genetic testing of foods, genetic diagnosis in the medical field It is expected to be applied to tailor-made medical care and contributes to the development of bio equipment.

  Moreover, according to the biorelevant molecular microarray of the present invention, it is possible to react not only in the closed system region but also in the open system region.

It is a top view of the barcode array of the present invention, and is a schematic diagram showing the basic configuration of the present invention FIG. 2 is a cross-sectional view taken along line AA of FIG. 1, and is a schematic diagram illustrating the barcode array of the present invention when configured as the first embodiment. It is the sectional view on the AA line of FIG. 1, and the schematic diagram which illustrates the barcode array of this invention in the case of being comprised as 2nd Embodiment FIG. 2 is a cross-sectional view taken along line AA in FIG. 1, and is a schematic diagram illustrating the barcode array of the present invention when configured as a third embodiment.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 1st member 2 2nd member 3 Reaction area | region 4 Sample supply port 5 Sample collection port 6 Groove 7 Partition 8 Linear convex part 9 Concave part 10 Linear groove part

Claims (7)

A biorelevant molecule microarray carrying a biorelevant molecule between a first member and a second member,
In either one of the first member and the second member, a biological-related molecular microarray in which a plurality of grooves are formed in parallel on the contact surface with respect to the other member, and a plurality of spaces serving as reaction regions are provided. A biorelevant molecule microarray carrying a biorelevant molecule between a first member and a second member,
A biological-related molecular microarray in which a plurality of grooves are formed in parallel on the contact surfaces of the first member and the second member, and a plurality of spaces serving as reaction regions are provided. A biorelevant molecule microarray carrying a biorelevant molecule between a first member and a second member,
In the first member, a plurality of linear protrusions are formed in parallel on the contact surface with respect to the second member,
In the second member, a plurality of linear grooves that can be fitted in one-to-one with the linear protrusions on the contact surface with respect to the first member are formed in parallel.
When the first member and the second member are brought into contact with each other while fitting the linear convex portion and the linear groove portion, a space serving as a reaction region is formed between the linear convex portion and the linear groove portion. Biologically associated molecular microarray configured to be formed.   The living body according to any one of claims 1 to 3, further comprising a sample supply port for allowing the reaction region to communicate with the outside and supplying a sample to the reaction region and a sample recovery port for recovering the sample from the reaction region. Related molecular microarrays.   The base material constituting at least one of the first member and the second member is a transparent silicon rubber such as polydimethylsiloxane or polymethyl methacrylate, or a plastic resin. The bio-related molecular microarray according to Item. A biorelevant molecular microarray carrying biorelevant molecules,
A bio-related molecular microarray having a microarray body in which a plurality of linear reaction regions are arranged side by side. The bio-related molecular microarray according to claim 6, wherein the substrate constituting the microarray body is a transparent silicon rubber or plastic resin such as polydimethylsiloxane or polymethyl methacrylate.

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