JP2008249677A - Device for introducing liquid, fixing holder, and analysis kit - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a device for introducing liquid that suppresses the generation of bubbles when introducing liquid, for example, containing a specimen to an analysis chip and facilitates the introduction operation of the liquid, and to provide a fixing holder that reliably fixes the device for introducing liquid and performs the introduction operation of liquid stably. <P>SOLUTION: The device for introducing liquid to a gap in an analysis chip having the gap between a carrier and a cover member is a device for introducing liquid, where a speed control means for controlling the introduction speed of liquid is arranged at the liquid passage section of the device. In the device for introducing liquid, the speed control means for controlling the introduction speed of liquid is arranged and has a function for controlling the introduction speed of liquid to ≤100 μL/minute. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a liquid introduction device for introducing, for example, a liquid preferably containing a test substance into a gap in an analysis chip having a gap between a carrier and a cover member. is there. The present invention also provides an analysis chip having a gap between a carrier and a cover member and a liquid introduction device for introducing a liquid into the analysis chip, and including a test substance. It is related with the fixed holder which can introduce | transduce stably and reliably.

  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, for nucleic acids, the relationship between various genes and the expression of their biological functions can be examined by methods utilizing complementarity between various nucleic acids / nucleic acids, such as Northern blotting or Southern blotting. In addition, the protein can be examined for the function and expression of the protein by utilizing 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 the gene expression of hundreds to tens of thousands, and is an analysis chip in which molecules such as DNA are arranged at high density on a base substrate such as glass or silicon. is there. By using such 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 biological, 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, that is, 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 base sequence data per gene. 16 to 20 25-mers are set, and a probe DNA is set by combining a 25-mer perfect match and a 1-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 a large number of genes can be schematically displayed in color. By such clustering, genes related to a certain disease can be identified.

  Analysis chips in which not only DNA but also proteins and saccharides are arranged on a substrate have come to be used as 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.

  When using an analysis chip such as DNA, it is necessary to apply (introduce) a liquid such as a prepared sample solution so as to spread over the entire region of the analysis chip where the selective binding substance is fixed. In order to spread a very small amount of the sample solution over the entire region of the analysis chip, there is a method in which a flat cover glass is previously applied to the surface of the chip and the sample solution is injected from the side by capillary action. In this case, it is necessary to stably hold a very small amount of sample solution on the chip while suppressing evaporation. However, in this method, the sample solution is not sealed during the reaction such as hybridization, and is always in the atmosphere. Evaporation into it will occur. Therefore, when it is necessary to carry out a long-term reaction such as 1 minute to overnight by this method, the sample solution remains in contact with the atmosphere for a long time. As a result, the solution evaporates and a stable result is obtained. There was a problem that it was not possible.

  As a means for solving such a problem of the evaporation of the sample solution, for example, a sealed container that can hold the DNA chip in a wet state is commercially available, and the evaporation can be suppressed by using this. However, when such a means is used, the experiment cost increases due to the purchase of a new sealed container, and the operation of stably installing the DNA chip in the sealed container is added. There was a problem that became complicated.

  In order to solve the above problems, an analysis chip has been proposed that includes a carrier and a cover member that covers the surface of the carrier and is bonded to the carrier, and further has a gap between the carrier and the cover member.

  This analysis chip can stably hold the sample solution on the carrier, suppress evaporation of the sample solution, and enclose fine particles in the space surrounded by the carrier and the cover member. It became possible to stir the solution containing the added test substance, and as a result, it was possible to achieve high efficiency of the reaction (see Non-Patent Document 1).

However, when introducing (applying) the sample solution into the gap between the carrier and the cover member, it is necessary to pay close attention to prevent the generation and invasion of bubbles. There was a problem that it was necessary.
Reiko Takizawa et al., Biotechnology Journal: July-August 2005, pages 418-420

  In an analysis chip having a gap between the carrier and the cover member, when a liquid such as a sample solution is introduced (applied) into the gap between the carrier and the cover member, the shape of the carrier or the cover member or the reaction efficiency Due to factors such as fine particles sealed in the space, air bubbles or air layers are likely to be generated, and the generation of such air bubbles does not allow the liquid such as the sample solution to permeate on the carrier. There is a problem that unevenness of reaction may occur, which is undesirable because it affects the analysis result. Furthermore, in order to prevent such intrusion of bubbles and air layers, it is necessary to pay close attention, and there is a problem that a skillful technique is necessary for the operation.

  In addition, the analysis chip having a gap between the carrier and the cover member and the liquid introduction device for introducing the liquid into the analysis chip are securely fixed, and the liquid containing the test substance can be stably and reliably It is important to be able to introduce it into

  Accordingly, an object of the present invention is to provide a liquid introduction device that suppresses the generation of bubbles when a liquid is introduced into an analysis chip and facilitates the operation of introducing the liquid.

  Another object of the present invention is to securely fix both the analysis chip having a gap between the carrier and the cover member and the liquid introduction device for introducing the liquid into the analysis chip so that the liquid is stable and stable. An object of the present invention is to provide a fixed holder that can be reliably introduced.

  The present invention aims to achieve the above object, and the liquid introduction device of the present invention is a device for introducing a liquid into the gap of an analysis chip having a gap between the carrier and the cover member. The liquid introducing device is characterized in that a speed adjusting means for adjusting the liquid introducing speed is arranged in the liquid passage portion of the device.

  According to a preferred aspect of the liquid introduction device of the present invention, the speed adjusting means for adjusting the liquid introduction speed has a function of controlling the liquid introduction speed to be 100 μL / min or less. It is.

  According to a preferred aspect of the liquid introduction device of the present invention, the liquid introduction device introduces the liquid into the void portion of the analysis chip by gravity and / or pressure difference by adding the liquid to the device. It is comprised as follows.

  According to a preferred aspect of the liquid introduction device of the present invention, the diameter of the liquid introduction portion of the device for introducing the liquid into the gap portion of the analysis chip is 0.001 mm or more and 0.8 mm or less.

  According to a preferred aspect of the liquid introduction device of the present invention, the speed adjusting means for adjusting the liquid introduction speed has a porous structure.

  According to a preferred aspect of the liquid introduction device of the present invention, the porous structure is composed of fibers and / or fine particles, and the fibers and / or fine particles constituting the porous structure are adhesive substances. And / or adhered or adhered by an adhesive substance.

  The liquid introduction device of the present invention can be combined with an analysis chip having a gap between the carrier and the cover member to form an analysis kit.

  The fixing holder of the present invention is a fixing holder for fixing both the analysis chip having a gap between the carrier and the cover member and the liquid introduction device, and the liquid introduction device Means for holding and fixing at least a part of the liquid introduction device is provided.

  According to a preferred aspect of the fixing holder of the present invention, the fixing holder includes means for holding and fixing at least a part of the liquid introduction device by an elastomer member.

  According to a preferred aspect of the fixed holder of the present invention, the fixed holder comprises means for making the holding position of the liquid introduction device movable in accordance with the position of the liquid inlet of the analysis chip. is there.

  According to a preferred aspect of the fixing holder of the present invention, the fixing holder can hold and fix a plurality of liquid introduction devices to a plurality of analysis chips.

  By using the liquid introduction device of the present invention, it is possible to suppress the generation of bubbles and air layers when introducing a liquid containing a sample into the gap of the analysis chip having a gap between the carrier and the cover member. In addition, the liquid can be easily introduced into the analysis chip without requiring any skill.

  In addition, with the fixing holder of the present invention, the analysis chip and the liquid introduction device for introducing the liquid into the analysis chip can be reliably fixed, and the liquid introduction operation can be performed stably.

  Hereinafter, the liquid introduction device and the fixed holder of the present invention will be described in more detail.

  The analysis chip in the present invention refers to the presence or absence of the test substance, the amount of the test substance, the property of the test substance, etc. by introducing (applying) a liquid such as a solution containing the test substance to the analysis chip. A chip used to measure Specific examples of such an analysis chip include a biochip for examining the amount of a specimen and the presence or absence of a specimen by a reaction between a 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 cell on the substrate surface Cell chip etc. in which is immobilized.

  In such an analysis chip, a liquid such as a solution (analyte solution) containing a test substance is introduced so as to spread over the entire area where the selective binding substance is provided on the analysis chip, or a small amount of an analyte solution, etc. It is preferable to stably hold the liquid on the analysis chip while suppressing evaporation. Therefore, for example, an analysis chip including a carrier as shown in FIG. 1 and a cover member bonded to a part thereof is preferably used.

  First, the analysis chip in which the liquid introduction device of the present invention is preferably used will be specifically described, but the present invention is not limited to these.

  FIG. 1 is a perspective view schematically showing an example of an analysis chip having a gap between a carrier and a cover member using the liquid introduction device of the present invention, and is cut along a plane along the line A1. It is the partial longitudinal cross-sectional view (b) which was made. In the example shown in FIG. 1, the carrier 1 is covered with a cover member 2 via an adhesive member 3 to form a gap 6 including a region where a selective binding substance is immobilized. The gap 6 is a closed space that does not communicate with the outside except that it communicates with the outside through one or a plurality of through holes 5. And the through-hole 5 has the liquid level parking chamber 4 with a diameter larger than a through-hole in the upper end part. In the use of the analysis chip shown in FIG. 1, the sample solution can be applied from any one or more of the through holes 5 using the liquid introduction device of the present invention, and the gap 6 is filled with the sample solution. To do. The through hole 5 is a liquid injection port. Thereafter, the sample solution can be easily hermetically sealed by closing the through hole 5 with a sealing member. As a result, the reaction between the specimen and the selective binding substance immobilized on the carrier 1 can be performed stably.

  FIG. 2 is a schematic perspective view (c) and a longitudinal sectional view (d) of an example of a carrier constituting an analysis chip having a gap between a carrier using the liquid introduction device of the present invention and a cover member. .

  FIG. 2 shows an example of the carrier shape of the analysis chip shown in FIG. 1. As shown here, a part of the carrier 1 has a concavo-convex structure, so that the detection sensitivity can be improved.

  FIG. 3 is a longitudinal sectional view schematically showing an example of an analysis chip having a gap portion between a carrier and a cover member using the liquid introduction device of the present invention.

  In the example shown in FIG. 3, the selective binding substance 7 such as DNA is immobilized on the upper surface of the convex portion of the carrier 1. Fine particles (in this case, spherical beads) 8 are placed in the gaps in the recesses of the carrier 1. The selective binding substance 7 and the fine particles (beads) 8 come into contact with a specimen solution (not shown) containing specimen DNA (test substance). The sample solution is held in the gap 6 defined by the carrier 1, the adhesive member 3, and the cover member 2.

  In the present invention, the carrier constituting the analysis chip can have a fine concavo-convex structure. As the shape of the concave portion and the convex portion, for example, the convex portion is preferably a columnar structure such as a prism, a cylinder, and a truncated cone. Moreover, it is preferable that the shape of the upper surface of a convex part is circular or square, such as a tri-octagon. The recesses or projections have a completely or substantially the same structure as shown in FIG. 2 and are preferably arranged alternately and regularly. In the case of such a regular arrangement, the shape of the concave portion is determined according to the shape of the convex portion.

  Examples of the material constituting the carrier include inorganic materials such as glass, ceramics, and silicon, and polymers such as polymethyl methacrylate (PMMA), polyethylene terephthalate, cellulose acetate, polycarbonate, polystyrene, polydimethylsiloxane, and silicon rubber. it can. The carrier is preferably composed of a synthetic polymer that is easy to mold, such as PMMA.

  Examples of the carrier molding method include a method such as an injection molding method or a hot embossing method when the carrier is a polymer, a method such as a sand blasting method when the carrier is glass or ceramic, and a case where the carrier is silicon. Examples include methods used in known semiconductor processes.

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

  As for the size of the convex part of the carrier, for example, the height is preferably about 10 to about 200 μm, and the width is preferably about 50 to about 150 μm. The distance between the convex portions is preferably, for example, 50 to 600 μm, and more preferably a size that allows a plurality of fine particles to enter. Further, as disclosed in Japanese Patent Application Laid-Open No. 2004-264289, the focus of the excitation light during detection is often adjusted on the upper surface of the flat part, and the height of the convex part differs from the height of the flat part. Then, since the focus of the excitation light on the top surface of the convex portion is blurred and the detection result may be blurred, the height of the convex portion may be the same height as the surrounding flat portion. preferable.

  The relationship between the convex part and the flat part is shown in FIG. 2 and FIG. 3, for example. Further, as shown in FIG. 3, the selective binding substance 7 is immobilized on the upper surface of the convex portion. Therefore, it is necessary to provide a space between the upper surface of the convex portion and the cover member 2 so that the selective binding substance 7 and the test substance can be combined. The size of such a space is preferably 1 to 500 μm in the height direction, for example. If the size of the space is smaller than this, the chance that the test substance contacts the immobilized selective binding substance 7 is extremely reduced, and the signal after hybridization becomes remarkably small. On the other hand, if the size of the space exceeds 500 μm When a small amount of sample is used, the concentration of the sample solution is reduced, the reactivity such as hybridization is lowered, and the detection signal is weakened.

  In order to immobilize the selective binding substance 7 on the upper surface of the convex part of the carrier, the upper surface of the convex part has a functional group (for example, an amino group, a hydroxy group, a carboxyl group, an aldehyde group that can be bonded to the selective binding substance 7). And epoxy groups). In order to introduce such a functional group, for example, the surface of the convex part of the carrier is subjected to plasma treatment or radiation treatment such as γ-ray or electron beam, and then further subjected to graft polymerization treatment to introduce a polar group. For example, a polycation such as poly-L-lysine or a silane coupling agent can be coated.

  Examples of the silane coupling agent used here include 3-aminopropyltriethoxysilane, 3-aminopropyltrimethoxysilane, 3-aminopropyldiethoxymethylsilane, and 3- (2-aminoethylaminopropyl) trimethoxy. Silane etc. are mentioned.

  As a preferred carrier that can be used in the present invention, for example, a carrier on which a selective binding substance is immobilized, the carrier is provided with uneven portions, and the selective binding substance has a plurality of convex and concave portions. JP-A 2004-264289 and Biotechnology Journal: 2005- 7 as a carrier that improves the S / N ratio of the detection result by immobilizing the selective binding substance immobilized on the part. August, pp. 418-420 (see Non-Patent Document 1), and the like.

  The cover member 2 used in the present invention covers at least part of the surface of the selective binding substance-immobilized carrier, and has a gap 6 between the substrate of the carrier 1 and the cover member 2. Can be adhered to the carrier 1. And the support | carrier 1 has the selective binding substance 7 fixed on the area | region which preferably exists in the surface and the said space | gap part 6 preferably. That is, preferably, the cover member 2 is bonded to the carrier 1 with the adhesive member 3 so that the region where the selective binding substance 7 is immobilized exists in the gap 6. The cover member 2 may be bonded in any manner as long as the gap 6 is formed, but is preferably bonded through an adhesive member 3 such as a double-sided tape or a resin composition.

  The cover member 2 can have one or more through holes 5 communicating with the gap 6, and preferably has a plurality of through holes. This through-hole can inject liquids such as fine particles, a sample to be tested, and a reaction buffer, and at the same time, is used for maintaining the pressure inside the analysis chip at atmospheric pressure. It is preferable that there are a plurality of through holes with respect to one gap portion 6, and in particular, the filling of the liquid is facilitated by using 3 to 6 through holes.

  When the cover member 2 has a plurality of through holes, their hole diameters may be the same or different, but one of the plurality of through holes is a liquid inlet and the other through hole is an air outlet. In the case of functioning as a liquid, it is preferable that the liquid injection port has a wide hole diameter necessary for liquid injection and the other through holes have a narrower hole diameter from the viewpoint of easy liquid application and liquid hermetic retention. Specifically, the through-hole size of the liquid injection port is preferably a hole diameter in the range of 0.01 to 2.0 mm, and the diameters of the other through-holes are preferably in the range of 0.01 to 1.0 mm.

  At least one of the through holes may be provided with a portion having a large hole diameter, that is, a so-called liquid level holding chamber 4 at the upper end by changing the hole diameter. Here, parking means to keep it in the necessary part. By providing the liquid level chamber 4, the rise of the liquid level of the liquid applied from the through hole 5 and filled in the gap portion 6 is suppressed, and it is easily and reliably performed when the through hole is sealed with the sealing member. And the outflow of liquid can be prevented. The shape of the liquid level holding chamber 4 can be, for example, a cylindrical shape, a prismatic shape, a conical shape, a pyramid shape, a hemispherical shape, or a shape similar to this. Among these, the columnar shape is a particularly preferable embodiment from the viewpoints of ease of manufacture and the high effect of suppressing the rise in the sample solution.

  With respect to the hole diameter (diameter) of the through-hole, for example, in the case of a combination of a cylindrical through-hole having a longitudinal cross-sectional shape and a liquid level holding chamber, the hole diameter (diameter) of the through-hole is 0.01 to 2 0.0 mm is preferable, and 0.3 to 1.0 mm is more preferable. By making the hole diameter of the through hole 0.01 mm or more, it becomes easy to apply the liquid. On the other hand, by setting the diameter of the through hole to 2.0 mm or less, it is possible to more effectively suppress the evaporation of the liquid before sealing after the application.

  The hole diameter (diameter) of the liquid level holding chamber 4 is preferably 1.0 mm or more. By setting the hole diameter of the liquid level holding chamber 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 holding effect can be obtained. The upper limit of the hole diameter of the liquid level holding chamber 4 can be, for example, 10 mm or less. Further, the depth of the liquid level holding chamber 4 can be set within a range of 0.1 to 5 mm, for example.

  Such a cover member 2 used in the present invention is preferably adhered to the above-mentioned selective binding substance-immobilized carrier with a removable strength and mode. When an analysis chip is used as a DNA chip, it is usually necessary to read the DNA chip with a dedicated scanner. However, when the cover member is adhered, it is difficult to set the cover on the dedicated scanner. Then, the cover member and the optical part of the scanner come into contact with each other, which may cause a failure. Even if reading through the cover member is possible, the reading value may be inaccurate. Therefore, it is preferable that the cover member is removable so that the cover member can be removed in the reading step.

  Furthermore, the fine particles 8 can be inserted into a space (gap portion) surrounded by the substrate of the carrier 1 and the cover member 2 as shown in FIG. The presence of the fine particles makes it possible to efficiently stir the injected liquid, resulting in a hybridization reaction promoting effect.

  Examples of the fine particle material include glass, ceramics such as yttria-stabilized zirconia, metals such as stainless steel, polymers such as nylon and polystyrene, and the like. Among these, ceramic fine particles are preferably used because they are physically and chemically stable and have a large specific gravity. More preferably used are fine particles of yttria-stabilized zirconium.

  In the present invention, in consideration of the convenience of the user, it is preferable that fine particles are sealed in advance in a space surrounded by the carrier and the cover member in the analysis chip to be used. If antistatic processing is performed, for example, by applying an antistatic agent to the cover member, the fine particles can be moved in the gap. As a result, it is possible to uniformly disperse the encapsulated fine particles in the voids by, for example, tilting the DNA chip before applying the liquid through the through-hole.

  By moving such fine particles during hybridization, the liquid is efficiently stirred. As means for moving the fine particles, preferably the method of rotating the analysis chip to drop the fine particles in the direction of gravity, the method of setting the analysis chip containing the fine particles in a shaker and shaking the substrate, the magnetic fine particles The method of moving fine particles using magnetic force is used, but the method of setting the chip on a shaker and rotating it in a horizontal plane has a large moving range of fine particles and moves without unevenness. It is preferably used because the liquid can be stirred. At this time, the rotational speed of the turning rotation is preferably 10 to 1000 rotations / minute, and more preferably 100 to 500 rotations / minute.

  The size (diameter) of the fine particles is preferably 10 μm to 500 μm, more preferably 50 μm to 300 μm. If the diameter is smaller than this, the effect of stirring may not be sufficiently obtained. Conversely, if the diameter is larger than this, it is necessary to enlarge the gap between the cover member and the substrate of the carrier in order to enclose the fine particles. As a result, the amount of liquid required increases. In addition, considering that a solution (liquid) such as a specimen is placed in a space (gap) surrounded by the substrate of the carrier and the cover member, and stirring is performed by moving the enclosed fine particles, the liquid and the fine particles are covered. It is preferable to close the through hole so as not to spill from the through hole of the member.

  As described above, the cover member can be detached without damaging the cover member and the substrate of the carrier as a mode of adhering the cover member to the carrier on which the selective binding substance is fixed. An aspect is preferable, for example, it can adhere | attach through adhesive layers, such as a double-sided tape and a resin composition.

  When a resin composition is used as the adhesive layer, a resin composition containing a polymer selected from the group consisting of acrylic polymers, silicone polymers, and mixtures thereof can be used as the resin composition. By using these resin compositions, it becomes possible to improve the sealing performance as compared with the double-sided tape, and also stable against long-term incubation as compared with the double-sided tape. It is particularly preferably used in an analysis system that requires this incubation. In particular, when a silicone-based elastomer is used as the adhesive member, the sealing property is good, and the cover member can be adhered in a state where it can be easily detached. Specific examples of such an elastomer include Dow Corning “Silgard” (registered trademark) and Shin-Etsu Chemical Co., Ltd., two-component RTV rubber for molding.

  The selective binding substance used in the present invention refers to a substance that can selectively bind to a test substance directly or indirectly. Examples include nucleic acids, proteins, saccharides and 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 and the like. Sugars include oligosaccharides and polysaccharides. Other antigenic compounds include peptides and small molecule compounds. Preferred selective compounds are nucleic acids and proteins (especially antibodies, antigens etc.). Such selective binding substances may be commercially available, or may be synthesized or prepared from natural sources such as living tissue or cells.

  For DNA such as a gene whose characteristics and primary structure (base sequence) are clear, a probe or primer can be prepared based on the sequence, and for example, the target DNA can be selected from 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, or a method such as restriction enzyme cleavage and mapping or sequencing.

  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. can do.

  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 with 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 and anti-peptide antibodies. These antibodies can be produced using a known method.

  Monoclonal antibodies include, for example, Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyzes, Plenum Press, US (1980); Ikuzaki Ikuo et al., Monoclonal Antibody Hybridoma and ELISA As for the antibody, reference can be made to, for example, Nao Matsuhashi et al., Introduction to Immunology Experiments (Biochemical Experiment Method 15), Japan Society Publication Center (1982)

  As for anti-peptide antibodies, for example, Takara Bio Co., Ltd. has commissioned synthesis, and it is also possible to obtain them using them. Briefly, the primary sequence of a protein is predicted to be mobile, hydrophilic / hydrophobic, secondary structure prediction and polarity prediction to predict the site located on the surface of the protein, and the immunized animal has an original sequence. 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 carrier, 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, 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 spotted on the substrate in order. The ink jet method is a method in which minute droplets are ejected from a nozzle by a piezoelectric element or the like and a selective binding substance is sprayed on a substrate of a carrier. Specifically, genes are ejected from a nozzle, and selective binding substances are aligned and arranged at high speed on a substrate of a carrier. Alternatively, when the selective binding substance is a nucleic acid, nucleotide synthesis can be sequentially performed on a carrier substrate by a photolithographic method.

  The liquid introduction device of the present invention is a device for introducing (applying) a liquid into a gap in the analysis chip having a gap between the carrier and the cover member as described above. Means for adjusting the liquid introduction speed in the solution passage section. A device here refers to an apparatus, a tool, a jig, or the like. Due to the shape and material of the carrier and cover member of the analysis chip as described above, the physical and chemical state of the surface, or the shape, material, physical property or aggregate state of the fine particles contained in the voids. Bubbles or air layers may be generated. The formation of bubbles or air layers in the liquid introduced into the gap corresponding to the detection unit means that the contact between the selective binding substance immobilized on the carrier and the substance in the test sample (analyte solution) is a bubble. Alternatively, since the binding is not partially or entirely inhibited by the air layer, there is a possibility of adverse effects such as poor sensitivity of detection results and poor reproducibility.

  By adjusting the liquid introduction speed by using the liquid introduction device of the present invention, it is possible to suppress the generation of bubbles or air layers when introducing the liquid typified by the sample solution into the gap. . That is, using the liquid introduction device of the present invention to suppress the generation of bubbles or air layers is a preferable aspect in the use of the analysis chip.

  Here, the bubble or the air layer is an aggregate of gas or air existing in the liquid, and the size per one is converted into a true sphere and has a diameter of 10 μm or more. By using a liquid introduction device, the number or size of these bubbles or air layers generated can be greatly reduced.

  The liquid introduction device of the present invention is capable of adjusting the liquid introduction speed into the void portion of the analysis chip because there is a means for adjusting the liquid introduction speed in the liquid passage section. When the liquid is introduced by the above, the liquid can be sufficiently infiltrated into the surface of the carrier, the cover member or the fine particles so that the liquid can be introduced to the carrier, the cover member or the fine particles, and the liquid can be introduced while releasing the air. it can. Furthermore, when the carrier has a fine concavo-convex structure, an air layer may be formed when the liquid is introduced due to the concavo-convex structure when the liquid is introduced. It is also possible to prevent the formation of an air layer due to the sneaking around of liquid.

  For these reasons, the liquid introduction device of the present invention can sufficiently suppress the generation of bubbles and air layers as a result. A preferable solution introduction rate in suppressing the generation of bubbles or air layers is 100 μL / min or less, and more preferably 75 μL / min or less. If the solution introduction rate is 100 μL / min or less, it is possible to sufficiently suppress the generation of bubbles and air layers in the analysis chip as described above, so that the solution introduction rate can be realized. A device is preferred.

  Further, the liquid introduction device of the present invention preferably has a shape in which the liquid is introduced by gravity and / or pressure difference by adding the liquid to the device. That is, when a liquid is added to the liquid addition part in the liquid introduction device, a pressure difference such as gravity is generated between the part where the liquid exists in the device and the part where the liquid is introduced into the gap part in the device. A shape in which liquid is automatically introduced. The pressure may be not only gravity but also air pressure and water pressure including positive pressure and negative pressure.

  As the shape of such a liquid introduction device, a shape as shown in FIG. 4 can be preferably used. FIG. 4 is a longitudinal sectional view schematically showing an example of the liquid introduction device of the present invention.

  As shown in FIG. 4, the liquid introduction device includes a liquid addition portion 9 that is a portion to which a liquid is added and a liquid passage portion 12, and the liquid passage portion 12 introduces the liquid into the gap portion of the analysis chip. And a speed adjusting means 10 for adjusting the liquid introduction speed. It is preferable that the liquid adding part 9 and the liquid introducing part 11 have a height difference when introducing the liquid. In particular, the liquid adding part 9 can be positioned higher than the liquid introducing part 11 when introducing the liquid. It is preferable that it is a shape.

  If the outer diameter of the liquid introduction part 11 of the liquid introduction device is smaller than the hole diameter of the liquid level holding chamber, the liquid introduction device is easily set. A range of 3 to 1.0 mm is more preferable.

  If the height difference is large, the liquid introduction speed tends to increase. If the height difference is small, the solution introduction speed tends to decrease. Therefore, the solution introduction speed can be adjusted to some extent depending on the shape. Further, the shape of the liquid adding portion 9 is preferably a shape that can store liquid, and can take various shapes such as a hemispherical shape, a conical shape, a pyramid shape, and a cylindrical shape, It is preferable that the size is such that more than the volume of the solution to be introduced can be stored in this portion. In addition, the liquid introduction part 11 preferably has a shape that can be inserted into the through hole of the cover member of the analysis chip described above, and in particular, the liquid introduction part 11 has a shape along the shape of the through hole. It is preferable to have a shape that closely adheres to the gap when inserted. This is because when a gap is generated, air enters from there and may cause bubbles and an air layer to be generated in the liquid introduced into the gap portion of the analysis chip.

  Moreover, as the shape of the liquid passage part 12 which connects the liquid addition part 9 and the liquid introducing | transducing part 11, various shapes, such as a cone shape, a column shape, a pyramid shape, and a prism shape, can be taken, for example, but liquid is especially The shape is preferably such that it can pass smoothly. In particular, since the shape of the liquid passage portion 12 determines the height difference between the liquid addition portion 9 and the liquid introduction portion 11, as described above, when the liquid velocity is adjusted to some extent by this height difference, an arbitrary It is necessary to design the shape so that the solution introduction speed is achieved. The material for producing the liquid introduction device having the shape as described above may be made of glass, metal, carbon, polymer, or the like. It is preferable to use a material, and examples of the material include polypropylene, polystyrene, polycarbonate, and polymethyl methacrylate.

  In addition, the liquid introduction device may be subjected to various treatments in order to change the surface properties, such as adsorption treatment, coating treatment, chemical treatment, heat treatment, cooling treatment, hydrolysis treatment, flame treatment, steam treatment, and chemical treatment. Various treatments such as pressure steam treatment, electron beam treatment, discharge treatment and vacuum treatment can be performed. In particular, a treatment method that imparts hydrophilicity to the surface is preferable in terms of smoothly introducing the solution.

  Commercially available products such as pipettes, pipette tips, columns, capillaries, tubes, wells, and dishes can be used as the liquid introduction device having the above-described shape.

  Further, in the liquid introduction device having the shape as described above, the diameter of the liquid introduction part 11 which is a part for introducing the liquid into the gap is preferably in the range of 0.001 to 0.8 mm, more preferably 0. The range is 0.005 to 0.5 mm. Here, the diameter of the liquid introduction part 11 refers to a value obtained by converting the area of the diameter part into a perfect circle. By reducing the diameter, it is possible to easily control the liquid introduction speed by the speed adjusting means for adjusting the liquid introduction speed as shown below. If the diameter is large, the liquid introduction speed tends to increase. If it is small, a tendency to reduce the liquid introduction rate can be produced.

  When the aperture is less than 0.001 mm, there is a tendency that the liquid cannot pass by adopting a structure for adjusting the liquid introduction speed described later.

  Further, in the liquid introduction device having the above-described shape, when the diameter of the liquid addition section 9 is larger than the diameter of the liquid introduction section 11, addition of the liquid becomes easy.

  In the liquid introduction device of the present invention, the most important thing is in the liquid passage part 12 between the part to which the liquid is added (liquid addition part 9) and the part to introduce the liquid into the gap part of the analysis chip (liquid introduction part 11). And a means for adjusting the liquid introduction speed. As a means for adjusting the liquid introduction speed, it is possible to adopt a configuration in which the liquid introduction speed is controlled by detecting the liquid introduction speed using a sensor or the like and mechanically feeding back the detected speed. However, this increases the structure, takes up space, and increases the cost.

  Therefore, it is preferable that the means for adjusting the introduction speed of the liquid take a simple structure in which the passage speed of the liquid is controlled by a physical barrier in the structure, and the passage speed of the liquid by such a physical barrier. As a structure for controlling the above, a structure in which the liquid flow path is simply narrowed is conceivable, and a porous structure is particularly preferable.

By adopting a porous structure, it becomes easy to arbitrarily control the liquid introduction speed by adjusting the size and shape of the pore diameter and the number and length of the pores. Regarding the pores of such a porous structure, the ratio of the area occupied by the pores on the cut surface of the porous structure is preferably in the range of 1 to 50%, more preferably in the range of 5 to 40%. It is preferable that the number of pores is in the range of 1 to 100000 pieces / mm ^2, more preferably in the range of 10 to 10,000 pieces / mm ^2. Moreover, it is preferable that the length of a hole is the range of 0.5-50 mm, More preferably, it is the range of 1-30 mm. The hole here refers to a penetrating structure through which liquid can pass.

  The porosity of the porous structure is preferably in the range of 1 to 50%, more preferably in the range of 2 to 40%. If the porosity exceeds 50%, the rate at which the liquid is introduced becomes difficult to control, and if the porosity is less than 1%, it is difficult to pass the solution.

The porosity is the apparent density ρ _a (g / cm ³ ) of the porous structure, that is, the area occupied by the porous structure is V (cm ³ ), and the weight of the structure forming the porous structure is measured. (G). W can be determined apparent density [rho _a by the dividing the V. Using this, the porosity F _V (%) can be determined as follows. That is, by using the volume V (cm ³ ) and W (g) used when the above apparent density was obtained, the specific gravity S _g (g / cm ³ ) of the fiber forming the sponge-like structure having a porous structure was further determined. And can be obtained by the following equation (1).
Porosity F _V (%) = [1− (W / S _g ) / V] × 100 (1)
In addition, since the speed adjusting means has a porous structure, it is possible to reduce the diameter of each hole of the porous structure, thereby removing bubbles previously present in the solution such as the sample solution to be added. It becomes possible to trap (capture).

  Examples of the porous structure as described above include a porous structure composed of fibers, fine particles, a net, and the like. In particular, fibers and fine particles can be preferably used from the viewpoint of ease of use and cost. About a fiber and microparticles | fine-particles, a hole is formed between them and can comprise a porous structure as a result. Materials used for fibers, fine particles and nets include glass, ceramics, stainless steel, iron, gold, silver, copper, magnets and other metals, nylon, polystyrene, chitin, chitosan, polymethyl methacrylate, polysulfone, polyethylene Examples thereof include polymers such as talates.

  In order to set the liquid introduction rate to 100 μL / min or less, depending on the type of material, the fibers preferably have an average single fiber diameter of 100 μm or less, and more preferably 50 μm or less. The lower limit of the fiber diameter is preferably 0.01 μm or more, more preferably 0.1 μm or more. The fine particles preferably have an average particle size of 400 μm or less, more preferably 300 μm or less. On the other hand, the lower limit is preferably 1 μm or more, more preferably 10 μm or more.

  Further, materials used as fibers and fine particles may be adhered or adhered to each other with an adhesive substance such as an adhesive or an adhesive substance such as silicone. Specific examples of such adhesive and sticky substances include polysaccharides such as dextran, starch, cellulose, and trehalose, water-soluble polymer substances such as alginic acid, polyvinyl alcohol, and polyethylene glycol, sodium dodecyl sulfate, and triton. (“Triton”) (registered trademark), surfactants such as “Tween” (registered trademark), and proteins such as albumin. After these substances are made into a solution such as an aqueous solution and passed through the fibers and fine particles to be adsorbed, the fibers and the fine particles can be adhered or adhered to each other by performing a drying treatment.

  As for the polymer material, the materials may be bonded to each other by melting and fusing the surfaces once by heat treatment or steam treatment. By performing the adhesion and / or adhesion treatment as described above, it is possible to prevent fibers and fine particles from spilling from the liquid introduction device during transportation.

  FIG. 5 is a schematic perspective view for explaining a usage mode of an example of the liquid introduction device of the present invention.

  As for the liquid introduction device having the above-described shape, for example, as shown in FIG. 5, in the analysis chip 14 having a gap between the carrier and the cover member, one of the through holes of the cover member of the present invention is used. The liquid introduction part 11 of the liquid introduction device 13 is inserted. Then, by adding the liquid to the liquid adding section 9 of the liquid introducing device 13, the liquid is introduced when the liquid passes through the liquid passing section 12 and passes through the means 10 for adjusting the liquid introduction speed. The speed is adjusted so that the liquid introduction speed is 100 μL / min or less, and the liquid is introduced without generating bubbles or an air layer in the gap portion as the detection portion. The lower limit of the liquid introduction rate is preferably 1 μL / min or more. When the liquid introduction rate is less than 1 μL / min, the liquid may evaporate.

  Also, when there are a plurality of through holes in the cover member, the air that has been present in the gap is discharged from the through holes other than the through holes into which the liquid has been introduced. Without generating an air layer. Therefore, by using the liquid introduction device of the present invention, a liquid such as a sample solution is provided in the void portion of the analysis chip 14 having a void portion between the carrier and the cover member without requiring any skill. Can be introduced.

  The liquid introduction device of the present invention may be disposable or repeatedly used. The present invention further includes an analysis chip having a gap between the carrier and the cover member, and the liquid introduction device. It can also take the form of an analytical kit. This analysis kit may further contain buffers, hybridization mixes, washings, measurement solutions, labels, labeling reagents (such as enzymes), "control" nucleic acids, software and other materials, reagents and specifications. .

  In addition, when introducing the liquid into the analysis chip, the analysis chip having a gap portion between the carrier and the cover member and the liquid introduction device for introducing the liquid into the gap portion are respectively provided with means for fixing. A holder can be used.

  The analysis chip in the present invention refers to the presence or absence of the test substance, the amount of the test substance, the property of the test substance, etc. by introducing (applying) a liquid such as a solution containing the test substance to the analysis chip. A chip used to measure Specific examples of such an analysis chip include a biochip for examining the amount of a specimen and the presence or absence of a specimen by a reaction between a selective binding substance immobilized on the substrate surface of a carrier 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 cell on the substrate surface Cell chip etc. in which is immobilized.

  The liquid introduction device of the present invention introduces a liquid into the gap of the analysis chip, and further includes means for adjusting the liquid introduction speed. By the presence of a speed adjusting means for adjusting the liquid introduction speed in the liquid passage part of the liquid introduction device, it is possible to adjust the liquid introduction speed into the gap of the analysis chip. When the liquid is introduced, the liquid can be sufficiently infiltrated into the surface of the carrier, the cover member, or the fine particles, and introduced while being familiar, and the liquid can be introduced while releasing air. . Furthermore, when the carrier has a fine concavo-convex structure, an air layer may be formed by introducing the liquid due to the concavo-convex when the liquid is introduced, but the liquid introduction speed is adjusted. Therefore, such a phenomenon can be prevented. For these reasons, the liquid introduction device of the present invention can sufficiently suppress the generation of bubbles and air layers as a result. A preferable solution introduction rate in suppressing the generation of bubbles or air layers is 100 μL / min or less, and more preferably 75 μL / min or less. By setting the solution introduction rate to 100 μL / min or less, it is possible to sufficiently suppress the generation of bubbles and air layers in the analysis chip as described above. Device is preferred.

  Further, the liquid introduction device of the present invention preferably has a shape in which the liquid is introduced by gravity and / or pressure difference by adding the liquid to the device. That is, by adding a liquid to the liquid addition portion in the liquid introduction device, gravity or the like is generated between a portion where the liquid is present in the liquid introduction device and a portion where the liquid is introduced into the gap portion in the liquid introduction device. This is a shape in which a liquid is automatically introduced due to the pressure difference. The pressure may be not only gravity but also air pressure and water pressure including positive pressure and negative pressure.

  The fixing holder of the present invention includes means for gripping and fixing at least a part of the liquid introduction device. As a means for gripping and fixing at least a part of the liquid introduction device provided in the fixing holder of the present invention, the liquid introduction portion (tip portion) of the liquid introduction device is a through hole of the cover member of the analysis chip. It is possible to maintain or maintain the state inserted in the.

  It is preferable that the liquid introduction device gripping portion provided in the fixed holder of the present invention can be set on the analysis chip without the liquid introduction portion (tip portion) of the liquid introduction device contacting the gripping portion.

  FIG. 6 is a partial perspective view schematically showing an example of means for fixing the liquid introduction device of the fixing holder of the present invention. A structure in which the liquid introduction device gripping portion 15 is narrower than the outer diameter of the liquid addition portion 9 of the liquid introduction device and thicker than the outer diameter of the liquid passage portion 12 (FIGS. 6E and 6F) or reinforcement The liquid introduction device gripping portion 15 is made narrower than the outer diameter of the liquid passage portion 12 by the elastomer member 16 on the plate 17 (FIGS. 6G and 6H), thereby gripping the liquid introduction device. The state can be maintained or maintained. By using the elastomer, the liquid introduction device can be easily and reliably held and fixed.

  The elastomer of the elastomer member referred to in the present invention has a rubbery property, that is, a property (rubber elasticity) that returns to the original shape when the load is removed after being deformed by applying a certain load to the material. Natural rubber, synthetic rubber, thermoplastic elastomer and the like are used. As the synthetic rubber, for example, styrene-butadiene rubber, butadiene rubber, acrylonitrile butadiene rubber, chloroprene rubber, butyl rubber, ethylene-propylene rubber, silicone rubber, and fluorine rubber are preferably used.

  The peculiar property of the thermoplastic elastomer is derived from a structure in which a soft segment (soft phase) composed of a soft rubber component and a hard segment (hard phase) composed of a hard resin component are separated. The soft segment exhibits the property of being softly compositionally deformed, and the hard segment prevents (restrains) plastic deformation like a crosslinking point of a synthetic rubber. The rubber elasticity similar to that of synthetic rubber is exhibited by these two functions. In the present invention, styrene, olefin, PVC, urethane, ester, and amide thermoplastic elastomers are preferably used.

  FIG. 7 is a schematic perspective view for explaining a usage mode of an example of the fixed holder of the present invention.

  When the liquid is introduced into the analysis chip using the liquid introduction device 13, the fixed holder of the present invention is held by the liquid introduction device holding portion 15 according to the position of the liquid inlet (through hole) of the analysis chip. It is preferable that the gripping position of the liquid introduction device 13 thus formed can be moved. At this time, as means for moving the gripping position of the liquid introduction device 13, a plurality of fixing screw holes 21 are provided in the stage 19 of the fixing holder, a long hole is formed in the stage, and the like. Accordingly, the receiving portion 18 of the fixing holder is moved, and the fixing screw 20 is fixed using a screw hole provided in the receiving portion 18 of the fixing holder. By being able to move the gripping position, it becomes possible to introduce liquid into various analysis chips.

  The fixed holder of the present invention can set a liquid introduction device for each of a plurality of analysis chips. By allowing a plurality of liquid introduction devices to be set, the time for introducing the liquid can be greatly reduced as compared with the case where the liquid is introduced one by one for the analysis chip.

  As a material for the fixing holder of the present invention, for example, resins such as polymethyl methacrylate, polyvinyl chloride, polypropylene, polycarbonate, and polymethylpentene, and metals such as aluminum, stainless steel, and iron are preferably used.

  By using the liquid introduction device and the fixed holder of the present invention as described above, the liquid is introduced into the gap portion of the chip such as the analysis chip having the gap portion between the carrier and the cover member. It can be performed. The measurement includes detecting the binding between the selective binding substance immobilized on the carrier and the substance in the test sample. When the selective binding substance is a nucleic acid, the measurement is based on DNA / DNA hybridization, DNA / RNA hybridization or RNA / RNA hybridization. When the selective binding substance is a protein, the measurement is based on an antigen-antibody reaction, that is, an immunological reaction. Conditions such as reaction temperature, time, and buffer are appropriately selected according to the type and chain length of the nucleic acid to be hybridized, the type of antigen and / or antibody involved in the immune reaction, and the like.

  Hybridization is generally performed under stringent conditions. Such conditions include, for example, 3 to 4 x SSC at a temperature of 30 to 50 ° C, 1 to 24 hours of hybridization in 0.1 to 0.5% SDS, followed by 2 x SSC and 0. Washing with a solution containing 1% SDS can be included. Here, 1 × SSC is a solution (pH 7.2) containing 150 mM sodium chloride and 15 mM sodium citrate.

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

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

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

  By using the liquid introduction device of the present invention, bubbles on the surface of the cover member in the gap portion of the analysis chip or in the vicinity thereof are substantially not generated. For example, DNA characterized by a carrier having an uneven structure Maintaining characteristics such as a good S / N ratio and high detection sensitivity inherent to the chip, improving variations in measurement values, and enabling accurate and highly reliable measurement.

  Next, the present invention will be described more specifically with reference to the following examples, but the present invention is not limited to these examples.

(Preparation of DNA immobilization carrier)
Three types of injection molds were produced using a LIGA (Lithographie Galvanforming Abforming) process, and polymethylmethacrylate (PMMA) carriers each having a shape as described later were obtained by an injection molding method. The average molecular weight of PMMA used in this example is 50,000, carbon black (# 3050B, manufactured by Mitsubishi Chemical) is contained in PMMA at a ratio of 1% by weight, and the carrier is black. When the spectral reflectance and spectral transmittance of this black carrier were measured, the spectral reflectance was 5% or less at any wavelength in the visible light region (wavelength of 400 nm to 800 nm), and in the same range of wavelengths, The transmittance was 0.5% or less. Neither spectral reflectance nor spectral transmittance had a specific spectral pattern (such as a peak) in the visible light region, and the spectrum was uniformly flat. Spectral reflectivity is obtained when specularly reflected light from a carrier is captured using a device (manufactured by Minolta Camera, CM-2002) equipped with an illumination / light-receiving optical system conforming to condition C of JIS Z 8722 (2000) The spectral reflectance of was measured.

  The shape of the carrier was 76 mm in length, 26 mm in width, and 1 mm in thickness, and the surface was flat except for the central part of the carrier. Each carrier was provided with a concave portion and a convex portion regularly arranged in the concave portion. Table 1 shows the size of the concave portion of each carrier, the number of convex portions, and the pitch (distance from the central portion of the convex portion to the adjacent central portion of the convex portion).

  When the height difference between the height of the upper surface of the convex portion of the concave and convex portions of each carrier (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.

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

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

This DNA was dissolved in pure water to a concentration of 0.3 nmol / μL to obtain a stock solution. When spotting on the carrier, PBS (NaCl 8 g, Na ₂ HPO ₄ · 12H ₂ O 2.9 g, KCl 0.2 g, KH ₂ PO ₄ 0.2 g in pure water was made up to 1 L. The sample is diluted with hydrochloric acid for pH adjustment, pH 5.5), diluted 10-fold so that the final concentration of the probe DNA is 0.03 nmol / μL, and the carboxylic acid on the carrier surface and the amino at the end of the 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.

  Then, these mixed solutions were spotted on the entire upper surface of the carrier convex portion by an arrayer (manufactured by Nippon Laser Electronics; Gene Stamp-II). Next, the carrier was placed in a sealed plastic container and incubated at a temperature of 37 ° C. and a humidity of 100% for 20 hours. Finally, it was washed with pure water, dried by centrifugation with a spin dryer.

(Adhesion of cover)
Three types of cover members (having an overhang structure on the outer peripheral portion) having four through-holes (diameter 0.8 mm) shown in FIG. 1 were prepared by injection molding according to the size of the recessed portion of the carrier. Immerse the cover member in a cleaning agent (Clean Ace (As One 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, PDMS polymer (manufactured by Toray Dow Corning Silicone) was applied to the carrier on which the probe DNA obtained above was immobilized, and the washed sub bar was adhered. The bonding condition is 2 hours at a temperature of 42 ° C.

(Preparation and encapsulation of fine particles)
Zirconia fine particles having an average particle diameter of 200 μm were 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 from the through hole of the cover member into the gap between the carrier and the cover member.

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

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

  Subsequently, a 9-base random primer (manufactured by Takara Bio Inc.) was dissolved at a concentration of 6 mg / ml, and 2 μl was added to the DNA solution purified after the PCR reaction. The solution was heated to a temperature of 100 ° C. and then rapidly cooled on ice. To this, 5 μl of a buffer attached to Klenow Fragment (manufactured by Takara Bio Inc.) and 2.5 μl of a dNTP mixture (dATP, dTTP, dGTP concentrations were 2.5 mM and dCTP concentrations were 400 μM, respectively) were added. Furthermore, 2 μl of Cy3-dCTP (manufactured by GE Healthcare Bioscience) was added. 10 U of Klenow Fragment was added to this solution and incubated at a temperature of 37 ° C. for 20 hours to obtain a specimen DNA labeled with Cy3. Since random primers are 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 area corresponding to the shorter base length was thinly smeared. This was purified by ethanol precipitation and dried.

  This labeled sample DNA is obtained by diluting 1% by weight BSA (bovine serum albumin), 5 × SSC (5 × SSC) with 20 × SSC (manufactured by Sigma) four times with pure water. In addition, a solution obtained by diluting 20 × SSC twice with pure water is expressed as 10 × SSC, and a solution diluted 100 times as 0.2 × SSC), 0.1 wt% SDS (sodium dodecyl sulfate), 0 A solution of 0.01 wt% salmon sperm DNA (each concentration is a final concentration) dissolved in 400 μl was used as a stock solution for hybridization.

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

(Hybridization)
Using a micropipette, the hybridization sample solution was injected from the through hole into the gap (reaction tank) between the carrier and the cover member by the methods of Examples 1 to 6 and Comparative Examples 1 and 2. Subsequently, silicon tape (As One) was used as a sealing material to close the four through holes. A hybridization chamber (Takara Hybridization chamber (manufactured by 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 carrier was set in the hybridization chamber. At this time, 15 μL of ultrapure water was dropped into the recesses at both ends of the position where the substrate was set, the hybridization chamber was closed, the six fixing screws were tightened, and the chamber was fixed at a temperature of 42 ° C. (FMS-1000 manufactured by Tokyo Rika Kikai Co., Ltd.) was placed and fixed on a shaker (MMS-310 manufactured by Tokyo Rika Kikai Co., Ltd.) The front surface of the thermostatic chamber was shielded with aluminum foil, Incubate for 16 hours at a temperature of 42 ° C with swirling at 250 rpm It was later. Incubation, the hybridization chamber was taken out of the substrate, after the cover member and the PDMS polymer adhered to the carrier substrate desorbed, washed and dried.

(Measurement)
The substrate after the above treatment was set on a DNA chip scanner (GenePix 4000B, manufactured by Axon Instruments), and measurement was performed with the laser output set to 33% and the photomultiplier voltage set to 500. Here, the fluorescence intensity is an average value of the fluorescence intensity in the spot, and the background noise is the fluorescence intensity of the convex portion where the probe DNA is not immobilized.

Example 1
A tip of a 10 μL disposable pipette tip (manufactured by Eppendorf) having a diameter of the liquid introduction portion of 0.3 mm was filled with 20 mg of zirconia fine particles having an average particle diameter of 200 μm to form a porous structure, thereby obtaining a liquid introduction device. The liquid introduction speed of the produced liquid introduction device was about 50 μL / min. The tip of the produced liquid introduction device (liquid introduction part) is inserted into the through-hole of the cover member of the DNA chip A produced above, and 110 μL of the hybridization sample solution is placed on the upper part of the liquid introduction device (liquid addition part) ). The added solution passed through the liquid passage part of the liquid introduction device by its own weight, and was introduced into the void part of the DNA chip from the liquid introduction part to fill the void part. The solution filled the void portion of the DNA chip in 2 minutes and a half. At this time, the number of bubbles measured with a CCD camera was zero. Further, when the hybridization was carried out, no defects or unevenness of the fluorescent spots were observed.

(Example 2)
The tip of a gel load chip (manufactured by Eppendorf) was cut to a diameter of 0.8 mm and a diameter of 0.1 mm, and the tip of the tip was filled with 20 mg of zirconia fine particles having an average particle diameter of 200 μm to form a porous structure. A liquid introduction device was obtained. The liquid introduction speed of the produced liquid introduction device was about 30 μL / min. The five DNA chips A produced above are set on the stage 19 of the fixed holder shown in FIG. 7, and the tip of the liquid introduction device produced above (liquid introduction part) is placed in each of the through holes of the cover members. Insert the liquid passage portion of the liquid introduction device into the liquid introduction device gripping portion 15 of the receiving portion 18 of the fixed holder, and set it so as to be substantially perpendicular to the DNA chip. 110 μL of the solution was added to the upper part (liquid addition part) of the liquid introduction device. The added specimen solution passed through the liquid passage part of the liquid introduction device by its own weight, and was introduced from the liquid introduction part into the void part of the DNA chip to fill the void part. As a result, the solution filled the void portion of the DNA chip in 5 minutes. At this time, the number of bubbles was measured with a stereomicroscope (Olympus SZX7) and found to be 0. Further, when the hybridization was carried out, no defects or unevenness of the fluorescent spots were observed.

(Example 3)
The tip of a 10 μL disposable pipette tip (manufactured by Eppendorf) having a diameter of the liquid inlet of 0.3 mm is filled with 40 mg of nylon fiber having an average diameter of 40 μm and an average length of 2 mm to form a porous structure. did. The liquid introduction speed of the produced liquid introduction device was about 50 μL / min. The tip of the produced liquid introduction device (liquid introduction part) is inserted into the through-hole of the cover member of the DNA chip A produced above, and 110 μL of the hybridization sample solution is placed on the upper part of the liquid introduction device (liquid addition part) ). The added specimen solution passed through the liquid passage part of the liquid introduction device by its own weight, and was introduced from the liquid introduction part into the void part of the DNA chip to fill the void part. The solution filled the void portion of the DNA chip in 3 minutes. At this time, the number of bubbles was measured with a stereomicroscope and found to be 0. Further, when the hybridization was carried out, no defect of the fluorescent spot or uneven fluorescence was observed.

Example 4
The tip of a 10 μL disposable pipette tip (manufactured by Eppendorf) having a diameter of 0.3 mm in the liquid inlet is filled with 20 mg of zirconia fine particles having an average particle diameter of 200 μm to form a porous structure, and a 10% dextran solution is further passed through 200 μL. After letting it liquid, it was dried at a temperature of 70 ° C. for 2 hours to obtain a liquid introduction device. The liquid introduction speed of the produced liquid introduction device was about 45 μL / min. The liquid introduction part (tip part) of the produced liquid introduction device was inserted into the through hole of the cover member of the DNA chip produced above, and 165 μL of the hybridization sample solution was added to the liquid addition part of the liquid introduction device. The added specimen solution passed through the liquid passage part of the liquid introduction device by its own weight, and was introduced from the liquid introduction part into the void part of the DNA chip to fill the void part. As a result, the solution filled the void portion of the DNA chip in 3 minutes. This was 0 when the number of bubbles was measured with a CCD camera. Further, when hybridization was performed, no defects or unevenness of the fluorescent spots were observed.

(Example 5)
The tip of a 10 μL disposable pipette tip (manufactured by Eppendorf) having a diameter of 0.3 mm in the liquid inlet is filled with 20 mg of zirconia fine particles having an average particle diameter of 200 μm to form a porous structure, and a 10% dextran solution is further passed through 200 μL. After letting it liquid, it was dried at a temperature of 70 ° C. for 2 hours to obtain a liquid introduction device. The liquid introduction speed of the produced liquid introduction device was about 45 μL / min. The receiving part of the fixing holder was fixed in accordance with the position of the through hole of the DNA chip B in Table 1. The five DNA chips B produced above are set on the stage of the fixed holder, the tip of the liquid introduction device produced above (liquid introduction part) is inserted into each through hole of the cover member, and further the liquid introduction The liquid passage portion of the device for use was set on the liquid introduction device gripping portion of the receiving portion of the fixed holder and set so as to be substantially perpendicular to the DNA chip. 165 μL of the hybridization sample solution was added to the upper part (liquid addition part) of the device for liquid introduction. The added specimen solution passed through the liquid passage part of the liquid introduction device by its own weight, and was introduced from the liquid introduction part into the void part of the DNA chip to fill the void part. This was 0 when the number of bubbles was measured with a CCD camera. Further, when hybridization was performed, no defects or unevenness of the fluorescent spots were observed.

(Example 6)
The tip of a 10 μL disposable pipette tip (manufactured by Eppendorf) having a diameter of 0.3 mm in the liquid inlet is filled with 20 mg of zirconia fine particles having an average particle diameter of 200 μm to form a porous structure, and a 10% dextran solution is further passed through 200 μL. After letting it liquid, it was dried at a temperature of 70 ° C. for 2 hours to obtain a liquid introduction device. The liquid introduction speed of the produced liquid introduction device was about 45 μL / min. The receiving part of the fixing holder was fixed in accordance with the position of the through hole of the DNA chip C in Table 1. The five DNA chips C produced above are set on the stage of the fixed holder, the tip of the liquid introduction device produced above (liquid introduction part) is inserted into each of the through holes of the cover members, and further the liquid introduction The liquid passage portion of the device for use was set on the liquid introduction device gripping portion of the receiving portion of the fixed holder and set so as to be substantially perpendicular to the DNA chip. 220 μL of the hybridization sample solution was added to the upper part (liquid addition part) of the liquid introduction device. The added specimen solution passed through the liquid passage part of the liquid introduction device by its own weight, and was introduced from the liquid introduction part into the void part of the DNA chip to fill the void part. This was 0 when the number of bubbles was measured with a CCD camera. Further, when hybridization was performed, no defects or unevenness of the fluorescent spots were observed.

(Comparative Example 1)
A 10 μL disposable pipette tip (manufactured by Eppendorf) having a diameter of the liquid introduction part of 0.3 mm was used as a liquid introduction device. The liquid introduction speed of the liquid introduction device was about 500 μL / min. The tip of the liquid introduction device (liquid introduction part) was inserted into the through-hole of the cover member of the DNA chip prepared above, and 165 μL of the hybridization sample solution was added to the upper part of the liquid introduction device (liquid addition part). . As a result, the solution filled the void portion of the DNA chip in 0.5 minutes. At this time, the number of bubbles measured with a stereomicroscope was 10. Further, when hybridization was performed, five defects of the fluorescent spot were confirmed, and uneven fluorescence was observed throughout.

(Comparative Example 2)
A gel load chip (manufactured by Eppendorf) having a diameter of the liquid introduction portion of 0.1 mm was directly used as a liquid introduction device. The liquid introduction speed of the liquid introduction device was about 200 μL / min. The tip of the liquid introduction device for liquid introduction (liquid introduction portion) is inserted into the through-hole of the cover member of the DNA chip prepared above, and 165 μL of the hybridization sample solution is placed on the top of the liquid introduction device (liquid addition portion). Added to. As a result, the solution filled the void portion of the DNA chip in 1 minute. At this time, the number of bubbles measured with a stereomicroscope was 7. Further, when hybridization was performed, two defects of the fluorescent spot were confirmed, and uneven fluorescence was also observed.

  The liquid introduction device of the present invention can suppress the generation of bubbles when introducing a liquid containing a specimen into a void portion of an analysis chip having a void portion between a carrier and a cover member, and The liquid introduction operation can be easily performed without requiring any skilled technique. As a result, characteristics such as a good S / N ratio and high detection sensitivity inherent to the analysis chip are maintained, and variations in measurement values are improved, thereby enabling accurate and reliable measurement. Therefore, 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.

  In addition, the fixing holder of the present invention can securely fix the analysis chip and the liquid introduction device for introducing the liquid to the analysis chip, and stably perform the liquid introduction operation.

FIG. 1 is a perspective view schematically showing an example of an analysis chip having a gap between a carrier and a cover member using the liquid introduction device of the present invention, and is cut along a plane along the line A1. It is the partial longitudinal cross-sectional view (b) which was made. FIG. 2 is a schematic perspective view (c) and a longitudinal sectional view (d) of an example of a carrier constituting an analysis chip having a gap between a carrier using the liquid introduction device of the present invention and a cover member. . FIG. 3 is a longitudinal sectional view schematically showing an example of an analysis chip having a gap portion between a carrier and a cover member using the liquid introduction device of the present invention. FIG. 4 is a longitudinal sectional view schematically showing an example of the liquid introduction device of the present invention. FIG. 5 is a schematic perspective view for explaining a usage mode of an example of the liquid introduction device of the present invention. FIG. 6 is a partial perspective view schematically showing an example of means for fixing the liquid introduction device of the fixing holder of the present invention. FIG. 7 is a schematic perspective view for explaining a usage mode of an example of the fixed holder of the present invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Carrier 2 Cover member 3 Adhesive member 4 Liquid level holding chamber 5 Through-hole 6 Cavity 7 Selective binding substance 8 immobilized on substrate 8 Fine particles (beads)
DESCRIPTION OF SYMBOLS 9 Liquid addition part 10 Speed adjustment means 11 for adjusting the introduction speed of a liquid 11 Liquid introduction part 12 Liquid passage part 13 Liquid introduction device 14 Analysis chip 15 which has a space | gap part between a support | carrier and a cover member 15 Liquid introduction device Grasping part 16 Elastomer member 17 Reinforcing plate 18 Receiving part 19 of fixing holder Stage 20 of fixing holder Fixing screw 21 Fixing screw hole

Claims (12)

  A device for introducing a liquid into the gap of the analysis chip having a gap between the carrier and the cover member, and a speed adjusting means for adjusting the liquid introduction speed into the liquid passage of the device A liquid introduction device arranged.   The liquid introduction device according to claim 1, wherein a speed adjusting means for adjusting the liquid introduction speed has a function of controlling the liquid introduction speed to be 100 μL / min or less.   The device for introducing a liquid according to claim 1 or 2, wherein the device is configured such that by adding a liquid to the device, the liquid is introduced into a gap of the analysis chip by gravity and / or a pressure difference.   The device for liquid introduction according to any one of claims 1 to 3, wherein the diameter of the liquid introduction portion of the device for introducing the liquid into the gap portion of the analysis chip is 0.001 mm or more and 0.8 mm or less.   The device for liquid introduction according to any one of claims 1 to 4, wherein the speed adjusting means for adjusting the liquid introduction speed has a porous structure.   The liquid introduction device according to claim 5, wherein the porous structure is composed of fibers and / or fine particles. The liquid introduction device according to claim 6, wherein fibers and / or fine particles constituting the porous structure are bonded or adhered to each other by an adhesive substance and / or an adhesive substance.   A fixing holder for fixing both of the analysis chip having a gap between the carrier and the cover member and the liquid introduction device according to any one of claims 1 to 7, wherein the liquid introduction device includes: A fixing holder comprising means for gripping and fixing at least a part of the liquid introduction device.   The fixing holder according to claim 8, further comprising means for gripping and fixing at least a part of the liquid introduction device by the elastomer member.   10. The fixed holder according to claim 8 or 9, further comprising means for making the gripping position of the liquid introduction device movable in accordance with the position of the liquid inlet of the analysis chip.   The fixing holder according to claim 8, wherein a plurality of liquid introduction devices can be gripped and fixed to a plurality of analysis chips.   An analysis kit comprising an analysis chip having a gap between the carrier and the cover member and the liquid introduction device according to claim 1.

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