JP2007304093A - Analytical chip - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an analytical chip and an analytical method, capable of preventing bubbles from generating, and capable of carrying out easily and stably a selective reaction of a specimen with an immobilized selective coupling substance, without disturbing the movement of a stirrer, when a solution containing an examined substance is applied on a selective coupling substance immobilizing carrier. <P>SOLUTION: This analytical chip comprises the carrier, having a recessed part on a surface, a cover member for covering the surface of the carrier and bonded to the carrier, an adhesive member interposed between the carrier and the cover member to bond the both, and the stirrer sealed in a cavity between the carrier and the cover member. In the analytical chip, a bonded point between the carrier and the cover member on the carrier surface is positioned in the outer region, separated from the outer circumferential part end face of the recessed part, and the thickness of the adhesive member is smaller than the smallest diameter of the stirrer. The analysis method that uses the chip is also disclosed. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention comprises a carrier on which a substance that selectively binds to a test substance (referred to herein as “selective binding substance”) is immobilized, and comprises a solution containing the test substance and the selective binding substance. The present invention relates to an analysis chip that can be used for analysis by reaction and an analysis method using the analysis chip.

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

  In recent years, a new analysis method called a DNA microarray method (DNA chip method) has been developed as a method for analyzing a large number of gene expressions at once, and applied to detection and quantification of proteins and sugar chains as well as DNA. A major feature of these methods is that a large number of DNA fragments, proteins, and sugar chains are aligned and fixed at high density on a flat glass substrate called a microarray or DNA chip. A sample labeled with a fluorescent dye or the like is used to hybridize a sample labeled with an expression gene of the cell to be studied on a flat substrate piece, and then complementary nucleic acids (DNA or RNA) are bound to each other. This method is applied to a method for reading at high speed and a method for detecting a response such as a current value based on an electrochemical reaction, and contributes to a quick estimation of the gene amount in a sample. In addition, the application field of DNA chips is greatly expected not only as a gene expression analysis for estimating the amount of expressed gene but also as a means for detecting a single nucleotide polymorphism (SNP) of a gene.

  When using a DNA chip, it is necessary to apply the prepared sample solution so that it spreads over the entire area on the DNA chip where the selective binding substance is provided. It is necessary to proceed with the hybridization with the sample solution stably while suppressing evaporation. As means for solving such a problem of the evaporation of the sample solution, a sealed container capable of keeping 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 problem that the experiment cost increases by purchasing a new sealed container and the operation of stably installing the DNA chip in the sealed container are added, and the experiment work becomes complicated. There is a problem of becoming.

  On the other hand, in Patent Document 1, a microparticle dispersion solution in which microparticles are added in advance to a sample DNA solution is applied to a DNA chip, covered with a cover glass, and sealed with a sealant, and the cover glass, DNA chip, and sealant To form a sealed void defined by: Thus, hybridization can be performed while preventing evaporation of the sample solution while stirring using the movement of the fine particles.

  However, the operation of the fine particle dispersion solution with a pipette is a complicated operation because the fine particles are likely to precipitate. In addition, in the operation of sealing the sample solution, there is a problem that the sample solution comes into contact with the sealant, and background noise increases due to contamination of the sealant with the sample solution, or the sample solution is lost due to solution leakage. Many. Therefore, there is a problem that a skilled technique is necessary to perform a good analysis according to the technique disclosed in Patent Document 1. Furthermore, there is a possibility that the fine particles adhere to the adhesion portion between the cover member and the chip and do not move, and as a result, the fine particles become an obstacle and the sample solution does not penetrate into the details.

Japanese Patent No. 3557419

  The present invention solves the above-mentioned problem, and prevents the generation of bubbles without impeding the movement of a stirrer when applying a solution containing a test substance on a selective binding substance-immobilized carrier. An object of the present invention is to provide an analysis chip and an analysis method capable of simply and stably performing a selective reaction between a specimen and an immobilized selective binding substance.

In order to solve the above problems, the present invention provides the following:
[1] a carrier having a recess on the surface;
A cover member covering the surface of the carrier and bonded to the carrier;
An adhesive member interposed between the carrier and the cover member to bond them;
An analysis chip comprising a stirrer enclosed in a gap between the carrier and the cover member,
The adhesion point between the carrier and the cover member on the carrier surface is located in an outer region away from the outer peripheral end surface of the recess, and the distance between the carrier surface and the carrier side surface of the cover member at the adhesion point is agitated. An analysis chip characterized by being shorter than the shortest diameter of the child.
[2] A convex portion is provided on the carrier-side surface outer periphery of the cover member, and the adhesive member is interposed between the carrier and a convex portion on the carrier-side surface outer periphery of the cover member. The analysis chip according to [1].
[3] The analysis chip according to [1] or [2], wherein the carrier has a selective binding substance immobilized on a surface of the carrier and located in the space. .
[4] The analysis chip according to any one of [1] to [3], wherein the cover member is detachably bonded to the carrier.
[5] Any of the above [1] to [4], wherein the adhesive member is a resin composition containing a polymer selected from the group consisting of a silicone polymer, an acrylic polymer, and a mixture thereof. 2. The analysis chip according to item 1.
[6] (A) A step of applying a test substance to the analysis chip according to any one of [1] to [5]; (B) a step of selectively binding the test substance to the carrier And (C) measuring the amount of the test substance bound to the carrier via the selective binding substance, and a method for analyzing the test substance.
[7] The analysis chip has a selective binding substance immobilized on a region on the surface of the carrier and located in the gap, and the step (C) includes the test substance and the selection. [6] The analysis method according to [6], which comprises interacting with a binding substance and binding the test substance to the carrier via the selective binding substance.

  According to the analysis chip and the analysis method of the present invention, a selective reaction between a specimen and an immobilized selective binding substance, represented by hybridization of a nucleic acid or the like, can be performed easily and stably. In particular, the present invention can be applied to a reaction with a small amount of sample solution, and a more accurate result can be obtained.

  The analysis chip of the present invention includes a carrier having a concave portion on the surface, a cover member that covers the surface of the carrier and is bonded to the carrier, an adhesive member that is interposed between the carrier and the cover member, and bonds them, And a stirrer enclosed in a gap between the carrier and the cover member.

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

As the carrier, it is necessary to use one having a concave portion on the surface. The cover member may cover at least a part of the surface of the carrier, and may be bonded so as to have a gap between the carrier and the cover member.
The carrier preferably has a selective binding substance immobilized on a region of the carrier located on the surface of the carrier. That is, preferably, the cover member is bonded to the carrier so that the region where the selective binding substance is immobilized exists in the gap. The cover member may be bonded in any manner as long as the gap is formed, but is preferably bonded via an adhesive member such as a double-sided tape or a resin composition. Specific examples of the carrier shape, the selective binding substance, the immobilization mode, the void shape, the adhesion mode, etc. will be described in detail later.

FIG. 1 is a perspective view showing an example of a schematic aspect of the analysis chip of the present invention, and FIG. 2 is a cross-sectional view of the analysis chip of FIG. 1 cut along a plane along arrow A1. In the example shown in FIGS. 1 and 2, the carrier 2 is covered with the cover member 1 via the adhesive member 3 to form a void 5 including the recess R <b> 1, and the void 5 is necessary in the present invention. Other than communicating with the outside through a plurality of through-holes 4 that can be provided, it is a closed space that does not communicate with the outside.
In the example of FIGS. 1 and 2, the adhesion point P1 between the carrier and the cover member on the surface of the carrier is located outside the recess R1. Although not shown, a stirrer is enclosed in the gap 5, and the thickness H1 of the adhesive member is shorter than the shortest diameter of the stirrer.

  In the analysis chip of the present invention, the adhesion point between the carrier and the cover member on the carrier surface needs to be located in a region other than the concave portion on the carrier surface. As described above, since the adhesion point is located in a region other than the concave portion, the stirrer does not adhere to the adhesive member, so that the fine particles freely move, and as a result, the generation of bubbles is prevented and the sample is immobilized. The selective reaction with the selective binding substance can be carried out simply and stably.

  Here, the bonding point between the carrier and the cover member means the position of the bonding member interposed between the carrier and the cover member. In the present invention, the bonding point on the carrier side is the position between the bonding member and the carrier surface. It is necessary to be located in a region outside the outer peripheral end face of the recess. Although the distance (for example, w of FIG. 2) from the outer peripheral part end surface of a recessed part will not be specifically limited if the stirrer to be used does not contact an adhesive member, it is preferable that it is 1-700 micrometers.

The distance between the carrier surface at the adhesion point and the carrier side surface of the cover member needs to be shorter than the shortest diameter of the stirrer enclosed in the gap 5. The distance between the carrier surface at the adhesion point and the carrier side surface of the cover member is the thickness of the adhesive member (for example, FIG. 2) when the cover member is flat as in the examples of FIGS. H1), and when the convex portion is provided on the outer periphery of the carrier side surface of the cover member as shown in FIG. 3 or FIG. 4, for example, the thickness of the adhesive member and the height of the convex portion (the upper surface of the convex portion) And the distance between the inner surface of the cover member) (for example, the height of H1 + H2 in FIGS. 3 and 4). The interval is not particularly limited as long as the stirrer does not enter, but is preferably 90% or less, more preferably 80% or less of the shortest diameter of the stirrer. Here, the shortest diameter of the stirring bar means the shortest diameter among the diameters that define the shape of the stirring bar.
In addition, as will be described later, when the stirring bar to be used is selected through a sieve or the like, there may be some width in the shortest diameter of the stirring bar. The interval can be shorter than the shortest diameter of the child.
The shortest diameter of the stirrer corresponds to the diameter when the shape of the stirrer is spherical, and corresponds to the diameter of the bottom surface when the shape of the stirrer is microrod (bar shape). Note that the shape of the stirring bar is not limited to a spherical shape or a microrod shape, as described later.

As described above, the bonding point is located in the outer region away from the outer peripheral end surface of the concave portion on the adhesive member and the carrier surface, so that three directions are defined by the concave portion on the carrier surface, the cover member, and the adhesive point. An enclosed space will be created.
Specifically, referring to FIG. 2, the outer peripheral end face P2 of the recess R1 is located on the inner side of the inner cross section P3 of the adhesive member, and is a portion P4 higher than the recess R1 adjacent to the recess R1 on the carrier surface. Occurs. As a result, a space S1 surrounded by three sides by the cross section P3 of the adhesive member, the carrier P4, and a part of the cover member is formed in the outer edge portion of the gap 5.
The thickness of the adhesive member in the analysis chip of the present invention is usually defined in the range of 1 μm to 100 μm, and is preferably 5 to 80 μm in order to have a constant adhesive strength. When using a flat cover member as in the example of FIGS. 1 and 2, the thickness of the adhesive member is the distance between the carrier surface at the adhesion point and the carrier side surface of the cover member, so the thickness of the adhesive member is agitated. It must be shorter than the shortest diameter of the child.

On the other hand, the shape of the analysis chip of the present invention is not particularly limited, and a flat one can be used as in the examples of FIGS. 1 and 2, and a convex portion is provided on the outer periphery of the carrier side surface of the cover member. Can be used. It is preferable to use the cover member provided with such convex portions because the positioning of the adhesive member is facilitated. In this case, the adhesive member can be interposed between the carrier and the convex portion of the cover member. That is, the adhesion point between the carrier and the cover member on the surface of the cover member is a part of the convex part, preferably the whole or part of the top surface of the convex part, particularly preferably the part or the whole including at least the inner peripheral part of the top surface of the convex part. Can be positioned. Also in this case, it is preferable that a space surrounded by three directions is generated by the carrier surface, the cover member (specifically, the carrier side surface and the convex side surface of the cover member), and the adhesive member. In particular, if the side surface of the convex portion of the cover member and the gap side end surface of the adhesive member form a flat surface, or the gap side end surface of the adhesive member is located slightly outside the side surface of the convex portion of the cover member, the fine particles And the adhesive member can be effectively avoided, which is preferable. Although it does not specifically limit about the shape of a convex part, It is preferable that it is the range of 0-150 micrometers as height of a convex part (H2 of FIG.3 and FIG.4). Further, the adhesive surface with the carrier is preferably the upper surface of the convex portion, in particular, the upper surface is preferably horizontal with respect to the carrier-side surface of the cover member, and most preferably the height of the convex portion is uniform. The shape of the inner side surface of the convex portion is not particularly limited, but is preferably a flat surface, and most preferably presents a flat surface that is substantially perpendicular to the carrier-side surface of the cover member. In addition, when providing a convex part in a cover member, about the thickness (H1 of FIG.3 and FIG.4) of an adhesive member, it is normally prescribed | regulated in the range of 1 micrometer-100 micrometers as above-mentioned, and in order to provide fixed adhesive strength 5 to 80 μm is preferable. An example of an analysis chip provided with such a cover member is shown in FIGS.
In the example shown in FIG. 3, a convex portion 1A is provided on the carrier-side surface outer periphery of the cover member, and a bonding point P5 between the carrier and the cover member is located on a part of the upper surface of the convex portion 1A. On the other hand, in the example shown in FIG. 4, a convex portion 1A is provided on the outer periphery of the carrier side surface of the cover member, and the entire upper surface of the convex portion 1A is an adhesion point P5 between the carrier and the cover member.

  In the analysis chip of the present invention, the cover member is preferably detachably bonded to the carrier. When the carrier is a DNA chip, it is usually necessary to read the DNA chip with a dedicated scanner. At that time, when the cover member is bonded, it is difficult to set it on the dedicated scanner. Even if it is set, if the scanning operation is performed, the cover member and the optical parts of the scanner may come into contact with each other, causing a failure. . Further, 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.

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

  When a double-sided tape is used as the adhesive member, it is preferable to use a double-sided tape having different adhesive strength on both sides. More specifically, it is preferable that the surface of the double-sided tape having different adhesive strength on both sides is bonded to the carrier side, and the surface having a strong adhesive force is bonded to the cover member side. By adopting such an aspect, when the cover member is peeled off, the double-sided tape is easily detached from the carrier at the same time in a state of being adhered to the cover member, so that in the reading step due to the remaining adhesive member on the carrier. Inconvenience can be avoided. As such a double-sided tape, Nitto Denko Corporation product number No. 535A, product numbers 9415PC and 4591HL manufactured by Sumitomo 3M Limited, and product numbers No. 7691 etc. are mentioned.

  When using a resin composition as an adhesive member, as the resin composition, a resin composition containing a polymer selected from the group consisting of acrylic polymers, silicone polymers, and mixtures thereof can be used. By using these for the adhesive layer, it becomes possible to improve the sealing performance as compared with the double-sided tape, and these can be preferably used. In particular, when such a resin composition is used as an adhesive member, it is more stable for long-term incubation than double-sided tape, and is particularly preferable in an analysis system that requires such long-term incubation. .

  In particular, when a silicone elastomer is used as the adhesive member, the sealing property is good and the cover can be adhered in a state where it can be easily detached. Specific examples of such an elastomer include Sylgard (Silgard is a registered trademark of Dow Corning) and two-component RTV rubber (for mold making) manufactured by Shin-Etsu Chemical Co., Ltd. In particular, it is preferable to use a silicone-based elastomer as an adhesive member because the cover can be adhered in a state in which the sealing property is good and can be easily detached. Specific examples of such an elastomer include Sylgard (registered trademark of Dow Corning) and two-component RTV rubber (for mold making) manufactured by Shin-Etsu Chemical Co., Ltd.

  The cover member is bonded to the carrier by using a dispenser (manufactured by Musashi Engineering Co., Ltd.), applying the adhesive member to a place away from the concave portion of the carrier, and placing the cover member over the probe DNA immobilization region. It can be carried out by applying a load from above the cover member and allowing it to stand and dry. At this time, the thickness of the adhesive member can be adjusted by the applied load. As the load at the time of adhesion, a range of 10 to 100 g weight can be preferably used.

  The analysis chip of the present invention further includes a stirrer enclosed in a gap defined by a structure including a carrier, a cover member, and an adhesive member. By including the stirrer, after the sample solution is applied, the analysis chip is swung, rotated, etc., to move the stirrer within the gap and achieve sufficient agitation of the sample solution for more precise analysis. It can be carried out.

  In the method disclosed in Patent Document 1 in which a specimen solution containing fine particles is applied, covered with a cover glass, and sealed with a sealant, the operation becomes complicated as described above, and background noise may increase. . However, in the case of the analysis chip of the present invention, since the cover member and the carrier are bonded and integrated, it is possible to enclose a stir bar in advance and to easily apply the sample solution. It is. Further, even in the operation of closing the through hole after applying the sample solution, there is an advantage that the background noise does not increase because the tape and the sealant do not come into contact with the sample solution.

The shape of the stirring bar is not particularly limited, and may be any shape such as a polygonal shape or a cylindrical shape other than the spherical shape. Further, the size of the stirring bar is not particularly limited as long as the distance between the carrier surface at the adhesion point and the carrier side surface of the cover member is shorter than the shortest diameter of the stirring bar. Preferably, the distance is within 90%, more preferably within 80% of the shortest diameter of the stirring bar. For example, in the case of spherical fine particles, the diameter can be 0.1 to 400 μm, preferably in the range of 0.1 to 300 μm. In the case of cylindrical fine particles, the length is 50 to 30000 μm, the bottom diameter is 10 to 400 μm, Preferably it can be set as the range of 10-300 micrometers. When the stirrer is a spherical fine particle and its size varies, the fine particle having a desired shortest diameter can be obtained by screening the fine particle that does not pass through a sieve of a specific size. it can. The size of the sieve can be arbitrarily selected. For example, by using a sieve having a size of 250 μm, 212 μm, 198 μm, 180 μm, 150 μm, 125 μm, or 90 μm, fine particles having the shortest diameter can be obtained. In addition, fine particles having a desired longest diameter can be obtained by selecting fine particles that pass through a sieve having a specific size. Note that two or more kinds of fine particles having different sizes and shapes can be used in combination as the stirring bar.
The material of the stirrer is not particularly limited, and glass, ceramic (for example, yttrium partially stabilized zirconia), metal (for example, gold, platinum, stainless steel), plastic (for example, nylon or polystyrene) can be used.

  The analysis chip of the present invention preferably has an embodiment in which a stirrer is enclosed in advance, but is not limited thereto, and may be an embodiment in which the stirrer is not enclosed. In this case, the analysis may be performed without using the stirrer, the stirrer may be mixed into the sample solution, and the stirrer may be filled in the gap simultaneously with the sample solution application, or before the sample solution is applied. Alternatively, a stirrer may be separately filled through the through hole later.

  The cover member preferably has one or more through holes communicating with the gap. In the use of the analysis chip shown in FIGS. 1 to 4, the sample solution is applied from one or more arbitrary positions of the through-hole 4 and filled into the gap 5. Thereafter, the sample solution can be easily hermetically sealed by closing the through hole 4 with a sealing member. As a result, the reaction between the specimen and the selective binding substance immobilized on the region R1 of the carrier can be performed stably. The number of through-holes having through-holes may be one or more, but is preferably a plurality, particularly 3 to 6, from the viewpoint of facilitating filling of a solution containing a test substance. As will be described later, when the gap is divided into a plurality of spaces that do not communicate with each other, it is preferable to have a plurality, particularly 3 to 6, through holes in each space. The diameter of the through-hole can be set as needed.However, when providing a plurality of through-holes, if some of them are used as apply ports and other through-holes are used as air vents, From the viewpoint of ease of application and hermetic retention of the solution, only the apply port has a wide hole diameter necessary for the application, for example, a diameter in the range of 0.01 to 2.0 mm, and other through holes have a narrower hole diameter, for example, It can be set to 0.01 to 1.0 mm.

  It is preferable that at least one of the through holes is provided with a portion having a large diameter at the upper end, that is, a so-called liquid level holding chamber, from the viewpoint of easily and reliably sealing the through hole. The shape of the chamber for holding the liquid surface can be appropriately selected from a cylindrical shape, a prismatic shape, a conical shape, a pyramidal shape, a hemispherical shape, etc., and particularly the ease of manufacture and the effect of suppressing the rise in the sample solution From the viewpoint of the height, etc., a cylindrical shape is preferable.

The diameter of the through hole can be determined from the viewpoint of easily applying the sample solution and more effectively suppressing evaporation of the sample solution before sealing and after sealing. For example, in the case of a combination of the cylindrical through-hole 4 having a longitudinal cross-sectional shape shown in FIG. 5 and the liquid level holding chamber 4A, the hole size (diameter) of the through-hole 4 is from 0.01. 2.0 mm is preferable, and 0.3 to 1.0 mm is more preferable.
On the other hand, the pore size (diameter) of the liquid level holding chamber 4A can be determined from the viewpoint of obtaining a sufficient liquid level holding effect, and is usually 1.0 mm or more, preferably 1.0 mm or more and 10 mm or less. be able to. Further, the depth of the liquid level holding chamber 4A is not particularly limited, but can be within a range of 0.1 to 5 mm.

  The shape of the cover member can be appropriately selected as a shape that covers at least a part of the surface of the base material and can be bonded so as to have a gap between the base material and the cover member. From the viewpoint of avoiding damage to the base material when the cover member is detached, a structure having a protruding portion at a portion farther from the base than a portion near the base at the outer peripheral portion, that is, an overhang structure is provided. It may be.

  The analysis chip of the present invention has a gap defined by a structure including a carrier, a cover member, and optionally an adhesive member, but the gap may be one space or a plurality of partitioned spaces. The plurality of partitioned spaces can be provided by a partition structure as shown in FIG. 5, for example. In the example shown in FIG. 5, the plurality of partitioned spaces 5 are provided by bonding the convex portions 2 </ b> B of the carrier and the cover member 1 via the adhesive members 3 </ b> B. In this example, the spaces 5 do not communicate with each other, and each has one or more through-holes 4 and a liquid level chamber 4A separately. As described above, by providing a plurality of partitioned spaces, it is possible to apply a plurality of types of sample solutions to one analysis chip, and therefore, it is possible to measure a plurality of types of samples simultaneously with one analysis chip. Can do.

  Further, the analysis chip of the present invention may have one cover member per one carrier, or may have two or more cover members. Specifically, as shown in FIGS. 6 and 7, a plurality of cover members 1 can be provided on a single carrier 2. Each of the plurality of cover members 1 can be provided on the carrier 2 via a separate adhesive member 3C. Preferably, each of the plurality of cover members 1 may have a gap 5 between the carrier 2 and one or more through holes communicating with each gap, and each cover member 1 Another region (recessed portion) R1 to which the selective binding substance is immobilized can be provided. By adopting such an aspect, it is possible to obtain an effect such as measuring a plurality of types of specimens simultaneously with one analysis chip, as in the aspect having a plurality of partitioned spaces. Furthermore, since the cover member can be detached independently for each region R1 by taking such an embodiment, for example, after analyzing one region R1, analysis is performed using another region R1. Independent use, such as doing.

  The material of the cover constituting the analysis chip of the present invention is not particularly limited, but a transparent material is preferable so that the state of the solution can be observed when the sample solution is applied. Such materials include glass or plastic. In particular, transparent resins such as polystyrene, polymethyl methacrylate, and polycarbonate can be preferably used from the viewpoint that structures such as through holes and liquid level holding chambers can be easily produced. The method for producing the cover is not particularly limited, and machining by cutting or injection molding is possible. From the viewpoint that mass production is possible, an injection molding method can be preferably used.

  The carrier constituting the analysis chip of the present invention is not particularly limited as long as it has a concave portion on the surface as described above, but a selective binding substance is immobilized on a part of at least one surface, preferably a part of the concave portion. It is preferable to have an area. In particular, it is preferable that the concave portion has one or more convex portions therein, and the selective binding substance is immobilized on the upper surface of the convex portion. By adopting such a structure, a non-specifically adsorbed specimen is not detected during detection, so that a noise is small and a result with a better S / N can be obtained as a result. The specific reason why the noise is reduced is as follows. That is, when a carrier having a selective binding substance immobilized on the upper surface of the convex portion is scanned using a device called a scanner, the laser beam is focused on the upper surface of the convex portion of the concavo-convex portion. This is because there is an effect that it is difficult to detect undesired fluorescence (noise) of the specimen adsorbed nonspecifically in the recess.

  Regarding the height of the convex portions provided in the concave portions, it is preferable that the height of the upper surface of each convex portion is substantially the same. Here, when the height is substantially the same, the selective binding substance is immobilized on the surface of the convex part, which is slightly different in height, and this is reacted with the fluorescently labeled sample, and the signal is detected when scanned with a scanner. The height at which the difference in strength of the level does not matter. Specifically, “the heights are substantially the same” means that the height difference is 50 μm or less. The difference in height is more preferably 30 μm or less, and even more preferably the same height. In addition, the same height as used in this application shall also include the error by the dispersion | variation which generate | occur | produces by production etc. If 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 is larger than 50 μm, the laser beam on the top surface of the convex portion with a shifted height will be blurred and fixed to the top surface of the convex portion. The signal intensity from the sample reacted with the selective binding substance may be weak, which is not preferable.

  In the concave portions, the arrangement of the convex portions when viewed from above the surface may be in a row or random. When the convex portions are arranged in rows, the rows can be equally spaced or can be arbitrarily spaced. In addition, it is possible to form sub-block regions arranged in a matrix by adjusting the spacing between the convex portions. In this case, the sub-block regions are used in combination with a plurality of size and / or shape stir bars corresponding to the spacing between the rows. be able to. However, in the case where the microrod-shaped stirrer is held in the concave portion, it is preferable that the convex portions are arranged in a row, particularly at regular intervals or at arbitrary intervals in a certain direction. Moreover, it is preferable that the upper surface of a convex part is substantially flat. Here, the fact that the top surface of the convex portion is substantially flat means that there is no unevenness of 20 μm or more. In addition, as long as the upper surface is flat, the shape of the whole convex part may take arbitrary shapes, such as trapezoid, cylinder shape, and polygonal column shape.

  In the analysis chip of the present invention, it is more preferable that a flat portion is provided in a portion other than the concave portion on the surface where the concave portion of the carrier is provided. In this case, the adhesion point is located on the flat portion. When providing a convex part in the recessed part of the support | carrier surface, it is preferable that the height of the upper surface of a convex part and the height of a flat part are substantially the same. That is, the difference between the height of the flat portion and the height of the upper surface of the convex portion is preferably 50 μm or less. If the difference between the height of the upper surface of the convex part and the height of the flat part exceeds 50 μm, the detectable fluorescence intensity may become weak, which is not preferable. The difference between the height of the flat portion and the height of the upper surface of the convex portion is more preferably 30 μm or less, and most preferably, the height of the flat portion and the height of the convex portion are the same.

  Specific examples of carriers that can be preferably used as components of the analysis chip of the present invention are illustrated in FIGS. In the example shown in FIGS. 8 and 9, the region R <b> 1 where the selective binding substance is immobilized is configured by a concave portion including a plurality of convex portions 6, and a flat portion 8 is provided around the region R <b> 1. A selective binding substance (for example, nucleic acid) is immobilized on the upper surface of the convex portion 6. By using this flat portion, it becomes possible to easily focus the light beam for measurement such as the excitation light of the scanner on the upper surface of the convex portion. More specifically, as shown in FIG. 10, when the scanner focuses the excitation light on the surface of the carrier, it is urged by a spring 13 to abut the carrier 2 against the jig 14, In many cases, the focal point is adjusted in advance by the lens 11 or the like so that the laser beam 12 is focused on the height of the abutting surface 15. By abutting the flat portion of the carrier of the analysis chip of the present invention against the surface 15 of the jig, the laser beam of the scanner can be easily focused on the upper surface of the convex portion of the carrier. In the example of FIG. 10, the carrier 2 is fixed so that the surface on which the selective binding substance is fixed is on the lower side.

  In the carrier of the analysis chip of the present invention, the convex part on which the selective binding substance in the concave part is immobilized means a part on which the selective binding substance (for example, nucleic acid) necessary as data is immobilized. In addition, there may be a convex portion where nothing is fixed, or a portion where only a dummy selective binding substance is fixed.

  When the carrier of the analysis chip of the present invention has a convex portion on which a selective binding substance is immobilized, the area of the upper surface of the convex portion is preferably substantially the same. Since the area of the upper surface of the convex portion is substantially the same, the area of the portion where the various selective binding substances are immobilized can be made the same, which is advantageous for later analysis. Here, the area of the upper part of the convex part is substantially the same means that the value obtained by dividing the largest upper surface area in the convex part by the smallest upper surface area is 1.2 or less.

The area of the upper surface of the convex portion on which the selective binding substance is immobilized is not particularly limited, but is 1 mm ² from the viewpoint that the amount of the selective binding substance can be reduced and handling is easy. Hereinafter, 10 μm ² or more is preferable.

  The height of the convex portion in the concave portion of the carrier preferably used in the present invention, that is, the difference in height between the upper surface of the convex portion and the bottom surface of the concave portion is preferably 10 μm or more and 500 μm or less, and particularly preferably 50 μm or more and 300 μm or less. If the height of the convex portion is lower than 10 μm, a non-specifically adsorbed specimen sample other than the spot may be detected, and as a result, the S / N may be deteriorated. On the other hand, when the height of the convex portion is higher than 500 μm, problems such as the convex portion being easily broken and broken may occur, which is not preferable.

  A preferable example of the relationship between the carrier, the cover member, the adhesive member, and the stirring bar in the analysis chip of the present invention will be described with reference to FIGS. In the example shown in FIGS. 11 and 12, the selective binding substance 9 such as DNA is immobilized on the upper surface 6 of the convex portion of the carrier 2. The stirrer (in this case, spherical beads) 7 is placed in the gap in the recess of the carrier 2. The selective binding substance 9 and the stirring bar 7 come into contact with a sample solution (not shown) containing the sample DNA (test substance). The sample solution is held in a gap defined by the carrier 2, the adhesive member 3, and the cover member 1. In the example of FIG. 11, the shortest distance between the upper surface 6 of the convex portion of the carrier and the cover member 1 is less than the diameter of the stirring bar 7. Thereby, the stirrer 7 cannot contact the upper surface 6 of the convex portion, and it is possible to prevent the selective binding substance 9 on the upper surface 6 of the convex portion from being damaged. When the stirrer has an aspherical shape such as an ellipse, for example, if the shortest distance between the upper surface of the convex portion and the container is less than the minimum diameter of the stirrer, the contact between the upper surface 6 of the convex portion and the stirrer It is possible to prevent the selective binding substance 9 from being damaged.

  The material of the carrier of the analysis chip of the present invention is not particularly limited, and examples thereof include inorganic materials such as glass, ceramic and silicon, polymers such as polyethylene terephthalate, cellulose acetate, polycarbonate, polystyrene, polymethyl methacrylate, and silicone rubber. it can. Among these, polymethyl methacrylate, polystyrene, polydimethylsiloxane (PDMS) elastomer, glass and silicon can be particularly preferably used. In addition to the above materials, additives such as carbon black can be blended in order to increase the S / N ratio of the optical measurement by making the carrier black.

  When the material is a polymer or the like, the carrier can be molded by an injection molding method, a hot embossing method, a method of polymerizing in a mold, or the like. Further, when the material is an inorganic substance such as glass or ceramic, it can be molded by a sandblast method, and when it is silicon, it can be molded by a known semiconductor process.

  The molded carrier can be subjected to various surface treatments as necessary before immobilizing the selective binding substance on the surface. Specific examples of such surface treatment include those described in JP-A No. 2004-264289.

  As the selective binding substance to be immobilized on the surface of the carrier, various substances capable of selectively binding directly or indirectly to the test substance can be used. Typical examples include nucleic acids, proteins, Mention may be made of sugars and other antigenic compounds. The nucleic acid may be DNA, RNA or PNA. A single-stranded nucleic acid having a specific base sequence selectively hybridizes and binds to a single-stranded nucleic acid having a base sequence complementary to the base sequence or a part thereof. Falls under the category of Examples of the protein include an antibody and an antigen-binding fragment of an antibody such as a Fab fragment or F (ab ′) 2 fragment, and various antigens. Since an antibody or an antigen-binding fragment thereof selectively binds with a corresponding antigen, and the antigen selectively binds with a corresponding antibody, it corresponds to a “selective binding substance”. As the saccharide, a polysaccharide is preferable, and various antigens can be exemplified. In addition, substances having antigenicity other than proteins and saccharides can be immobilized. Particularly preferred as “selective binding substances” are nucleic acids, antibodies and antigens. The selective binding substance used in the present invention may be a commercially available substance or may be obtained from a living cell or the like. Moreover, you may fix a cell on the surface of a support | carrier as a selective binding substance.

  Preparation of DNA or RNA from living cells can be carried out by a known method, for example, DNA extraction by the method of Blin et al. (Blin et al., Nucleic Acids Res. 3: 2303 (1976)) or the like. Extraction can be performed by the method of Favaloro et al. (Favaloro et al., Methods Enzymol. 65: 718 (1980)). Examples of the nucleic acid to be immobilized include linear or circular plasmid DNA and chromosomal DNA, DNA fragments obtained by digesting these with restriction enzymes or chemically, DNA synthesized with enzymes in a test tube, or chemically synthesized oligos. Nucleotides and the like can also be used.

  The test substance used in the analysis method using the analysis chip of the present invention includes nucleic acids to be analyzed, for example, genes such as pathogenic bacteria and viruses, causative genes of genetic diseases, etc., a part thereof, and various antigenic properties. Examples include, but are not limited to, biological components, antibodies against pathogenic bacteria, viruses, and the like. Examples of specimens containing these test substances include body fluids such as blood, serum, plasma, urine, feces, spinal fluid, saliva, various tissue fluids, various foods and beverages, and dilutions thereof. It is not limited to these. Moreover, the nucleic acid to be the test substance may be labeled with a nucleic acid extracted from blood or cells by a conventional method, or may be amplified by a nucleic acid amplification method such as PCR using the nucleic acid as a template. In the latter case, the measurement sensitivity can be greatly improved. When a nucleic acid amplification product is used as a test substance, the amplified nucleic acid can be labeled by performing amplification in the presence of a nucleoside triphosphate labeled with a fluorescent substance or the like. When the test substance is an antigen or antibody, the test substance antigen or antibody may be directly labeled by a conventional method, or the test substance antigen or antibody may be bound to a selective binding substance. Thereafter, the carrier is washed, and a labeled antibody or antigen that reacts with the antigen or antibody with an antigen-antibody is reacted, and the label bound to the carrier can be measured.

  The method for analyzing a test substance of the present invention comprises (A) a step of applying a test substance to the analysis chip of the present invention; (B) a step of selectively binding the test substance to the carrier; and (C And (b) measuring the amount of the test substance bound on the carrier via the selective binding substance.

  In the step (A), the test substance subjected to labeling and amplification as described above is made into a solution such as an aqueous solution or a suitable buffer solution, if necessary, and injected into the concave portion of the carrier with a normal instrument such as a pipette. Can be performed. In particular, when using an analysis chip having a cover member having a through hole, the injection can be performed from the through hole.

  When using an analysis chip having a cover member having a through-hole, after injecting a test substance into the through-hole, the sealing member is affixed in such a manner that a part or all of the through-hole is sealed. It is desirable to seal by. Preferred examples of the sealing member include flexible tapes such as Kapton (trademark, polyimide film, manufactured by Toray DuPont), polyester, cellophane, or vinyl chloride adhesive tape. Not limited, it is possible to use any non-flexible plate-like adhesive member, and an atypical sealing agent can be used, but the effect of the present invention by the liquid level chamber can be improved. From the viewpoint of obtaining, a flexible tape and a plate-like member are preferable, and from the viewpoint of easy operation, a flexible tape is more preferable.

  When a tape or plate-like member is used as the sealing member, the number of sheets used is arbitrary. Specifically, all the through-holes on the cover member may be sealed with a single sealing member, and a plurality of through-holes are sealed with a plurality of sealing members, respectively. Also good. In addition, when a plurality of cover members are provided on a single carrier as described above, separate sealing members may be used for each of the cover members, and the through holes on the plurality of cover members are combined together. You may seal with the sealing member of a sheet. Usually, it is preferable to use one sealing member per one cover member because simple and reliable sealing can be achieved.

  A specific example of sealing will be described with reference to FIG. In the example of FIG. 12, after applying a sample solution (not shown) from the through hole 4, a flexible adhesive tape 10 is applied so as to cover the entire surface of the liquid level holding chamber 4A, and the through hole is formed. It is sealed. According to such an embodiment, it is possible to achieve sealing that is simple and does not cause leakage of the sample solution or measurement error.

  The step (B) can be performed in the same manner as the operation in the conventional analysis chip. In the step (B), preferably, the analysis chip having a selective binding substance immobilized on the surface of the carrier and located in the void is used as the analysis chip. It can be performed by interacting with a binding substance and binding the test substance to the carrier via the selective binding substance. The reaction temperature and time are appropriately selected according to the chain length of the nucleic acid to be hybridized, the type of antigen and / or antibody involved in the immune reaction, etc. In the case of nucleic acid hybridization, usually 30 ° C. to 70 ° C. In the case of an immune reaction, it is usually from room temperature to about 40 ° C. for about 1 minute to several hours. In the step (B), if necessary, the analysis chip can be swung, rotated, etc., to promote selective binding. After the selective bonding is completed, the cover member is usually detached and can be used for the next step (C).

  The step (C) can also be performed in exactly the same manner as in the conventional analysis chip. For example, the amount of the test substance that is appropriately fluorescently labeled and bound to the selective binding substance can be measured by reading the amount of fluorescence with a known scanner or the like.

  In the analysis method of the present invention, when a nucleic acid is immobilized as a selective binding substance, a nucleic acid having a sequence complementary to this nucleic acid or a part thereof can be measured. When an antibody or antigen is immobilized as a selective binding substance, the antigen or antibody that immunoreacts with this antibody or antigen can be measured. Note that “measurement” in this specification indicates both detection and quantification.

  The analysis kit of the present invention includes the analysis chip of the present invention having a through-hole and a sealing member that is attached to the cover member and seals the through-hole. As the said sealing member, the thing similar to what was demonstrated in the process (A) of the analysis method of the said this invention can be included. The analysis kit of the present invention can be used according to the analysis method of the present invention.

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

Example 1
(Preparation of DNA immobilization carrier)
A resin composition was prepared by mixing 99 parts by mass of PMMA (polymethyl methacrylate) having an average molecular weight of 50,000 and 1 part by mass of carbon black (trade name # 3050B, manufactured by Mitsubishi Chemical Corporation).

  A mold for injection molding was prepared using a known method of LIGA (Lithographie Galvanoformung Abformung). Using this mold, the resin composition was injection molded to obtain a black carrier having a shape as described later.

  The shape of the carrier was generally as shown in FIG. 13, the size was 76 mm long, 26 mm wide, and 1 mm thick, and the surface was flat except for the central portion R1 of the carrier. The central portion R1 of the carrier is provided with a rectangular concave portion having a length and width of 10 mm and a depth of 150 μm. In this concave portion, 64 convex portions 6 having a diameter of 130 μm, a height of 150 μm, and a top surface diameter of 130 μm ( (8 × 8) provided (FIG. 13 is a schematic diagram, so the number of convex portions shown in the figure is smaller than the actual number). The difference in height between the height of the upper surface of the convex portion of the concavo-convex portion (the average value of the heights of the 64 convex portions) and the flat portion 8 was measured to be 3 μm or less. Further, when the variation in the height of the upper surfaces of the 64 convex portions (the difference between the height of the upper surface of the highest convex portion and the height of the upper surface of the lowest convex portion) was measured, it was 3 μm or less. Furthermore, the pitch L1 of the convex portion (distance from the central portion of the convex portion to the adjacent central portion of the convex portion) was 560 μm, and the distance from the convex portion on the outermost side to the peripheral edge portion of the concave portion was 790 μm.

  When the spectral reflectance and the spectral transmittance of this black carrier were measured, the spectral reflectance was 5% or less at any wavelength in the visible light region (wavelengths from 400 nm to 800 nm), and in the same range of wavelengths. The transmittance in the thickness direction was 0.5% or less. Neither spectral reflectance nor spectral transmittance had a specific spectral pattern (such as a peak) in the visible light region, and the spectrum was uniformly flat. Note that the spectral reflectance is the spectrum when specularly reflected light from a carrier is captured using an apparatus (manufactured by Minolta Camera, CM-2002) equipped with an illumination / light receiving optical system that conforms to condition C of JIS Z 8722. The reflectance was measured.

(Immobilization of probe DNA)
A DNA having the sequence shown in SEQ ID NO: 1 and aminated at the 5 ′ end was synthesized. This DNA was immobilized according to the scheme shown in FIG.

  The carrier 2 was immersed in a 10N aqueous sodium hydroxide solution at 70 ° C. for 12 hours. This was washed with pure water, 0.1N HCl aqueous solution, and pure water in this order to generate carboxyl groups on the surface of the carrier.

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

(Preparation of sample DNA)
As the sample DNA, DNA having a base sequence of SEQ ID NO: 4 (968 bases) capable of hybridizing with the probe DNA immobilized on the DNA immobilization carrier was used. The method for preparing this DNA is shown below.

  DNAs having the nucleotide sequences of SEQ ID NO: 2 and SEQ ID NO: 3 were synthesized. These were each dissolved in pure water to obtain an aqueous solution having a concentration of 100 μM. Subsequently, pKF3 plasmid DNA (Takara Bio Inc. product number; 3100) (SEQ ID NO: 5: 2246 bases) was prepared, and this was used as a template. Amplification was performed by Polymerase Chain Reaction.

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

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

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

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

(Preparation of analysis chip)
A cover member having four through-holes 4 shown in FIGS. 13 and 14 and having a convex portion having a height of 100 μm on the outer periphery of the carrier-side surface was produced by cutting a PMMA plate having a thickness of 1 mm. The convex part of this cover member was produced so that the adhesion point was located at a distance of 500 μm outside (W in FIG. 13) from the end face of the concave part of the carrier surface.
Using silicone elastomer (Silgard) 3 as an adhesive member, an adhesive member was applied to a place away from the concave portion of the carrier at a discharge pressure of 0.45 MPa using a dispenser. The cover member was placed so as to cover the probe DNA immobilization region, a load of 50 g was applied from above the cover member, and the cover member was allowed to stand until the adhesive member spread over the entire adhesive surface. After confirming that the adhesive member spreads over the entire surface, the load was removed, and the adhesive member was allowed to stand in an oven at 42 ° C. for 2 hours until the adhesive member was completely solidified.
The thickness of the adhesive member was measured using a micrometer. Specifically, the thickness was calculated by subtracting the thickness of the carrier and the cover member convex portion measured in advance from the thickness of the prepared analysis chip. The thickness of the adhesive member on each side (H in FIG. 13) was 14 μm, 10 μm, 9 μm, and 12 μm, respectively (see FIG. 14), which was almost the desired 10 μm thickness. That is, the distance between the carrier surface and the carrier-side surface of the cover member was approximately 110 μm.
Through the through hole of the cover member, a zirconia stirrer having a diameter of 150 μm to 180 μm was added into the gap, and the analysis chip shown in FIGS. 13 and 14 was obtained.

(Hybridization)
The sample DNA was hybridized to the analysis chip obtained above. Specifically, 50 μl of hybridization solution was injected from the through hole 4 using a micropipette. Then, the through-hole was plugged with Kapton tape (manufactured by ASONE Corporation, model number 5-5018-01). However, as the Kapton tape, four pieces cut into 3 mm squares were used, and each one through hole was closed. Thereafter, the analysis chip was incubated at 42 ° C. for 16 hours. After the incubation, the cover member and the double-sided tape were detached from the carrier, and the carrier was washed and dried.

(Evaluation)
In the above hybridization process, the inside of the void of the analysis chip was observed, and the following items were evaluated.
(1) Is the stirrer attached to the adhesive member by stirring?
(2) When the sample solution is applied, is the solution spread throughout the gap?
(3) Whether the stirrer moves smoothly around the gap during stirring.
(4) Check if bubbles are generated inside the gap during hybridization.
As a result of evaluation in this example, it was as follows. No adhesion of the stirrer was observed on the adhesive member. When the sample solution was applied to the obtained analysis chip, the sample solution was spread throughout the gap, and no gap was found in the details. After applying the sample solution, the stirrer moved smoothly in the gap, and it was confirmed that the stirring in the gap was performed efficiently. Observation of the inside of the gap after hybridization revealed no generation of bubbles in the gap.

The fluorescence intensity of the three convex portions (spots 1 to 3) on which the probe DNA of the analysis chip was immobilized was measured, and the hybridization result between the sample DNA and the probe DNA was evaluated. The carrier after the above treatment was set on a DNA chip scanner (Axon Instruments GenePix 4000B), and measurement was performed with the laser output set to 33% and the photomultiplier voltage set to 500. The results are shown in Table 1. The three fluorescence intensities shown here are values of the fluorescence intensities after hybridization at three different convex portions.
As a result of the measurement, sufficient fluorescence intensity was obtained at all three locations, and there was no variation in hybridization reaction due to differences in spot locations.

Comparative Example 1
The analysis chip as shown in FIG. 16 is prepared by operating in the same manner as in Example 1 except that the adhesion point between the carrier and the cover member is located in contact with the concave portion of the carrier. Visual observation of the inside of the void, hybridization, and measurement of fluorescence intensity were performed. The results are shown in Table 1.
When a stirrer was added in the gap before the sample solution was applied, the stirrer adhered to the adhesive member was fixed, and the stirrer that was immobilized when the sample solution was injected became an obstacle, and the sample solution It did not spread throughout the area.
In addition, a gap was generated between the adhering stirrer and the adhesive member, which was caused by this, and bubbles were generated in the entire gap after hybridization.
Moreover, the fluorescence intensity decreased as a whole, and a variation in fluorescence intensity depending on the spot location was observed.

Comparative Example 2
The adhesion point between the carrier and the cover member is located away from the concave portion of the carrier, but analysis is performed in the same manner as in Example 1 except that the diameter of the stirrer is smaller than the gap between the carrier and the cover member. A chip was prepared, and the inside of the void was visually observed, hybridization, and fluorescence intensity were measured. The results are shown in Table 1.
As a result, as compared with Example 1, when the stirrer was added into the gap, the stirrer entered the gap between the carrier and the cover member, and the stirrer adhered to the adhesive member portion. For this reason, the stirrer attached when applying the sample solution became an obstacle, and the sample solution did not spread over the entire region.
In addition, a gap generated around the attached stirring bar was caused, and bubbles were generated in the entire gap after hybridization.
In addition, a decrease in fluorescence intensity was observed as a whole, and a variation in fluorescence intensity was observed depending on the spot location.

Comparative Example 3
An analysis chip was prepared in the same manner as in Example 1 except that the stirring bar was not sealed in the gap between the carrier and the cover member, and hybridization and fluorescence intensity were measured. The results are shown in Table 1.
When the stir bar was not sealed, it was confirmed that the sample solution spread throughout the gap. In addition, no bubbles were observed in the voids after hybridization. However, since there was no stirrer, stirring in the gap was not sufficiently performed, and the variation in fluorescence intensity depending on the spot location was remarkable. In addition, a decrease in fluorescence intensity was observed depending on the spot location.

Example 2
The cover member was bonded by applying a load of 20 g from above, and the analysis chip in which the thickness of the adhesive member on each side (H in FIG. 13) became 24 μm, 26 μm, 28 μm, and 25 μm, respectively, Except that a zirconia stirrer having a diameter of 180 μm to 212 μm was added into the gap through the through hole of the cover member, the same operation as in Example 1 was performed, and FIG. 13 and FIG. 14 (however, H in FIG. Were replaced with the above-mentioned numerical values.) An analysis chip as shown in FIG. 2 was prepared, and visual observation in the void, hybridization, and measurement of fluorescence intensity were performed. The results are shown in Table 1.

  The stirrer did not adhere to the adhesive member before the sample solution was applied. When the sample solution was applied to the analysis chip, the sample solution spread throughout the gap, and the stirrer moved without stagnation within the gap, confirming that stirring in the gap was performed efficiently. Observation of the inside of the gap after hybridization revealed no generation of bubbles in the gap. As for the fluorescence intensity at three different convex portions obtained after hybridization, sufficient fluorescence intensity was obtained at all three locations, and there was no variation in hybridization reaction due to differences in spot locations.

Example 3
The cover member was bonded by applying a load of 10 g from above, and an analysis chip in which the thickness of the adhesive member on each side (H in FIG. 13) became 52 μm, 55 μm, 56 μm, and 54 μm, respectively, Except that a zirconia stirrer having a diameter of 212 μm to 250 μm was added into the gap through the through hole of the cover member, the same operation as in Example 1 was performed, and FIG. 13 and FIG. 14 (however, H in FIG. Were replaced with the above-mentioned numerical values.) An analysis chip as shown in FIG. 2 was prepared, and visual observation in the void, hybridization, and measurement of fluorescence intensity were performed. The results are shown in Table 1.

  The stirrer did not adhere to the adhesive member before the sample solution was applied. When the sample solution was applied to the analysis chip, the sample solution spread throughout the gap, and the stirrer moved without stagnation within the gap, confirming that stirring in the gap was performed efficiently. Observation of the inside of the gap after hybridization revealed no generation of bubbles in the gap. As for the fluorescence intensity at three different convex portions obtained after hybridization, sufficient fluorescence intensity was obtained at all three locations, and there was no variation in hybridization reaction due to differences in spot locations.

Example 4
The cover member was bonded by applying a load of 50 g from above, and an analysis chip in which the thickness of the adhesive member on each side (H in FIG. 13) became 10 μm, 12 μm, 13 μm, and 11 μm, respectively, Except that a zirconia stirrer having a diameter of 180 μm to 198 μm was added into the gap through the through hole of the cover member, the same operation as in Example 1 was performed, and FIGS. 13 and 14 (however, H in FIG. 14) Were replaced with the above-mentioned numerical values.) An analysis chip as shown in FIG. 2 was prepared, and visual observation in the void, hybridization, and measurement of fluorescence intensity were performed. The results are shown in Table 1.

  The stirrer did not adhere to the adhesive member before the sample solution was applied. When the sample solution was applied to the analysis chip, the sample solution spread throughout the gap, and the stirrer moved without stagnation within the gap, confirming that stirring in the gap was performed efficiently. Observation of the inside of the gap after hybridization revealed no generation of bubbles in the gap. As for the fluorescence intensity at three different convex portions obtained after hybridization, sufficient fluorescence intensity was obtained at all three locations, and there was no variation in hybridization reaction due to differences in spot locations.

Example 5
The adhesion point of the convex part of the cover member is located at a distance of 700 μm outside from the end face of the concave part of the carrier surface (W in FIG. 13), and the cover member is adhered by applying a load of 50 g from above, The thickness of the adhesive member on the side (H in FIG. 13) is 11 μm, 12 μm, 12 μm, and 11 μm, respectively, and the diameter is 125 μm to 150 μm in the gap through the through hole of the cover member. Except that the zirconia stirrer was added, the same operation as in Example 1 was performed, and analysis as shown in FIGS. 13 and 14 (where H in FIG. 14 is replaced with the above-mentioned numerical values). A chip was prepared, and the inside of the void was visually observed, hybridization, and fluorescence intensity were measured. The results are shown in Table 1.

  The stirrer did not adhere to the adhesive member before the sample solution was applied. When the sample solution was applied to the analysis chip, the sample solution spread throughout the gap, and the stirrer moved without stagnation within the gap, confirming that stirring in the gap was performed efficiently. Observation of the inside of the gap after hybridization revealed no generation of bubbles in the gap. As for the fluorescence intensity at three different convex portions obtained after hybridization, sufficient fluorescence intensity was obtained at all three locations, and there was no variation in hybridization reaction due to differences in spot locations.

Example 6
The adhesion point of the convex part of the cover member is located at a distance of 300 μm outside from the end face of the concave part of the carrier surface (W in FIG. 13), and the cover member is adhered by applying a load of 50 g from above, Zirconia having a diameter of 180 μm to 212 μm in the gap through the through-holes of the cover member, using an analysis chip in which the thickness of the adhesive member on the side (H in FIG. 13) was 11 μm, 12 μm, 13 μm, and 11 μm, respectively. The analysis chip as shown in FIG. 13 and FIG. 14 (however, H in FIG. 14 is replaced with the above-mentioned numerical value) is operated in the same manner as in Example 1 except that a stirrer is added. The sample was prepared, and visual observation inside the void, hybridization, and measurement of fluorescence intensity were performed. The results are shown in Table 1.

  The stirrer did not adhere to the adhesive member before the sample solution was applied. When the sample solution was applied to the analysis chip, the sample solution spread throughout the gap, and the stirrer moved without stagnation within the gap, confirming that stirring in the gap was performed efficiently. Observation of the inside of the gap after hybridization revealed no generation of bubbles in the gap. As for the fluorescence intensity at three different convex portions obtained after hybridization, sufficient fluorescence intensity was obtained at all three locations, and there was no variation in hybridization reaction due to differences in spot locations.

FIG. 1 is a perspective view schematically showing an example of the analysis chip of the present invention. FIG. 2 is a partial cross-sectional view of the analysis chip of the present invention shown in FIG. 1 cut along a plane along arrow A1. FIG. 3 is a partial cross-sectional view showing an example of the analysis chip of the present invention. FIG. 4 is a partial cross-sectional view showing an example of the analysis chip of the present invention. FIG. 5 is a longitudinal sectional view schematically showing an example of the analysis chip of the present invention having a partition structure. FIG. 6 is a perspective view schematically showing another example of the analysis chip of the present invention. FIG. 7 is a partial cross-sectional view of the analysis chip of the present invention shown in FIG. 6 cut along a plane along arrow A2. FIG. 8 is a perspective view schematically showing an example of a carrier constituting the analysis chip of the present invention. FIG. 9 is a longitudinal sectional view of the carrier illustrated in FIG. FIG. 10 is a longitudinal sectional view schematically showing an example of a jig and a scanner for reading a reaction result using the analysis chip of the present invention. FIG. 11 is a cross-sectional view schematically showing an example of the analysis chip of the present invention. FIG. 12 is a longitudinal sectional view schematically showing another example of the analysis chip of the present invention. FIG. 13 is a longitudinal sectional view schematically showing an example of the analysis chip in the example. FIG. 14 is a plan view of the cover member of the analysis chip of FIG. 13 as viewed from above. FIG. 15 is a schematic diagram showing the immobilization of probe DNA in Examples and Comparative Examples of the present application. FIG. 16 is a cross-sectional view schematically showing an analysis chip in a comparative example.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Cover member 1A Convex part 2 of cover member Carrier 3, 3C Adhesive member 4 Through-hole 4A Liquid level parking chamber 5 Air gap (space)
6 Convex part on carrier surface 7 Stirrer 8 Flat part 9 Selective binding substance 10 Sealing member (tape)
DESCRIPTION OF SYMBOLS 11 Objective lens 12 Laser excitation light 13 Spring 14 for abutting microarray to jig | tool Jig 15 Jig | butting surface P1 Carrier-side adhesion point P2 Outer peripheral part end surface P3 of concave part of carrier surface Inner cross section P4 of adhesive member Surface portion P5 on the outer side of the concave portion P5 Adhesive point S1 on the cover member side Space R1 Area where the selective binding substance is immobilized (concave portion)
L1 convex pitch

Claims (7)

A carrier having a recess on the surface;
A cover member covering the surface of the carrier and bonded to the carrier;
An adhesive member interposed between the carrier and the cover member to bond them;
An analysis chip comprising a stirrer enclosed in a gap between the carrier and the cover member,
The adhesion point between the carrier and the cover member on the carrier surface is located in an outer region away from the outer peripheral end surface of the recess, and the distance between the carrier surface and the carrier side surface of the cover member at the adhesion point is agitated. An analysis chip characterized by being shorter than the shortest diameter of the child.   The convex part is provided in the carrier side surface outer periphery of the said cover member, The said adhesive member is interposed between the said carrier and the convex part of the carrier side surface outer periphery of the said cover member. The analysis chip described.   The analysis chip according to claim 1, wherein the carrier has a selective binding substance immobilized on a region of the carrier located on the surface of the carrier.   The analysis chip according to claim 1, wherein the cover member is detachably bonded to the carrier.   The analysis according to any one of claims 1 to 4, wherein the adhesive member is a resin composition containing a polymer selected from the group consisting of a silicone-based polymer, an acrylic polymer, and a mixture thereof. Chip. (A) A step of applying a test substance to the analysis chip according to any one of claims 1 to 5;
(B) selectively binding the test substance to the carrier; and (C) measuring the amount of the test substance bound to the carrier via the selective binding substance. And a method for analyzing a test substance.   The analysis chip has a selective binding substance immobilized on a region of the carrier located on the surface of the carrier, and the step (C) includes the test substance and the selective binding substance. And the test substance is bound to the carrier via the selective binding substance.

Images