JP2008249465A - Probe array and its manufacturing method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a simply-manufacturable probe array capable of reducing consumption of a liquid sample, and its manufacturing method. <P>SOLUTION: This probe array is equipped with a planar probe fixing sheet 2A comprising a base material 21A and a probe immobilization layer 22A; a transparent planar thermally-fusible first cover sheet 3A positioned on the probe immobilization layer 22A side of the probe fixing sheet 2A; and a planar thermally-fusible second cover sheet 4A positioned on the base material 21A side of the probe fixing sheet 2A. Full fringe parts of the first cover sheet 3A and the second cover sheet 4A are thermally fused, and a clearance for constituting a liquid introduction space 6A is provided between the probe immobilization layer 22A of the probe fixing sheet 2A and the first cover sheet 3A. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a probe array and a manufacturing method thereof.

  In recent years, probe arrays such as a DNA chip and a protein chip have been developed that can detect a target substance by immobilizing a probe capable of reacting with a target substance (for example, a biological substance such as DNA or protein) on a plate-like carrier such as glass or silicon. ing.

  Detection of the target substance by the probe array is performed by adding a liquid sample containing the target substance onto a plate-like carrier, covering with a cover glass to prevent drying, reacting the probe with the target substance, washing, and A substance other than the reacted target substance is removed, and a labeling substance (for example, a fluorescent substance, an enzyme, etc.) previously bound to the target substance is detected.

  However, in the conventional probe array, the liquid sample is sucked with a micropipette, added uniformly to the plate carrier, covered with a cover glass, and after the reaction, the cover glass is removed and the plate carrier is washed uniformly. Therefore, workability was poor and a relatively large amount of liquid sample was required.

Under such circumstances, a probe array has been developed in which a plurality of types of probes are fixed in a stripe pattern on the inner wall of a light-transmitting cylindrical capillary (see Patent Document 1).
Japanese Patent Laid-Open No. 11-75812

  According to the probe array, the target substance can be detected by a simple operation of inflow and outflow of a liquid sample, a cleaning liquid, and the like with respect to the hollow portion of the cylindrical capillary. However, the amount of liquid sample required is still large, and the manufacturing method of the probe array itself is not easy.

  The present invention has been made in view of such a situation, and an object of the present invention is to provide a probe array that can reduce the amount of liquid sample used and can be easily manufactured, and a method for manufacturing the same. .

  In order to achieve the above object, first, the present invention is a flat probe fixing sheet having a probe fixing layer on one side, and is heat-sealable located on the probe fixing layer side of the probe fixing sheet. A first planar cover sheet having transparency; and a second planar cover sheet that is located on the opposite side of the probe immobilization layer of the probe immobilization sheet and that can be heat-sealed. At least both edges of the cover sheet and the second cover sheet are heat-sealed, and a liquid introduction space is formed between the probe fixing layer of the probe fixing sheet and the first cover sheet. There is provided a probe array characterized in that there is a void to be formed (claim 1).

  Secondly, the present invention provides a planar probe fixing sheet having a probe fixing layer on both sides, and a planar first sheet which is located on one side of the probe fixing sheet and has heat-sealable transparency. And a flat second cover sheet that is located on the other surface side of the probe fixing sheet and has heat-transmissible transparency, the first cover sheet and the second cover sheet At least both edges of the cover sheet are heat-sealed, between the probe fixing layer of the probe fixing sheet and the first cover sheet, and between the probe fixing layer of the probe fixing sheet and the second There is provided a probe array characterized in that there are air gaps constituting liquid introduction spaces between the cover sheets (claim 2).

  In the above inventions (Inventions 1 and 2), the first cover sheet and the second cover sheet are heat-sealed at all peripheral edges except for one place, and are not heat-sealed. May be a liquid injection part (Claim 3), and other than the two places, it is heat-sealed at the entire periphery, and the non-heat-fused parts are the liquid injection part and the discharge part. (Claim 4).

  Thirdly, the present invention relates to a planar probe fixing sheet having a probe fixing layer on one side or both sides, and a planar shape having heat-transparent transparency located on the probe fixing layer side of the probe fixing sheet. Cover sheet (one or two sheets), at least both edges of the probe fixing sheet and the cover sheet are heat-sealed, and the probe fixing layer of the probe fixing sheet and the cover sheet There is provided a probe array characterized in that there is an air gap constituting a liquid introduction space between them (claim 5).

  Fourthly, in the present invention, a planar first cover sheet having transparency that can be heat-sealed is stacked on the probe-immobilized layer side of a planar probe-immobilized sheet having a probe-immobilized layer on one side, On the opposite side of the probe fixing layer of the probe fixing sheet, a flat second cover sheet that can be heat-sealed is overlaid, and at least both edges of the first cover sheet and the second cover sheet are heat-fused. A probe array manufacturing method is provided, characterized in that a gap forming a liquid introduction space is formed between the probe fixing layer of the probe fixing sheet and the first cover sheet. Item 6).

  Fifthly, in the present invention, a planar first cover sheet having transparency that can be heat-sealed is superimposed on one surface side of a planar probe fixing sheet having probe fixing layers on both sides, and the probe A flat second cover sheet having transparency that can be heat-sealed is stacked on the other surface side of the fixing sheet, and at least both edges of the first cover sheet and the second cover sheet are heat-fused. A liquid introduction space between the probe fixing layer of the probe fixing sheet and the first cover sheet and between the probe fixing layer of the probe fixing sheet and the second cover sheet. Provided is a method for manufacturing a probe array, characterized in that a gap is formed (claim 7).

  Sixth, the present invention provides a flat cover sheet (1 or 2) having transparency that can be heat-sealed on the probe-fixing layer side of a flat probe-fixing sheet having a probe-fixing layer on one or both sides. And at least both edges of the probe fixing sheet and the cover sheet are heat-sealed, so that a gap constituting a liquid introduction space is formed between the probe fixing layer of the probe fixing sheet and the cover sheet. A method for manufacturing a probe array is provided.

  According to the said invention (inventions 6-8), a probe array can be simply manufactured by heat sealing | fusion. Further, since the sheet-like members are stacked and heat-sealed, the volume of the liquid introduction space in the obtained probe array becomes extremely small. According to such a probe array, the detection operation can be efficiently performed with a small amount of sample solution.

  According to the present invention, a probe array can be easily manufactured by thermal fusion. Moreover, since the volume of the liquid introduction space in the obtained probe array is extremely small, the detection operation can be performed efficiently with a small amount of sample solution.

Hereinafter, embodiments of the present invention will be described.
[First Embodiment]
1A is a cross-sectional view of the probe array 1A according to the first embodiment of the present invention (XX cross-sectional view of FIG. 1B), and FIG. 1B is a probe array 1A according to the same embodiment. FIG. As shown in FIG. 1, the probe array 1A according to the present embodiment includes a planar probe fixing sheet 2A, a planar first cover sheet 3A, and a planar second cover sheet 4A. Yes.

  The probe fixing sheet 2A includes a base material 21A and a probe fixing layer 22A laminated on the base material 21A. The substrate 21A in the present embodiment is not particularly limited as long as the probe-immobilized layer 22A can be formed and is insoluble in a liquid sample, a cleaning solution, or the like.

  Examples of the material of the base material 21A include, for example, polyolefins such as polyethylene and polypropylene, polyesters such as polyethylene terephthalate and polybutylene terephthalate, polyvinyl chloride, polystyrene, polyurethane, polycarbonate, polymethyl methacrylate, ethylene vinyl acetate copolymer, Sheet made of resin such as ethylene (meth) acrylic acid copolymer, ethylene (meth) acrylic acid ester copolymer, ionomer resin, or laminated sheet thereof; Sheet or plate made of metal or alloy such as aluminum or stainless steel A glass plate; a ceramic plate; and a composite material thereof.

  The color of the base material 21A is preferably a color that easily recognizes a labeling substance (for example, a fluorescent substance) that is bound to the target substance bound to the probe of the probe-immobilized layer 22A, for example, white. preferable.

  The thickness of the base material 21A is not particularly limited as long as the liquid introduction space 6A described later is formed, but is usually 5 to 200 μm, preferably 10 to 50 μm.

  The probe immobilization layer 22A is obtained by immobilizing a probe on a layer made of a material (probe immobilization material) for immobilizing a probe. The probe can be immobilized using, for example, chemical adsorption or chemical reaction by electrostatic bonding, covalent bonding, protein-protein interaction, protein-small molecule interaction, or the like. Moreover, it can fix using the physical adsorption effect | action of the particle | grains which have a pore.

  For example, when electrostatic coupling is used, it is preferable to select a polycationic substance as the probe immobilization material. Examples of polycationic substances include polymers having cationic groups; homopolymers or copolymers of basic amino acids such as polylysine, polyarginine, a copolymer of lysine and arginine, or derivatives thereof; polyethyleneimine, etc. And a polycationic polymer. Among these, poly L-lysine, polyethyleneimine and the like are preferably used.

  Further, for example, when utilizing a physical adsorption action, it is preferable to select inorganic particles, particularly porous inorganic particles, as the probe immobilization material. Examples of such inorganic particles include silica particles, silicon carbide, silicon nitride, boron carbide, and alumina. The average secondary particle size of the inorganic particles is preferably 0.1 to 20 μm, and particularly preferably 0.5 to 10 μm.

When the inorganic particles are porous, BET specific surface area is preferably 100~1000m ² / g, it is particularly preferably 200~600m ² / g. The pore volume is preferably 0.5 to 5 ml / g, particularly preferably 1 to 3 ml / g. Furthermore, the average pore diameter is preferably 30 to 500 mm, and particularly preferably 50 to 300 mm.

  When using inorganic particles as a probe immobilization material, by coating inorganic particles and a solvent, if desired, in a binder such as a thermoplastic resin, a thermosetting resin, an active energy ray curable resin, The probe immobilization layer 22A can be formed.

  The thickness of the layer made of the probe-immobilizing material is not particularly limited as long as the thickness is such that the probe is reliably fixed, but is usually 0.5 to 500 μm, preferably 5 to 300 μm.

  The probes are preferably fixed in a plurality of spots as a probe group. The type of probe included in each probe group is usually identified based on the fixed position of each probe group. Each probe group may be fixed in any form as long as the type and position of the probe are associated with each other, and each probe group may be fixed in a streak shape, for example. The number of probe groups to be fixed is not particularly limited, and can be changed as appropriate. Further, the arrangement of each probe group, the size of the spot, and the like can be changed as appropriate.

  One probe group usually includes a plurality of probes of the same type. The probes included in each probe group are biological materials such as nucleic acids, proteins, antigens, antibodies, enzymes, sugar chains, and the like. The types of probes included in different probe groups may be the same or different types, but it is preferable that a plurality of types of probes are included in the entire probe group. By fixing a plurality of types of probes, a plurality of types of target substances can be detected simultaneously in parallel.

  The first cover sheet 3A is made of a material that can be fused by heat or ultrasonic waves and has transparency. Since the first cover sheet 3A has transparency, a labeling substance (for example, a fluorescent substance) bound to the target substance bound to the probe of the probe fixing sheet 2A is recognized from the outside of the first cover sheet 3A. can do. That is, the transparency of the first cover sheet 3A is not particularly limited as long as the labeling substance can be recognized.

  As a material constituting the first cover sheet 3A, for example, polyolefin such as polyethylene and polypropylene, polyacrylonitrile, ethylene-methacrylic acid copolymer, polystyrene, polyvinyl chloride, polyvinylidene chloride, polyamide and the like are heat-fusible. Sheet made of a resin having polyester, polyester such as polyethylene terephthalate, polybutylene terephthalate, polyethylene naphthalate, nylon, cellophane, ethylene-vinyl acetate copolymer, polyvinyl chloride, polypropylene, polystyrene, polycarbonate, polysulfone, polyether ether ketone , Polyethersulfone, polyphenylene sulfide, polyetherimide, polyimide, fluororesin, polyamide, acrylic resin, norbornene resin, etc. To become the base sheet, laminated sheet or the like coated with a resin having a sheet stack or the heat-fusible and made of a resin having the heat-fusible can be mentioned.

  The thickness of the first cover sheet 3A is not particularly limited as long as it is a thickness at which a liquid introduction space 6A described later is formed, but is usually 1-1000 μm, preferably 10-500 μm.

  The second cover sheet 4A is made of a material that can be fused by heat or ultrasonic waves. As the second cover sheet 4A, the same cover sheet as the first cover sheet 3A can be used. However, the second cover sheet 4A need not have transparency.

  In the probe array 1A according to the present embodiment, the first cover sheet 3A and the second cover sheet 4A are arranged such that the probe fixing sheet 2A is accommodated therein (so that the probe fixing sheet 2A is not sandwiched). However, it is heat-sealed at the entire periphery. That is, the thermal fusion part 5A is formed on the entire periphery of the probe array 1A. The width of the heat-sealed portion 5A is not particularly limited as long as the first cover sheet 3A and the second cover sheet 4A are securely bonded, but is usually 0.2 to 10 mm, preferably 2 to 5 mm.

  As shown in FIG. 1A, a gap exists between the probe fixing layer 22A of the probe fixing sheet 2A and the first cover sheet 3A, and this gap constitutes a liquid introduction space 6A. This gap is naturally formed as the probe array 1A is manufactured.

  In order to manufacture the probe array 1A, the first cover sheet 3A is stacked on the probe fixing layer 22A side of the probe fixing sheet 2A, and the second cover sheet is stacked on the substrate 21A side of the probe fixing sheet 2A. The peripheral portions of the first cover sheet 3A and the second cover sheet 4A are heat-sealed. The first cover sheet 3A and the second cover sheet 4A are both flat, but as a result of pressurizing their peripheral portions for heat fusion, the portions other than the peripheral portions (center portion) are slightly Lift up. Thereby, a very thin gap, that is, a liquid introduction space 6A having a small volume is formed between the probe fixing layer 22A of the probe fixing sheet 2A and the first cover sheet 3A.

  Although the volume of the liquid introduction space 6A depends on the size of the probe array 1A, it is preferably 1 to 50 μl, and particularly preferably 3 to 10 μl. When the volume of the liquid introduction space 6A is within such a range, a necessary and sufficient liquid sample can be brought into contact with the probe of the probe immobilization layer 22A, and the amount of the liquid sample used can be reduced as compared with the conventional case.

  In addition, the probe array 1A according to the present embodiment can be simply manufactured by simply heat-sealing the first cover sheet 3A, the probe fixing sheet 2A, and the second cover sheet as described above. it can.

  The probe fixing sheet 2A can be manufactured by a conventional method. For example, the surface of the base material 21A is coated with a probe fixing material by an applicator, blade coater, roll coater, bar coater, die coater, screen printer, spray, etc. On the other hand, a desired probe may be spotted as a probe group and immobilized.

  As shown in FIG. 1B, the planar shape of the probe array 1A according to the present embodiment is a rectangle, but is not limited to this as long as the target substance can be detected.

  Detection of the target substance using the probe array 1A can be performed, for example, as follows.

  As a first step, a liquid sample containing a target substance bound with a labeling substance is put into a syringe, the needle of the syringe is punctured into the first cover sheet 3A, the liquid sample is allowed to flow into the liquid introduction space 6A, and the target substance and Contact the probe. During this time, the hole formed by the puncture of the syringe needle may be closed with an adhesive tape or the like.

  The type of the target substance is not particularly limited, and examples thereof include biological substances such as nucleic acids, proteins, antigens, antibodies, enzymes, sugar chains, etc. Examples of combinations of probes and target substances include, for example, nucleic acid / complementary Nucleic acid, receptor protein / ligand, enzyme / substrate, antibody / antigen and the like. The nucleic acid includes DNA, RNA, and analogs or derivatives thereof (for example, peptide nucleic acid (PNA), phosphorothioate DNA, etc.).

  Examples of the labeling substance include fluorescent substances such as fluorescein, rhodamine and phycoerythrin; enzymes such as alkaline phosphatase and horseradish peroxidase; chemiluminescent substances such as luminol, lucigenin and acridium ester; and bioluminescent substances such as luciferase and luciferin. It is done. The solvent of the liquid sample is, for example, water, a buffer solution, or an organic solvent, and can be appropriately selected according to the type of the target substance.

  As a second step, after the liquid sample is caused to flow out of the liquid introduction space 6A through the hole formed by the needle puncture of the syringe or the hole formed by the needle puncture of the syringe separately, the liquid is used using the syringe. A cleaning solution is allowed to flow into and out of the introduction space 6A to clean the probe. Thereby, substances other than the target substance reacted with the probe can be removed.

  As a third step, a labeling substance that is bound to the target substance is detected. When the labeling substance is a fluorescent substance, the target substance can be detected by irradiating the probe fixing layer 22A with excitation light and detecting the fluorescence emitted from the probe fixing layer 22A with a fluorescence detector. When the labeling substance is an enzyme, the target substance can be detected by an enzyme color reaction. Here, since the first cover sheet 3A has transparency, it is possible to carry out detection without removing the probe fixing sheet 2A without changing the probe array 1A having the above-described configuration.

  In the above embodiment, the first cover sheet 3A and the second cover sheet 4A are heat-sealed at the entire periphery, but the present invention is not limited to this. For example, like the probe array 1A 'shown in FIG. 2, the first cover sheet 3A and the second cover sheet 4A may be heat-sealed at the entire peripheral edge except for one place. In this case, the part which is not heat-sealed comprises the liquid injection | pouring part 7A. In such a probe array 1A ′, it is not necessary to make a hole in the first cover sheet 3A with a syringe needle when in use, and the liquid is allowed to flow into the liquid introduction space 6A by inserting the needle into the liquid injection portion 7A. it can.

  Further, for example, like the probe array 1A ″ shown in FIG. 3, the first cover sheet 3A and the second cover sheet 4A may be heat-sealed at the entire peripheral edge except for two places. . In the present embodiment, portions that are not heat-sealed are formed at opposing positions of the probe array 1A ″. In this case, the portions that are not heat-sealed constitute the liquid injection portion 7A and the discharge portion 8A.

  In such a probe array 1A ″, when used, it is not necessary to make a hole in the first cover sheet 3A with a syringe needle, and the liquid sample is caused to flow into the liquid introduction space 6A by inserting the needle into the liquid injection portion 7A. . At this time, since the air in the liquid introduction space 6A is discharged from the discharge portion 8A, the liquid sample can flow smoothly. Also, when cleaning the probe, the liquid in the liquid introduction space 6A can be discharged from the discharge portion 8A, so that the cleaning liquid can flow smoothly.

  The planar shape of the heat-sealing part 5A may be a strip having substantially the same width as shown in FIG. 1B and FIG. 2, or as shown in FIG. The width may gradually narrow toward the discharge part 8A.

[Second Embodiment]
4A is a cross-sectional view of the probe array 1B according to the second embodiment of the present invention (XX cross-sectional view of FIG. 4B), and FIG. 4B is a probe array 1B according to the same embodiment. FIG. As shown in FIG. 4, the probe array 1B according to the present embodiment includes a planar probe fixing sheet 2B, a planar first cover sheet 3B, and a planar second cover sheet 4B. Yes.

  The probe fixing sheet 2B includes a base material 21B, a probe fixing layer 22B stacked on one surface of the base material 21B, and a probe fixing layer 22B ′ stacked on the other surface of the base material 21B. The

  The material of the base material 21B and the material of the probe immobilization layers 22B and 22B 'are the same as those of the base material 21A and the probe immobilization layer 22A in the first embodiment. In this embodiment, since the probe immobilization layers 22B and 22B ′ are formed on both surfaces of the base material 21B, the number of types is twice that of the case where the probe immobilization layer is formed on one surface of the base material. It is possible to immobilize the probes, or it is possible to miniaturize the probe fixing sheet and thus the probe array.

  The material of the first cover sheet 3B is the same as that of the first cover sheet 3A in the first embodiment. Further, since the second cover sheet 4B needs to have transparency like the first cover sheet 3B, the material of the second cover sheet 4B is the first cover sheet 3A in the first embodiment. It is the same.

  The probe array 1B according to the present embodiment can be manufactured by the same method as the probe array 1A according to the first embodiment. As a result, a gap constituting the liquid introduction space 6B is formed between one probe fixing layer 22B of the probe fixing sheet 2B and the first cover sheet 3B, and the other probe fixing of the probe fixing sheet 2B is fixed. A gap constituting the liquid introduction space 6B ′ is formed between the layer 22B ′ and the second cover sheet 4B.

  The probe fixing sheet 2B can be manufactured by the same method as the probe fixing sheet 2A in the first embodiment except that the probe fixing layers 22B and 22B 'are formed on both surfaces of the base material 21B.

  The detection of the target substance using the probe array 1B can be performed in the same manner as the method in the probe array 1A according to the first embodiment, but the inflow and outflow of the liquid are divided into the two liquid introduction spaces 6B and 6B ′. Needs to be done.

  Also in the probe array 1B according to the present embodiment, there are one or two portions that are not thermally fused as in the probe array 1A according to the first embodiment. A discharge part may be formed.

[Third Embodiment]
FIG. 5A is a cross-sectional view of the probe array 1C according to the third embodiment of the present invention (XX cross-sectional view of FIG. 5B), and FIG. 5B is a probe array 1C according to the same embodiment. FIG. As shown in FIG. 5, the probe array 1C according to the present embodiment includes a planar probe fixing sheet 2C, a planar first cover sheet 3C, and a planar second cover sheet 4C. Yes.

  The probe fixing sheet 2C includes a base material 21C and a probe fixing layer 22C laminated on the base material 21C. The material of the base material 21C and the material of the probe fixing layer 22C are the same as those of the base material 21A and the probe fixing layer 22A in the first embodiment.

  The material of the first cover sheet 3C is the same as that of the first cover sheet 3A in the first embodiment, and the material of the second cover sheet 4C is the second material in the first embodiment. It is the same as the cover sheet 4A.

  In the probe array 1C according to the present embodiment, the first cover sheet 3C and the second cover sheet 4C are heat-sealed at the entire peripheral edge so as to sandwich the edge of the probe fixing sheet 2C. . Otherwise, the probe array 1C can be manufactured in the same manner as the probe array 1A according to the first embodiment.

  Detection of a target substance using the probe array 1C can be performed in the same manner as in the probe array 1A according to the first embodiment.

  Also in the probe array 1C according to the present embodiment, there are one or two portions that are not thermally fused as in the probe array 1A according to the first embodiment. A discharge part may be formed. Further, similarly to the probe array 1B according to the second embodiment, the probe fixing layers 22C are formed on both surfaces of the base material 21C, and each probe fixing layer 22C of the probe fixing sheet 2C and the first cover are formed. A gap constituting the liquid introduction space 6C may be formed between the sheet 3C and the second cover sheet 4C.

[Fourth Embodiment]
FIG. 6A is a cross-sectional view of the probe array 1D according to the fourth embodiment of the present invention (XX cross-sectional view of FIG. 6B), and FIG. 6B is a probe array 1D according to the same embodiment. FIG. As shown in FIG. 6, the probe array 1D according to the present embodiment includes a planar probe fixing sheet 2D and a planar cover sheet 3D.

  The probe fixing sheet 2D includes a base material 21D and a probe fixing layer 22D laminated on the base material 21D. The material of the base 21D is the same as the base 21A in the first embodiment.

  On the other hand, the probe immobilization material of the probe immobilization layer 22D needs to be able to immobilize the probe and to have heat fusion properties. Examples of the probe immobilization material include polyolefins such as polyethylene and polypropylene, polyacrylonitrile, ethylene-methacrylic acid copolymer, polystyrene, polyvinyl chloride, polyvinylidene chloride, polyamide and the like.

  The material of the cover sheet 3D is the same as that of the first cover sheet 3A in the first embodiment.

  In the probe array 1D according to the present embodiment, the cover sheet 3D and the probe fixing layer 22D of the probe fixing sheet 2D are heat-sealed at the entire periphery. And the space | gap which comprises liquid introduction space 6D is formed between probe fixed layer 22D and cover sheet 3D.

  In order to manufacture the probe array 1D, the cover sheet 3D is overlaid on the probe fixing layer 22D side of the probe fixing sheet 2D, and in this state, the peripheral portions of the cover sheet 3D and the probe fixing layer 22D are heat-sealed. By this manufacturing method, the liquid introduction space 6D is naturally formed.

  Detection of a target substance using the probe array 1D can be performed in the same manner as the method in the probe array 1A according to the first embodiment.

  Also in the probe array 1D according to the present embodiment, there are one or two portions that are not thermally fused as in the probe array 1A according to the first embodiment. A discharge part may be formed. Similarly to the probe array 1B according to the second embodiment, the probe immobilization layer 22D is formed on both surfaces of the base material 21D, and the cover sheet 3D is provided so as to cover each probe immobilization layer 22D. Between each probe fixing layer 22D of the probe fixing sheet 2D and the cover sheet 3D, a gap constituting the liquid introduction space 6D may be formed.

[Fifth Embodiment]
FIG. 7A is a sectional view of a probe array 1E according to the fifth embodiment of the present invention (XX sectional view of FIG. 7B), and FIG. 7B is a probe array 1E according to the same embodiment. FIG. As shown in FIG. 7, the probe array 1E according to this embodiment includes a planar probe fixing sheet 2E and a planar cover sheet 3E.

  The probe fixing sheet 2E includes a base material 21E and a probe fixing layer 22E laminated on the base material 21E. However, the probe immobilization layer 22E is not formed on two opposite edge portions (edge portions extending in the longitudinal direction in the present embodiment) of the base material 21E. The material of the probe fixing layer 22E is the same as that of the probe fixing layer 22A in the first embodiment.

  On the other hand, the material of the base material 21E needs to have heat-fusibility. As a material constituting the base material 21E, the same material as the first cover sheet 3A in the first embodiment can be used. However, the color of the base material 21E does not need to be transparent and is preferably white.

  The material of the cover sheet 3E is the same as that of the first cover sheet 3A in the first embodiment.

  In the probe array 1E according to the present embodiment, the cover sheet 3E and the base material 21E of the probe fixing sheet 2E are heat-sealed at two opposite edges. Two opposing edge portions that are not heat-sealed are openings, and constitute a liquid injection portion and a discharge portion, respectively. And the space | gap which comprises the liquid introduction space 6E is formed between the probe fixed layer 22E and the cover sheet 3E.

  In order to manufacture the probe array 1E, the cover sheet 3E is stacked on the probe fixing layer 22E side of the probe fixing sheet 2E, and in this state, both edges of the cover sheet 3E and the base material 21E are heat-sealed. By this manufacturing method, the liquid introduction space 6E is naturally formed.

  Detection of a target substance using the probe array 1E can be performed in the same manner as in the probe array 1A ″ (see FIG. 3) described above.

  Also in the probe array 1E according to the present embodiment, the cover sheet 3E and the probe fixing sheet 2E may be heat-sealed at the entire peripheral edge similarly to the probe array 1D according to the fourth embodiment. Similarly to the probe array 1B according to the second embodiment, probe immobilization layers 22E are formed on both surfaces of the substrate 21E, and a cover sheet 3E is provided so as to cover each probe immobilization layer 22E. Between each probe fixing layer 22E of the probe fixing sheet 2E and the cover sheet 3E, a gap constituting the liquid introduction space 6E may be formed.

[Sixth Embodiment]
8A is a cross-sectional view of the probe array 1F according to the sixth embodiment of the present invention (XX cross-sectional view of FIG. 8C), and FIG. 8B is a probe array 1F according to the same embodiment. FIG. 8C is a plan view of the probe array 1 </ b> F according to the same embodiment. As shown in FIG. 8, the probe array 1F according to the present embodiment includes a planar probe fixing sheet 2F and a planar cover sheet 3F.

  The probe fixing sheet 2F includes a base material 21F and a probe fixing layer 22F laminated on the base material 21F. However, the probe immobilization layer 22F is formed at two opposite edge portions of the base material 21E (edge portions extending in the longitudinal direction in the present embodiment) and the center line portion of the base material 21E. Absent. That is, the probe immobilization layer 22F is divided into two so that the slit 9F is formed therebetween.

  The width of the slit 9F is preferably 0.05 to 1 mm, particularly preferably 0.1 to 0.5 mm.

  The material of the base material 21F is the same as that of the base material E in the fifth embodiment, and the material of the probe immobilization layer 22F is the same as that of the probe immobilization layer 22A in the first embodiment. The material of the cover sheet 3F is the same as that of the first cover sheet 3A in the first embodiment.

  In the probe array 1F according to the present embodiment, the cover sheet 3F and the base material 21F of the probe fixing sheet 2F are heat-sealed at the entire periphery. However, the portion of the slit 9F between the two probe-immobilized layers 22F is opened without being heat-sealed, and constitutes a liquid injection portion 7F and a discharge portion 8F. On the other hand, a gap constituting the liquid introduction space 6F is formed between the probe fixing layer 22F and the cover sheet 3F.

  In order to manufacture the probe array 1F, the cover sheet 3F is overlaid on the probe fixing layer 22F side of the probe fixing sheet 2F, and in this state, the cover sheet 3F and the base material 21F are all pressed with such a pressure that the slit 9F is not crushed. The peripheral part is heat-sealed. By this manufacturing method, the liquid injection part 7F and the discharge part 8F and the liquid introduction space 6F are formed.

  Detection of a target substance using the probe array 1F can be performed in the same manner as in the probe array 1A ″ (see FIG. 3) described above.

  Also in the probe array 1F according to the present embodiment, as in the probe array 1B according to the second embodiment, the probe immobilization layers 22F are formed on both surfaces of the base material 21F, and each probe immobilization layer 22F is covered. Thus, a cover sheet 3F may be provided, and a gap constituting the liquid introduction space 6F may be formed between each probe fixing layer 22F of the probe fixing sheet 2F and the cover sheet 3F.

[Seventh Embodiment]
9A is a cross-sectional view of the probe array 1G according to the seventh embodiment of the present invention (XX cross-sectional view of FIG. 9B), and FIG. 9B is a probe array 1G according to the same embodiment. FIG. As shown in FIG. 9, the probe array 1G according to this embodiment includes a planar probe fixing sheet 2G and a planar cover sheet 3G.

  The probe fixing sheet 2G includes a base material 21G and a probe fixing layer 22G laminated on the base material 21G. However, the probe immobilization layer 22G is not formed on the periphery of the base material 21G. The material of the probe immobilization layer 22G is the same as that of the probe immobilization layer 22A in the first embodiment.

  On the other hand, the material of the base material 21G needs to have heat-fusibility. As a material constituting the base material 21G, the same material as the first cover sheet 3A in the first embodiment can be used. However, the color of the base material 21G does not need to be transparent and is preferably white.

  The cover sheet 3G in the present embodiment has a concave portion other than the peripheral edge portion. The depth of the concave portion needs to be larger than the thickness of the probe fixing layer 22G and such a depth that the cover sheet 3G and the probe fixing layer 22G do not come into contact after heat sealing. Specifically, the depth of the concave portion is preferably 5 to 100 μm, and particularly preferably 10 to 50 μm. In this invention, the sheet | seat which has the above recessed parts shall also be contained in a planar category. The material of the cover sheet 3G is the same as that of the first cover sheet 3A in the first embodiment.

  In the probe array 1G according to the present embodiment, the cover sheet 3G and the base material 21G of the probe fixing sheet 2G are heat-sealed at the entire peripheral edge (portion other than the concave portion of the cover sheet 3G). And the recessed part (The space | gap between the probe fixed layer 22G and the cover sheet 3G) of the cover sheet 3G comprises the liquid introduction space 6G.

  In order to manufacture the probe array 1G, the cover sheet 3G is overlaid on the probe fixing layer 22G side of the probe fixing sheet 2G so that the concave portion is on the probe fixing sheet 2G side. The entire peripheral edge of each is heat-sealed. By this manufacturing method, the liquid introduction space 6G is formed.

  Detection of a target substance using the probe array 1G can be performed in the same manner as in the probe array 1A according to the first embodiment.

  Also in the probe array 1G according to the present embodiment, as in the probe array 1A according to the first embodiment, there are one or two portions that are not heat-sealed, and a liquid injection unit or a liquid injection unit and A discharge part may be formed. Further, similarly to the probe array 1B according to the second embodiment, probe immobilization layers 22G are formed on both surfaces of the base material 21G, and a cover sheet 3G is provided so as to cover each probe immobilization layer 22G. A liquid introduction space 6G may be formed between each probe fixing layer 22G of the probe fixing sheet 2G and the cover sheet 3G.

  The embodiment described above is described for facilitating understanding of the present invention, and is not described for limiting the present invention. Therefore, each element disclosed in the above embodiment is intended to include all design changes and equivalents belonging to the technical scope of the present invention.

  For example, the probe arrays 1 </ b> A to 1 </ b> D according to the above-described embodiment may be heat-sealed only at both edges instead of being heat-sealed at the entire peripheral edge.

  EXAMPLES Hereinafter, although an Example etc. demonstrate this invention further more concretely, the scope of the present invention is not limited to these Examples etc.

[Example 1]
(1) Production of probe fixing sheet A white polyethylene terephthalate film (hereinafter referred to as “PET film”) having a thickness of 50 μm, which is a base material, is cut into a size of 30 mm × 10 mm, and poly L-lysine as a probe fixing material It was immersed in a solution (concentration: 0.01%, solvent: 0.1 × PBS) and shaken for 1 hour. Subsequently, it was washed vigorously 4 times in ultrapure water to wash away excess poly L-lysine. Then, the PET film to which poly-L-lysine was adhered was dried at 60 ° C. for 4 hours in a vacuum oven, and poly-L-lysine (thickness 5 μm) was adhered to the PET film.

  The PET film coated with poly-L-lysine, which is a probe-immobilizing material as described above, was cut into a size of 30 mm × 3 mm, and positive control probe DNA and negative control probe DNA (concentration: 100 pmol) on the coated surface. / ΜL, solvent: 0.2 × SSC) was spotted 1 μL each. In addition, 200-500 mer poly (dA) was used as the positive control probe DNA, and 200-500 mer poly (dT) was used as the negative control probe DNA.

  Next, the spotting solution was dried and irradiated with 600 mJ of ultraviolet rays using a UV crosslinker (UV irradiation device) to obtain a probe fixing sheet having a probe fixing layer on which the probe DNA was fixed.

(2) Preparation of probe array As a cover sheet, a transparent three-layer film (PET: 12 μm / silica vapor deposited PET: 12 μm / cast polypropylene (CPP): 50 μm, 40 mm × 15 mm) having a heat fusion layer made of polypropylene on one side Two sheets were prepared.

  The probe fixing sheet prepared in the above (1) was sandwiched between the two cover sheets with their heat-fusible layers inside. At this time, the probe fixing sheet was arranged at the center of the cover sheet. In this state, the entire peripheral edge of the two cover sheets was heat-sealed with a width of 5 mm, and the end 3 mm of the heat-sealed part was cut. In this way, a probe array as shown in FIG. 1 was obtained.

(3) Hybridization A microsyringe was filled with a hybridization solution containing the target oligonucleotide (target oligonucleotide concentration: 1 pmol / μL, yeast tRNA concentration: 1 μg / μL, solvent: 3 × SSC containing 0.2% SDS). . As the target oligonucleotide, 22-mer poly (dT) in which biotin was bound to the 5 ′ end was used.

  The needle of the microsyringe was punctured at the end of the cover sheet on the probe immobilization layer side of the probe array produced in (2), and the liquid sample was injected into the liquid introduction space of the probe array. The hole formed in the cover sheet by needle puncture was closed with an adhesive tape and heated overnight in a constant temperature bath at 40 ° C.

(4) Post-hybridization washing Take out the probe array from the thermostat, remove the hybridization solution, and wash off the non-specifically adsorbed target oligonucleotide. Remove the adhesive tape and cover the probe-immobilized layer side. A needle of a syringe is punctured at the end of the sheet facing the hole, and washing buffer 1 (2 × SSC, 0.1% SDS) is slowly injected into the liquid introduction space, and then washing buffer 2 (1 × SSC) Was slowly injected, and further washing buffer 3 (0.2 × SSC) was slowly injected, and finally ultrapure water was slowly injected to finish the cleaning operation.

(5) Blocking and detection of target oligonucleotide A blocking solution (1% casein, 3 × SSC) was injected into the solution introduction space of the probe array after washing with a syringe, and blocking was performed at room temperature for 30 minutes. After draining the blocking solution with ultrapure water, a streptavidin / alkaline phosphatase complex solution (stock solution was 0.2 M NaCl, 0.1 M Tris-HCl (pH 7.4), 0.05% Triton-X, 1% casein). The solution was diluted 2000 times with a solution) and reacted at room temperature for 30 minutes.

  After discharging the streptavidin / alkaline phosphatase complex solution from the solution introduction space, a syringe is used in the solution introduction space to remove the streptavidin / alkaline phosphatase complex that did not bind to biotin bound to the target oligonucleotide. Buffer A (0.2M NaCl, 0.1M Tris-HCl (pH 7.4), 0.05% Triton-X) was injected to perform washing, and then Buffer B (0.2M Washing was performed by injecting NaCl, 0.1 M Tris-HCl (pH 7.4)).

  Thereafter, the solution introduction space was filled with a substrate solution (10 mL of buffer B, 9 μL of BCIP (5-bromo-4-chloro-3-indolyl phosphate) solution and 18 μL of NBT (nitroblue tetrazolium) solution), and 3 hours at room temperature. The color reaction was allowed to stand.

  As a result, a clear signal appeared in the portion where the positive control probe DNA that was complementary to the target oligonucleotide was immobilized, but in the portion where the negative control probe DNA that was not complementary to the target oligonucleotide was immobilized. No signal appeared.

[Example 2]
To 100 parts by mass of acrylic resin (manufactured by Nippon Carbide Industries, KP-1374, solid content (acrylic resin content) 33.0% by mass), silica particles (manufactured by Nippon Silica Industry, Nipgel BY-600, solid content) 100% by mass) and 57 parts by mass of ethyl acetate were dispersed to prepare a coating solution. The silica particles have an average secondary particle size (measurement method: Coulter counter method) of 6 μm, a BET specific surface area (measurement method: nitrogen adsorption method) of 450 m ² / g, and a pore volume (measurement method: measurement method: The nitrogen adsorption method) was 1.3 ml / g, and the average pore diameter (measurement method: nitrogen adsorption method) was 10 mm.

  The above coating solution was applied with an applicator on a white 50 μm thick easy-adhesive polyester film (Toyobo Co., Ltd., Cosmo Shine A4300) as a base material, dried at 100 ° C. for 5 minutes, and a thickness of 20 μm. A layer made of a probe-immobilizing material was formed.

  After cutting the easy-adhesive polyester film having the layer made of the probe-immobilized material as described above into a size of 30 mm × 3 mm, the probe DNA is immobilized in the same manner as in Example 1 to obtain a probe-immobilized sheet. It was.

  Using the obtained probe fixing sheet, a probe array was prepared in the same manner as in Example 1, and the target oligonucleotide was detected in the same manner as in Example 1. As a result, a clear signal appeared in the portion where the positive control probe DNA that was complementary to the target oligonucleotide was immobilized, but in the portion where the negative control probe DNA that was not complementary to the target oligonucleotide was immobilized. No signal appeared.

Example 3
A white three-layer film (PET: 12 μm / silica vapor-deposited PET: 12 μm / cast polypropylene (CPP): 50 μm) having a heat fusion layer made of polypropylene on one side was prepared as a substrate for the probe fixing sheet.

  As shown in FIG. 10, a poly L-lysine solution (concentration: 0.01%, solvent: 0.1 × PBS) as a probe-immobilizing material is pattern-coated on the surface of the heat-sealing layer of the substrate as shown in FIG. Then, it was dried in a vacuum oven at 60 ° C. for 4 hours, and poly L-lysine (thickness 5 μm) was adhered to the heat-sealing layer of the base material 21 (reference numeral 22 in FIG. 10 represents a layer made of a probe-immobilized material). Show).

  The three-layer film coated with poly-L-lysine, which is a probe-immobilizing material as described above, is cut into a size of 30 mm × 13 mm as shown in FIG. Was fixed to obtain a probe fixing sheet 2.

  As a cover sheet, a transparent three-layer film (PET: 12 μm / silica vapor-deposited PET: 12 μm / cast polypropylene (CPP): 50 μm, 30 mm × 13 mm) having a heat fusion layer made of polypropylene on one side was prepared. The surface of the probe fixing sheet on the probe fixing layer side is aligned with the surface of the cover sheet on the heat sealing layer side, and both edges extending in the longitudinal direction of both sheets are heat-sealed with a width of 5 mm, and The end part 3 mm of the heat fusion part was cut. In this way, a probe array as shown in FIG. 7 was obtained.

  Using the obtained probe array, the target oligonucleotide was detected in the same manner as in Example 1. However, the inflow and outflow of liquid were performed from the opening part of the probe array which is not heat-sealed. As a result, a clear signal appeared in the portion where the positive control probe DNA that was complementary to the target oligonucleotide was immobilized, but in the portion where the negative control probe DNA that was not complementary to the target oligonucleotide was immobilized. No signal appeared.

Example 4
A white three-layer film (PET: 12 μm / silica vapor-deposited PET: 12 μm / cast polypropylene (CPP): 50 μm) having a heat fusion layer made of polypropylene on one side was prepared as a substrate for the probe fixing sheet.

  The coating solution prepared in the same manner as in Example 2 was pattern-coated on the surface of the heat-fusible layer of the substrate as shown in FIG. 10, dried at 100 ° C. for 5 minutes, and fixed to a probe having a thickness of 20 μm. A layer 22 made of a chemical material was formed.

  As shown in FIG. 11, the three-layer film formed with the probe-immobilizing material layer was cut into a size of 30 mm × 13 mm as described above, and then the probe DNA was immobilized in the same manner as in Example 1. A probe fixing sheet 2 was obtained.

  Using the obtained probe fixing sheet, a probe array was prepared in the same manner as in Example 3, and the target oligonucleotide was detected in the same manner as in Example 1. As a result, a clear signal appeared in the portion where the positive control probe DNA that was complementary to the target oligonucleotide was immobilized, but in the portion where the negative control probe DNA that was not complementary to the target oligonucleotide was immobilized. No signal appeared.

  The present invention is useful for simple production of a probe array and reduction of the amount of liquid sample used.

(A) is sectional drawing of the probe array which concerns on 1st Embodiment, (b) is a top view of the probe array which concerns on the same embodiment. It is a top view of the probe array which concerns on other embodiment. It is a top view of the probe array which concerns on other embodiment. (A) is sectional drawing of the probe array which concerns on 2nd Embodiment, (b) is a top view of the probe array which concerns on the embodiment. (A) is sectional drawing of the probe array which concerns on 3rd Embodiment, (b) is a top view of the probe array which concerns on the same embodiment. (A) is sectional drawing of the probe array which concerns on 4th Embodiment, (b) is a top view of the probe array which concerns on the embodiment. (A) is sectional drawing of the probe array which concerns on 5th Embodiment, (b) is a top view of the probe array which concerns on the same embodiment. (A) And (b) is sectional drawing of the probe array which concerns on 6th Embodiment, (c) is a top view of the probe array which concerns on the embodiment. (A) is sectional drawing of the probe array which concerns on 7th Embodiment, (b) is a top view of the probe array which concerns on the embodiment. It is a top view of the probe fixing sheet before cutting produced in the Example. It is a top view of the probe fixing sheet produced in the Example.

Explanation of symbols

1A, 1A ′, 1A ″, 1B, 1C, 1D, 1E, 1F, 1G... Probe array 2, 2A, 2B, 2C, 2D, 2E, 2F, 2G ... Probe fixing sheets 21, 21A, 21B, 21C, 21D, 21E, 21F, 21G ... base materials 22A, 22B, 22B ', 22C, 22D, 22E, 22F, 22G ... probe immobilization layer 22 ... layers 3A, 3B, 3C made of probe immobilization material, first cover Sheets 3D, 3E, 3F, 3G ... cover sheets 4A, 4B, 4C ... second cover sheets 5A, 5B, 5C, 5D, 5E, 5F, 5G ... heat-sealing portions 6A, 6B, 6B ', 6C, 6D , 6E, 6F, 6G ... Liquid introduction space 7A, 7F ... Liquid injection part 8A, 8F ... Discharge part 9F ... Slit

Claims (8)

A planar probe fixing sheet having a probe fixing layer on one side;
A flat first cover sheet having transparency that can be heat-sealed, located on the probe-fixing layer side of the probe-fixing sheet;
A second cover sheet, which is located on the opposite side of the probe-immobilized layer of the probe-immobilized sheet, and which can be heat-sealed, and
At least both edges of the first cover sheet and the second cover sheet are heat-sealed,
A probe array, wherein a gap constituting a liquid introduction space exists between the probe fixing layer of the probe fixing sheet and the first cover sheet. A planar probe fixing sheet having probe fixing layers on both sides; and
A planar first cover sheet having transparency that can be heat-sealed, located on one surface side of the probe fixing sheet;
A second cover sheet having a transparent shape that can be heat-sealed, located on the other surface side of the probe fixing sheet;
At least both edges of the first cover sheet and the second cover sheet are heat-sealed,
Liquid introduction spaces are formed between the probe fixing layer of the probe fixing sheet and the first cover sheet and between the probe fixing layer of the probe fixing sheet and the second cover sheet, respectively. A probe array characterized by the presence of voids.   The 1st cover sheet and the 2nd cover sheet are heat-sealed in all the peripheral parts except one place, and the part not heat-sealed serves as a liquid injection part. The probe array according to claim 1, wherein the probe array is characterized.   The 1st cover sheet and the 2nd cover sheet are heat-sealed in all the peripheral parts except two places, and the places which are not heat-sealed serve as a liquid injection part and a discharge part. The probe array according to claim 1, wherein the probe array is provided. A planar probe fixing sheet having a probe fixing layer on one side or both sides; and
A flat cover sheet that is located on the probe fixing layer side of the probe fixing sheet and has heat-transparent transparency, and
At least both edges of the probe fixing sheet and the cover sheet are heat-sealed,
A probe array, wherein a gap constituting a liquid introduction space exists between the probe fixing layer of the probe fixing sheet and the cover sheet. A flat first cover sheet having transparency that can be heat-sealed is superimposed on the probe fixing layer side of a flat probe fixing sheet having a probe fixing layer on one side, and the probe fixing sheet is fixed to the probe. On the opposite side of the layer, a flat second cover sheet that can be heat-sealed is overlaid,
At least both edges of the first cover sheet and the second cover sheet are heat-sealed, so that a liquid introduction space is provided between the probe fixing layer of the probe fixing sheet and the first cover sheet. A method of manufacturing a probe array, comprising forming a gap. A planar first cover sheet having transparency that can be heat-sealed is superposed on one surface side of a planar probe fixing sheet having probe fixing layers on both sides, and the other surface side of the probe fixing sheet In addition, a flat second cover sheet having transparency that can be heat-sealed is overlaid,
At least both edge portions of the first cover sheet and the second cover sheet are heat-sealed, so that the probe fixing layer of the probe fixing sheet and the first cover sheet, and the probe fixing sheet A method for producing a probe array, comprising: forming a void that constitutes a liquid introduction space between the probe-immobilized layer and the second cover sheet. On the probe fixing layer side of the flat probe fixing sheet having the probe fixing layer on one side or both sides, a planar cover sheet having transparency that can be heat-sealed is overlaid,
Heat-sealing at least both edges of the probe fixing sheet and the cover sheet, thereby forming a gap that constitutes a liquid introduction space between the probe fixing layer of the probe fixing sheet and the cover sheet. A method for producing a probe array.

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