WO2015147004A1 - Puce analytique - Google Patents

Puce analytique Download PDF

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Publication number
WO2015147004A1
WO2015147004A1 PCT/JP2015/058967 JP2015058967W WO2015147004A1 WO 2015147004 A1 WO2015147004 A1 WO 2015147004A1 JP 2015058967 W JP2015058967 W JP 2015058967W WO 2015147004 A1 WO2015147004 A1 WO 2015147004A1
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WO
WIPO (PCT)
Prior art keywords
substrate
cover
reaction
test substance
groove
Prior art date
Application number
PCT/JP2015/058967
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English (en)
Japanese (ja)
Inventor
黒田　俊彦
北村　義之
Original Assignee
東レ株式会社
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Publication date
Application filed by 東レ株式会社 filed Critical 東レ株式会社
Priority to JP2015518698A priority Critical patent/JPWO2015147004A1/ja
Publication of WO2015147004A1 publication Critical patent/WO2015147004A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • B01L3/5085—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates
    • B01L3/50853—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates with covers or lids
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0689—Sealing
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/14—Process control and prevention of errors
    • B01L2200/141—Preventing contamination, tampering
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/04—Closures and closing means
    • B01L2300/041—Connecting closures to device or container
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B01L2300/0636—Integrated biosensor, microarrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0819—Microarrays; Biochips
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0822—Slides
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0848—Specific forms of parts of containers
    • B01L2300/0854—Double walls

Definitions

  • the present invention relates to an analysis chip having a plurality of reaction units.
  • the analysis chip has a substrate on which a selective binding substance (nucleic acid, protein, lipid, sugar, etc.) that is a substance that selectively binds to a test substance is immobilized, and the selective binding substance on the substrate It is used for subjecting a test substance to a hybridization reaction in a normal solution and analyzing the presence / absence, state or amount of the substance contained in the test substance from the reaction result.
  • a selective binding substance nucleic acid, protein, lipid, sugar, etc.
  • microarray In which molecules such as DNA, proteins, and sugar chains are arranged at high density on a substrate for the purpose of simultaneously measuring many tens to tens of thousands of gene expressions. There is something.
  • Chain detection and quantification are possible, and for example, systematic and comprehensive gene expression analysis in animal models of various diseases and cell biology phenomena can be performed.
  • a DNA microarray (DNA chip) is used for detection / quantification of nucleic acids based on a nucleic acid / nucleic acid hybridization reaction.
  • a DNA chip for example, a DNA chip on which a large number of DNA fragments are aligned and fixed at high density is used.
  • a DNA chip hybridizes on a flat substrate a sample labeled with a gene expressed in a cell to be studied with a fluorescent dye, and binds complementary nucleic acids (DNA or RNA) to each other.
  • DNA chips are highly expected not only in gene expression analysis by detection and quantification of expressed genes but also in application fields such as detection of single nucleotide substitutions (SNPs) of genes.
  • the analysis chip is used not only for nucleic acids such as DNA but also for testing and analyzing proteins and saccharides.
  • proteins such as antibodies, antigens, and enzyme substrates are immobilized on the substrate.
  • Analytical chips such as the above-mentioned DNA chip have been used for research purposes so far, but in recent years, efforts toward realization of testing and diagnosis using genes and proteins have become active.
  • the analysis chip is used for inspection / diagnosis applications, since operation reliability and reproducibility are strictly required, considerable skill is required for manual operation.
  • group medical examinations such as health examinations and medical checkups
  • the number of specimens to be processed becomes enormous, and therefore a system capable of measuring many specimens at a time is essential. Therefore, an analysis chip capable of testing a plurality of specimens with one sheet and an apparatus capable of automatically analyzing the analysis chip are required and are being developed.
  • it is required to reduce the amount of sample used for analysis.
  • the volume of the sample used for analysis is small. It becomes microliter order, and handling of the sample becomes difficult.
  • automated analyzers are very useful in that many samples can be analyzed at once and high reproducibility is obtained.
  • it is difficult to recover when an error occurs during operation. If it cannot be recognized, there is a risk that an erroneous test result may be obtained.
  • an analysis chip having a plurality of reaction units for example, when a sample solution is spilled, there is a risk of mixing into adjacent reaction units and providing an erroneous test result. Therefore, it is desired to develop an analysis chip that can obtain a correct analysis result that is extremely unlikely to cause the above-described error.
  • Patent Document 1 discloses a microwell array including a container in which a plurality of independent wells are arranged in an array and a cover that can cover the container. A small amount of liquid (specimen solution) exceeding the volume of the well is spotted on the well, then the cover is welded and excess liquid is pushed out of the well, and the liquid is sealed so that almost no air remains in the well. These samples and reagents are reacted in a minute space.
  • Patent Document 2 uses unnecessary force or a complicated system to seal the well liquid-tight when heating and stirring the sample in the well of the multi-well plate, and then cooling to open the seal.
  • a pad used to seal a well having an opening on the upper surface of a multi-well plate, such as a microtiter plate, that can be released at will without being disclosed.
  • Patent Document 3 includes a substrate and a cover that can be in close contact with a part of the substrate, the inner surface of the cover is hydrophilic, and the cover has at least one hole therethrough, and the substrate Includes a plurality of concavo-convex structures, and a selective binding substance is immobilized on the upper surface of the convex portion.
  • the multiwell plate cover of Patent Document 2 is detachable.
  • the cover is designed to have a plurality of elastic compressible ridges adjusted so as to be placed on the opening of a multiwell plate such as a microtiter plate in order to facilitate attachment / detachment of the cover, and occurs when the cover is attached / detached. It can be said that the structure is not considered at all with respect to liquid leakage and liquid mixing into adjacent wells.
  • the cover of the microarray of Patent Document 3 is attached to react a test substance and a selective binding substance in a state where a stirring bead is enclosed, and a liquid leak or an adjacent array is removed when the cover is removed. No particular consideration is given to the mixing of the liquid into the liquid.
  • This invention solves the said subject, and it aims at providing the analysis chip
  • the present inventors have intensively studied an analysis chip having a plurality of reaction parts composed of a substrate and a cover, and as a result, provided a groove on at least one of the substrate and the cover, When removing the cover from the substrate by forming a void that surrounds the outside of the opening of the reaction part in a state where the opening of the reaction part containing the solution is covered with a cover to form a closed space
  • the present inventors have found that liquid leakage that frequently occurs and mixing of the test substance solution into the adjacent reaction part does not occur.
  • the present invention includes the following (1) to (5).
  • An analysis chip comprising: A selective binding substance that selectively binds to a test substance is fixed to a bottom surface or a side surface that forms the closed space of at least one of the substrate or the cover, and the closed space is opened in a state where the cover is attached to the substrate.
  • An analysis chip having a groove that forms a surrounding void.
  • analysis chip according to (2) the 1 contact angle of the solution theta containing the test substance on the contact surface, when the substrate or the angle side P 2 forms a contact surface P 1 and the groove of the cover and theta 2, theta 1 and sum of theta 2 is less than 180 °, analysis chip according to (2).
  • the risk of contamination of the test substance solution due to liquid leakage during analysis can be dramatically reduced. Therefore, it becomes possible to accurately detect or quantify the test substance contained in the test substance solution by the analysis using the analysis chip.
  • the risk of contamination of the test substance solution due to liquid leakage can be drastically reduced, which is useful when measuring multiple specimens such as health checkups and medical checkups at once.
  • FIG. 1 The figure which showed an example of the chip
  • A The top view of an example of the board
  • B Sectional drawing of an example of the board
  • C Sectional drawing when a cover is attached to the substrate shown in (a) or (b) when the selective binding substance is immobilized on the substrate.
  • FIG. 1 The figure which showed an example of the chip
  • A The top view of an example of the cover in which the groove
  • B Sectional drawing of an example of the cover in which the groove
  • C Sectional drawing when the cover shown in (a) and (b) is attached to the substrate when the selective binding substance is immobilized on the substrate.
  • (B) The top view of an example of the cover provided with the groove
  • FIG. 6 is a diagram showing an example of an analysis chip used in Examples 1 to 5.
  • A Sectional view when a cover without a groove is mounted on a substrate in which a selective binding substance is immobilized on the upper surface of the convex part and a groove is provided in a shape surrounding the concave opening of the reaction part .
  • B A selective binding substance is immobilized on the upper surface of the convex portion, and the groove is formed in a shape surrounding the opening of the concave reaction portion on the substrate provided with the groove in a shape surrounding the concave opening of the reaction portion. Sectional drawing at the time of mounting
  • a cover provided with a water repellent surface on the contact surface with the cover, and a substrate having a selective binding substance immobilized on the upper surface of the convex portion, and a groove provided in a shape surrounding the concave opening of the reaction portion. Sectional drawing at the time of mounting
  • a substrate provided with a groove in a shape surrounding the concave opening of the reaction part is provided with a convex part on the surface of the concave part provided on the surface, and the selective binding substance is immobilized on the upper surface of the convex part. Sectional drawing at the time of mounting
  • a substrate provided with grooves in a shape surrounding the concave opening of the reaction part is provided with a convex part on the surface of the concave part provided on the surface, and the selective binding substance is immobilized on the upper surface of the convex part.
  • the analysis chip injects a solution containing a test substance (sometimes referred to as a “test substance solution” in this specification) into the chip, the presence or absence of the test substance, and the amount of the test substance. Used to measure the properties of the test substance.
  • a biochip that measures the amount and presence of a test substance by a reaction between the test substance and a selective binding substance immobilized on the surface of the carrier 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 cell carrier Examples thereof include a cell chip immobilized on the surface.
  • the analysis chip used in the present invention includes a substrate and a cover.
  • a selective binding substance is fixed to at least one of the substrate and the cover, and a concave shape into which a solution containing a test substance is injected into the substrate.
  • the reaction part is formed.
  • the concave reaction part forms a space composed of a side surface and a bottom surface, and when a selective binding substance is immobilized on the substrate, it selectively binds to all or part of the bottom surface or side surface of the reaction part. Sexual substances are immobilized.
  • the selective binding substance is immobilized on the cover, the selective binding property is applied to all or part of the surface located in the closed space formed when the concave reaction portion of the substrate is covered with the cover. The substance is immobilized.
  • the analysis chip of the present invention may have one or a plurality of convex portions on the bottom surface or side surface of the concave reaction portion of the substrate, and the selective binding substance may be immobilized on the upper surface of the convex portion.
  • a concave portion is formed on the surface of the cover located in the closed space formed when the concave reaction portion of the substrate is covered with the cover, and one or a plurality of convex portions are provided on the concave plane.
  • the selective binding substance may be immobilized on the upper surface of the convex portion (for example, FIG. 20).
  • the analysis chip with this structure is used for analysis of the test substance, and when detecting the signal, the detection noise is greatly reduced by focusing the scanner on the convex surface where the selective binding substance is immobilized.
  • the signal / noise ratio (S / N ratio) can be increased.
  • the selective binding substance in the present invention means various substances that can selectively bind to a test substance directly or indirectly.
  • Typical examples of the selective binding substance that can bind to the test substance include nucleic acids, proteins, peptides, saccharides, and lipids.
  • the nucleic acid includes DNA and RNA, and may be PNA or LNA.
  • DNA include, but are not limited to, chromosomal DNA, viral DNA, cDNA obtained by reverse transcription of DNA or RNA such as bacteria or mold, and fragments that are a part thereof.
  • RNA messenger RNA, ribosomal RNA, small RNA or micro RNA, fragments that are a part of these, and the like can be used, but are not limited thereto.
  • chemically synthesized DNA or RNA 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.
  • the nucleic acid may be derived from natural products such as living cells, or may be synthesized by a nucleic acid synthesizer.
  • Preparation of DNA or RNA from living cells can be carried out by known methods, for example, DNA extraction by the method of Blin et al. (Blin et al., Nucleic Acids Res. 3: 2303 (1976)), etc. About extraction, it can carry out by the method of Favaroro et al. (Favaroro et al., Methods Enzymol. 65: 718 (1980)).
  • nucleic acid to be immobilized examples include linear or circular plasmid DNA or chromosomal DNA, DNA fragments obtained by cutting or chemically cutting them with restriction enzymes, DNA synthesized by enzymes in a test tube, or chemically synthesized oligonucleotides Etc. can also be used.
  • Examples of the protein include antibody and antigen-binding fragments of antibodies such as Fab fragments and F (ab ′) 2 fragments, 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”.
  • sugars include sugar chains such as various monosaccharides, oligosaccharides, and polysaccharides.
  • the lipid may be a simple lipid or a complex lipid.
  • antigenic substances other than the nucleic acids, proteins, saccharides, and lipids can be immobilized. Moreover, you may fix a cell on the surface of a support
  • DNA, RNA, protein, peptide, sugar, sugar chain, and lipid are particularly preferable.
  • test substance used in the present invention examples include nucleic acids to be measured (target nucleic acids), for example, genes such as pathogenic bacteria and viruses, causative genes of genetic diseases, and parts thereof, various biological components having antigenicity, pathogenic bacteria, Examples include antibodies against viruses and the like, but are not limited thereto.
  • target nucleic acids for example, genes such as pathogenic bacteria and viruses, causative genes of genetic diseases, and parts thereof, various biological components having antigenicity, pathogenic bacteria, Examples include antibodies against viruses and the like, but are not limited thereto.
  • test substance used in the present invention blood, serum, plasma, urine, feces, spinal fluid, saliva, various body fluids such as various tissue fluids, various foods and drinks, and dilutions thereof can be used. It is not limited to these.
  • the nucleic acid as the test substance may be a nucleic acid extracted from blood or cells by a conventional method, labeled with a fluorescent substance, or may be amplified by a nucleic acid amplification method such as PCR using the nucleic acid as a template. Good.
  • 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.
  • 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.
  • 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 test substance labeled with a fluorescent substance by removing the phosphate group at the 5 ′ end of the nucleic acid by alkaline phosphatase, for example is reacted with a selective binding substance
  • a method of measuring the bound label, or a method of measuring the label of the detection probe by capturing a test substance with a selective binding substance (capture probe) and then binding a detection probe labeled with a fluorescent substance to the test substance. (Sandwich hybridization method) is preferably used.
  • FIG. 1 examples of the analysis chip used in the present invention will be described with reference to FIGS. 1 to 11, 15, and 16.
  • the analysis chip used in the present invention includes a substrate 1 and a cover 5 as shown in FIGS. 1 to 6, 15, and 16.
  • the substrate 1 is provided with a concave reaction portion 2.
  • a closed space is formed by the bottom and side surfaces of the reaction unit 2 and the surface of the cover 5 by covering the concave reaction unit of the substrate with the cover, that is, covering the cover with the cover.
  • the closed space refers to a space blocked from the outside, and is a state in which liquid or gas existing in the closed space does not substantially go out of the space.
  • the number of the concave reaction parts 2 provided on the analysis chip can be arbitrarily set, but is preferably plural. For example, 2, 4, 8, 12, 16, 24 36, 48 or 96.
  • a solution containing a test substance is placed in a microtiter plate or the like and dispensed into an analysis chip using, for example, a multi-pipette such as a 4-series, 6-series, 8-series, or 12-series, the reaction part 2 having a concave shape.
  • a multi-pipette such as a 4-series, 6-series, 8-series, or 12-series
  • the reaction part 2 having a concave shape.
  • An analysis chip having a substrate having a plurality of concave reaction portions is useful in a case where a sample containing a plurality of test substances is analyzed at
  • the analysis chip of the present invention is used by injecting a solution containing a test substance into the concave reaction part 2.
  • the shape of the opening of the concave reaction part 2 is not particularly limited.
  • a solution containing a test substance in an amount that does not completely fill the concave reaction part 2 is dropped and sealed with the cover 5 to form a concave shape.
  • the reaction part 2 is stirred as a closed space, it is preferable that the space or bubbles left in the concave reaction part 2 that is not filled with the test substance solution be easy to move.
  • the outer peripheral shape of the bottom surface of the reaction unit 2 is a quadrangle, hexagon, circle, or ellipse because the space or bubbles left in the reaction unit 2 having a concave shape can be easily moved.
  • the outer peripheral shape of the bottom surface of the concave reaction part 2 is a polygon, the corners of the polygon are R-processed, or the concave reaction part not filled with the solution containing the test substance It is preferable because the space or bubbles left in 2 are easy to move.
  • the analysis chip of the present invention is provided with a groove on at least one of the substrate 1 or the cover 5.
  • 1 to 4 show the case where the groove 3 is provided in the substrate 1
  • FIGS. 5, 15 and 16 show the case where the groove 5 is provided in the cover 5, and FIG.
  • channel 10 are each provided is shown, respectively.
  • the groove is provided at a position surrounding a closed space formed in a state where the concave reaction portion 2 of the substrate 1 is covered with the cover 5.
  • the concave reaction portion 2 of the substrate 1 is sealed with a cover 5 to form a closed space.
  • the groove 3 and the cover 5 examples of FIGS. 1 to 4
  • the groove 10 and the substrate 1 examples of FIGS. 5, 15, and 16
  • the groove 3 and the groove 10 are integrated.
  • the gap 8 is formed as a closed space having a shape surrounding the outer periphery of the opening in an annular shape. That is, the concave reaction part becomes a closed space by covering the substrate with the cover.
  • channel becomes a closed space and the space
  • the substrate 1 and the cover 5 are in contact with each other through the respective contact surfaces 6.
  • the selective binding substance that selectively binds to the test substance may be fixed to at least one of the substrate 1 or the cover 5.
  • FIGS. 1 to 4 (b), (c), (e), FIGS. 5 and 15 (c), (e), FIGS. 6 and 16 (c) are selective binding substances to the substrate 1.
  • 1 to 5 and 15 (d) and (f) and FIGS. 6 and 16 (d) show the case where the selective binding substance is immobilized on the cover 5.
  • the selective binding substance is immobilized at an arbitrary position surrounded by the closed space formed by the concave reaction portion 2 and the cover 5 of the substrate 1.
  • At least one of the contact surface of the substrate and the cover in a state where the cover is attached to the substrate may be water-repellent.
  • FIGS. 1 to 4 (e) and (f) are applied to the contact surface of the cover 5
  • FIGS. 5 and 15 (e) and (f) are applied to the contact surface of the substrate 1 for water repellent treatment.
  • the analysis chip on which the water repellent surface 18 is formed is shown.
  • a step 9 may be provided in the opening of the substrate 1 as a liquid level parking structure. It is preferable to provide the step (counterbore) 9 because the overflow of the solution when the cover 5 is attached to the substrate 1 can be effectively prevented.
  • the cover 5 can be attached to and detached from the analysis chip of the present invention. After the reaction such as the hybridization reaction and the antigen-antibody reaction is completed, the cover 5 is detached, and the inside of each reaction part is washed, so that the excess reagent contained in the solution containing the test substance or non-specific binding occurs. The reagent is washed away, and the amount of the substance to be detected can be accurately measured.
  • the analysis may be performed with the cover 5 attached.
  • the opening of the reaction part 2 is covered with a cover to form a closed space.
  • a closed space is formed in the concave reaction portion 2 of the substrate 1.
  • the opening of the reaction unit 2 may be collectively covered with the cover 5 that can cover all the reaction units 2 of the substrate 1, or the reaction unit 2 may be partially covered with the cover that can partially cover the reaction unit 2.
  • a part of the part may be covered.
  • the reaction part 2 After injecting the solution 12 containing the test substance into the reaction part 2, the reaction part 2 is made a closed space, and the test substance and the selective binding substance 4 are reacted by stirring the solution 12 containing the test substance.
  • the reaction at this time is, for example, hybridization when the test substance is a nucleic acid, and antigen-antibody reaction when the test substance is a protein.
  • the injection of the solution 12 containing the test substance into the reaction part 2 of the analysis chip is performed so as to leave a part of the space of the reaction part 2, and the reaction part 2 contains the test substance containing the test substance.
  • a space not filled with 12 is formed.
  • the analysis chip is stirred by a horizontal circular motion or the like to form a concave shape.
  • the space moves in the reaction unit 2, the movement of the solution 12 containing the test substance is promoted, and the reaction rate between the test substance and the selective binding substance 4 can be increased.
  • the analysis chip is agitated by horizontal circular motion or the like, the space formed in the reaction unit 2 may exist as a single space in the reaction unit 2 or may be divided into a plurality of spaces, that is, a plurality of spaces. May exist as bubbles.
  • the proportion of the space not filled with the test substance solution in the entire space of the reaction unit 2 is preferably 10% or more and 90% or less, and 15% or more and 80% or less. More preferably, it is 20% or more and 70% or less. If the proportion of this space is less than 10%, when the analysis chip is stirred, the movement of the test substance solution in the space of the reaction unit 2 becomes insufficient, and the test substance solution may not be substantially stirred. On the other hand, if it exceeds 90%, the chance that the solution containing the test substance comes into contact with the area where the selective binding substance is immobilized decreases, leading to a decrease in the progress of the reaction.
  • the solution containing the test substance adheres to the cover 5 by stirring.
  • a solution containing the test substance may leak out from the gap between the contact surfaces 6 of the substrate 1 and the cover 5 due to capillary action.
  • FIG. 7 shows an example when the analysis chip having no groove on the substrate 1 and the cover 5 is stirred by a horizontal circular motion.
  • FIG. 8 and FIG. 9 show one embodiment of the analysis chip of the present invention.
  • at least one of the substrate 1 and the cover 5 has a groove, and the groove can prevent leakage of liquid due to capillary action.
  • the groove provided in the analysis chip of the present invention preferably has an angle at the boundary line between the contact surface of the substrate and the cover and the side surface of the groove when the cover is mounted on the substrate.
  • the corner in the present invention refers to an intersection line formed by the intersection of two surfaces.
  • the contact surface 6 of FIG. 1 and the side surface 7 of the groove intersect, and when a groove is provided on the cover, the contact surface 6 of FIG. Corners are formed by intersecting the side surfaces 11.
  • the two surfaces may be flat surfaces, curved surfaces, or any combination of a flat surface and a curved surface.
  • the sectional view of the groove has a shape as shown in FIGS.
  • FIGS. 10A to 10F the groove has a corner.
  • FIGS. 10A to 10C show examples in which the side surface of the groove is a curved surface
  • FIGS. 10D to 10G show examples in which the side surface of the groove is a flat surface.
  • a boundary line between the contact surface of the substrate or cover provided with the groove and the side surface of the groove which is a curved surface as shown in FIGS. 10 (a) to 10 (c) or a flat surface as shown in FIGS. 10 (d) to 10 (g).
  • corners are formed.
  • a chip for analysis in which the reaction part is covered with a cover to make a closed space, when the cover is detached from the substrate, a narrow space is formed between the contact surface of the substrate and the cover.
  • Capillary action may occur, liquid may enter the space, and the liquid may leak out of the container.
  • the solution containing the test substance leaks from a narrow gap between the contact surfaces of the substrate and the cover.
  • a groove with a corner is formed on the outer periphery of the reaction portion, and leakage of liquid due to capillary action can be prevented at this corner portion.
  • FIG. 10 (h) when the contact surface of the opening and the side surface of the groove do not intersect, the groove does not have a corner and the opening is rounded. It is not enough to stop the leak.
  • the distance between the contact surfaces of the substrate and the cover becomes small immediately after the reaction portion of the analysis chip substrate is agitated in a state where the reaction portion is covered with a cover and closed, and then the cover is removed from the substrate.
  • the solution containing the test substance enters between the contact surfaces by capillary force, and easily leaks out of the concave reaction part.
  • the degree of leakage of the solution is large, the solution reaches the adjacent reaction part, and as a result, mixing occurs.
  • the angle theta 2 formed by the contact angle theta 1 of the solution containing the test substance, and the side surface P 2 of the contact surface P 1 and the groove of the substrate or the cover Is preferably less than 180 °.
  • Figure 11 represents a contact angle theta 1 of the solution to the contact surface, the side surface P 2 of the contact surface P 1 and the groove of the substrate or the cover and the angle theta 2 formed by the relationship between the leakage of the solution between the contact surfaces Yes.
  • the sum of ⁇ 1 and ⁇ 2 is 180 ° or more, so that the leakage of the solution due to the capillary force cannot be stopped, and the liquid leaks into the groove tends to occur.
  • the sum of ⁇ 1 and ⁇ 2 is less than 180 °, and liquid leakage to the groove due to capillary force can be prevented.
  • ⁇ 2 is preferably 120 ° or less, and more preferably 110 ° or less. More preferably, it is 100 degrees or less. Further, it can be said that the smaller the value of ⁇ 2 is, the higher the effect of preventing the liquid leakage due to the capillary force is.
  • ⁇ 2 is about 90 °.
  • ⁇ 1 is preferably 90 ° or less.
  • the contact surface of the substrate and / or cover of the analysis chip of the present invention is preferably water repellent.
  • the contact angle of the solution containing the test substance with respect to the contact surface is increased, and leakage due to capillary action can be more reliably prevented.
  • the water repellency is simply the property of repelling water, and can be expressed quantitatively by, for example, the contact angle of water.
  • the contact angle is a quantification of the degree of surface wetting, such that a clean glass surface wets well with water, while a fluorine-coated surface repels water.
  • the width-height method ( ⁇ / 2 method) is generally used as a method for measuring the contact angle. Assuming that the shape of the minute droplet is a part of a sphere, if the radius of the droplet 14 is r and the height is h as shown in FIG. 13, the relationship with the contact angle ⁇ is is there.
  • the contact angle ⁇ can be calculated by the following formula 3.
  • Contact angle can be measured using a dedicated contact angle meter, for example, manufactured and sold by Kyowa Interface Chemical Co., Ltd., Cruz (Germany), and the like.
  • the contact angle of the contact surface of the substrate or cover is preferably 60 ° or more, more preferably 70 ° or more, and further preferably 80 ° or more. More preferably, it is 90 ° or more.
  • the substrate or cover having a water repellent contact surface can be obtained, for example, by a method of manufacturing the substrate or cover itself with a water repellent material or a method of covering the contact surface of the substrate or cover with a water repellent material.
  • a material having high water repellency for example, tetrafluoroethylene (PTFE), tetrafluoroethylene / verfluoroalkyl vinyl ether copolymer (PFA), tetrafluoride.
  • PTFE tetrafluoroethylene
  • PFA verfluoroalkyl vinyl ether copolymer
  • a substrate or a cover may be manufactured using a fluorine compound such as ethylene / hexafluoropropylene copolymer (FEP) or silicone.
  • FEP ethylene / hexafluoropropylene copolymer
  • the method of coating the contact surface with a water repellent material includes, for example, a method of attaching a material having a water repellent surface to the contact surface of the substrate or cover, a method of coating the contact surface of the substrate or cover with the water repellent material, etc. Is included.
  • a commercially available adhesive tape whose surface is a fluorine compound may be used.
  • 903UL / No. 9030UL Nonto Denko
  • fluororesin film adhesive tape No. 8410 Teeraoka Seisakusho
  • fluororesin adhesive tape ASF-110FR (Chuko Kasei Kogyo), etc.
  • ASF-110FR Cho Kasei Kogyo
  • a commercially available coating agent capable of imparting water repellency is spray coating, dip coating, dip spin coating, roll coating, spin flow coating, What is necessary is just to coat by the coating using a brush or a brush.
  • paint processing agents that can impart water repellency include AsahiGuard E-SERIES (Asahi Glass), Novec (TM) high-functional coating agent (Sumitomo 3M), SIFEL2000 series for adhesion and coating, and fluorine-based antifouling additive KY.
  • FG-5040 and FG-4010 Each series (Fluorotechnology) of FG-5040 and FG-4010 is preferably used.
  • Various water-repellent coating agents for automobiles may be used. Further, a method of imparting a fine structure imitating the surface of a lotus leaf to the surface by coating is also preferably used.
  • the opening is covered with a cover, and the analysis chip is moved by rotation, vibration, or a combination thereof, thereby stirring the solution.
  • the rotational motion includes horizontal circular motion in which the analysis chip itself rotates around the rotational axis by circular motion or elliptical motion, revolving motion that revolves around the rotational axis outside the analytical chip, and a combination of rotation and revolution. For example, revolving movement.
  • the vibration a method of vibrating the analysis chip itself or the test substance solution with an ultrasonic vibrator or a piezoelectric element is used.
  • the horizontal circular motion of the present invention refers to a rotational mode that is performed so that circular motion of the same radius having each unique center of rotation is made at any point on the analysis chip.
  • FIG. 14 shows an example of the horizontal circular motion.
  • point A rotates on a circular orbit having a radius r centered on OA at a predetermined number of revolutions, and point B similarly has a radius r centered on OB.
  • a straight line AB connecting arbitrary points A and B on the analysis chip 17 is always parallel in an arbitrary trajectory of circular motion.
  • the straight line AB is parallel even when the analysis chip 17 is located at any one of the position P1, the position P2, the position P3, and the position P4.
  • the analysis chip When the analysis chip is moved in a horizontal circular motion, the analysis chip is preferably installed so that the surface on which the selective binding substance is immobilized is parallel or substantially parallel to the rotation surface.
  • the solution in each reaction part can be stirred under the same conditions by horizontally moving, so that the selective binding substance in each reaction part And the test substance can be performed under the same conditions, and variation in reaction between reaction parts can be reduced.
  • the revolution method in which the analysis chip is revolved while rotating, or by the revolution method in which the rotation center is outside the analysis chip the plurality of concave reaction parts are agitated under different conditions. As a result, there may be a variation in reaction between the concave reaction parts.
  • the direction of the rotation surface when the analysis chip is horizontally moved is not particularly limited.
  • the horizontal or substantially horizontal direction the direction inclined 15 degrees from the horizontal direction, the direction inclined 30 degrees from the horizontal direction, or 45 degrees from the horizontal direction.
  • An inclined direction, a direction inclined by 60 degrees from the horizontal direction, a direction inclined by 75 degrees from the horizontal direction, a vertical or substantially vertical direction, and the like can be used.
  • a preferable direction of the rotation surface is horizontal or substantially horizontal.
  • the substantially horizontal direction means a direction close to horizontal toward the surface on which the selective binding substance of the analysis chip is immobilized, for example, a direction inclined in a range of 0 to 3 degrees with respect to the horizontal plane. Is preferred.
  • substantially vertical direction means a direction that is nearly perpendicular to the surface on which the selective binding substance of the analysis chip is immobilized, for example, a direction tilted in the range of 0 to 3 degrees with respect to the vertical surface. Is preferred.
  • the horizontal circular motion of the analysis chip may be performed intermittently, for example, by changing the rotational speed even if the rotational speed is constant, or by stopping for a certain time during the horizontal circular motion.
  • the direction of rotation is not particularly limited, and may be clockwise, counterclockwise, or a combination thereof.
  • the time for the horizontal circular movement during the reaction is not particularly limited, and can be appropriately determined within a range sufficient for the selective binding substance and the test substance to react.
  • the test substance when it is a nucleic acid, it can be set according to the time required for the hybridization reaction with the probe nucleic acid that is a selective binding substance.
  • the solution stirring method of the present invention by applying a centrifugal acceleration of 1 ⁇ g or more during horizontal circular motion, the selective reaction between the test substance and the selective binding substance is effectively promoted, and the test substance is shortened. It has a feature that can be detected or quantified in time.
  • the time for horizontal circular movement is preferably short.
  • the reaction time is preferably 3 hours or more and 4 hours or less, more preferably 2 hours or less, further preferably 1 hour or less, and 0.5 hours or less. It is particularly preferred.
  • centrifugal acceleration is the amount of centrifugal force applied to an object in the form of rotational motion, expressed in the form of acceleration, and is the absolute value of the distance from the center of rotation and the square of the angular velocity of rotational motion.
  • Proportional. Centrifugal acceleration in the present invention refers to centrifugal force, that is, relative centrifugal acceleration (RCF), and is calculated by the following equation (4).
  • RCF 1118 ⁇ R ⁇ N 2 ⁇ 10 ⁇ 8 (Formula 4)
  • RCF Relative centrifugal acceleration ( ⁇ g)
  • R radius of rotation (cm)
  • N Number of rotations (rpm).
  • centrifugal acceleration When the analysis chip of the present invention is moved in a horizontal circular motion, it is preferable to give a centrifugal acceleration of 1 ⁇ g or more.
  • the lower limit of the centrifugal acceleration is preferably 5 ⁇ g or more, more preferably 10 ⁇ g or more.
  • the upper limit of the centrifugal acceleration is not particularly limited, but is preferably 50 ⁇ g or less, more preferably 40 ⁇ g or less, and further preferably 30 ⁇ g or less.
  • the range of centrifugal acceleration is preferably 1 ⁇ g to 50 ⁇ g, more preferably 5 ⁇ g to 40 ⁇ g, and still more preferably 10 ⁇ g to 30 ⁇ g.
  • the target centrifugal acceleration can be given by appropriately setting the rotation speed and the rotation radius. Therefore, the number of rotations and the radius of rotation can be selected according to the specifications of the stirring device for stirring the analysis chip. For example, when the rotation radius is small, a large centrifugal acceleration can be applied by increasing the rotation speed.
  • Rotational radius can be selected as appropriate to obtain a desired centrifugal acceleration in combination with the rotational speed.
  • Preferable radius ranges are 0.1 mm or more and less than 20 mm, 0.2 mm or more and less than 19 mm, 0.3 mm or more and less than 18 mm, 0.4 mm or more and less than 17 mm, and 0.5 mm or more and less than 16 mm.
  • the radius of rotation is larger than 20 mm, the centrifugal force becomes dominant, and the tendency that the space not filled with the test substance solution is pressed against the outer periphery of the concave reaction part becomes strong, and the stirring efficiency is reduced. Stirring unevenness may occur.
  • the rotation speed of the horizontal circular motion can be appropriately selected as a value that can obtain a desired centrifugal acceleration in combination with the rotation radius.
  • the preferable range of the rotational speed of the horizontal circular motion is, for example, 500 rpm to 10000 rpm, 600 rpm to 9000 rpm, 700 rpm to 8000 rpm, 800 rpm to 7000 rpm, 900 rpm to 6000 rpm, 1000 rpm to 5000 rpm.
  • a smaller radius of rotation is preferable in that the reaction apparatus and the stirring apparatus can be miniaturized and the apparatus embodying the solution stirring method of the present invention can be made compact.
  • the stirrer for stirring the analysis chip of the present invention is not particularly limited as long as it can give a centrifugal acceleration of 1 ⁇ g or more by a combination of the rotational speed of the horizontal circular motion and the rotational radius.
  • a plate shaker can be suitably used.
  • the device is preferably a device that can control the rotational speed, drive time, etc. of the horizontal circular motion from the outside.
  • the substrate or cover of the analysis chip of the present invention is located on the bottom or side surface of the concave reaction part of the substrate or in a closed space formed when the concave reaction part of the substrate is covered with the cover.
  • One or a plurality of convex portions for immobilizing the selective binding substance may be provided on the surface of the cover.
  • the heights of the plurality of convex portions are preferably substantially the same.
  • the height of the convex part can be set arbitrarily within the range where the reaction part can form a closed space with the cover attached to the substrate and does not exceed the height (depth) of the reaction part (closed space). Can do.
  • the detection noise can be greatly increased by focusing the scanner on the top of the convex part on which the selective binding substance is immobilized.
  • the signal / noise ratio (S / N ratio) can be increased.
  • the material of the substrate and cover used in the analysis chip of the present invention is not particularly limited.
  • the materials preferably used are various polymers such as silicone rubber, such as glass, ceramic, or silicone resin, polyethylene terephthalate, cellulose acetate, polycarbonate, polystyrene, polymethyl methacrylate (PMMA), polyolefin, polydimethylsiloxane (PDMS) elastomer. is there.
  • polymethyl methacrylate, polystyrene, polydimethylsiloxane (PDMS) elastomer, glass, or silicone resin can be preferably used.
  • the substrate or cover of the analysis chip of the present invention is preferably made of a material that can reduce autofluorescence when the selective binding substance is immobilized.
  • a material that can reduce autofluorescence when the selective binding substance is immobilized is black.
  • the substrate and cover constituting the analysis chip of the present invention can be manufactured by various known manufacturing methods.
  • the material when the material is a polymer or the like, it can be produced by injection molding, cutting, hot embossing, polymerization in a mold, or the like.
  • the material when the material is an inorganic substance such as glass or ceramic, it can be manufactured by a sandblast method, and when it is a silicone resin, it can be manufactured by a known semiconductor process.
  • the ridge line portion may be chamfered (so-called thread chamfering), but the substrate or cover of the analysis chip of the present invention has C0.2 or less. C chamfering or R chamfering of R0.2 or less may be applied.
  • the substrate or cover produced as described above can be subjected to various surface treatments as necessary prior to immobilizing the selective binding substance on the surface.
  • Examples of such surface treatment include those described in JP-A No. 2004-264289.
  • an S / N ratio (signal-to-noise ratio) can be used as an index indicating the sensitivity of signal detection.
  • S / N 2 as a detection limit.
  • concentration or amount of a test substance with an S / N ratio of 2 to 3 is adopted as the detection limit. If the S / N is 2 or more, the detection is more reliable than the detection limit. It can be judged (for example, Makoto Niwa, “If you know this, statistical methods for chemistry-how to handle correct data”, 2008, Chemistry Doujin, page 101).
  • the analysis chip of the present invention can efficiently advance the selective reaction between the immobilized selective binding substance and the test substance, and can detect or quantify the test substance in a short time.
  • the reaction time which conventionally required a reaction time of 6 to 20 hours can be greatly shortened. Therefore, for example, in the examination / diagnosis area (for example, infectious diseases such as influenza and sepsis examination / diagnosis, etc.) that require rapid analysis of a large number of specimens, and in the examination center, a vast number of specimens are processed. In this case, it is preferable to apply the analysis chip of the present invention.
  • Example 1 Preparation of analysis chip substrate
  • Substrate B1 (FIGS. 18 (a) and 18 (b)) having a convex portion on the bottom surface of the concave reaction portion and not having a groove on the outer peripheral portion, and a substrate having a groove on the outer peripheral portion as follows.
  • A (FIGS. 18C and 18D) were respectively produced.
  • Two types of molds for injection molding are prepared using a known method of LIGA (Lithographie Galvanforming Abforming), and a substrate made of polymethylmethacrylate (PMMA) having a shape as described later is obtained by the injection molding method. It was.
  • the average molecular weight of PMMA used was 50,000, and carbon black (# 3050B manufactured by Mitsubishi Chemical) was contained in PMMA at a ratio of 1% by weight to make the substrate black.
  • the spectral reflectance and spectral transmittance of this black substrate were measured, the spectral reflectance was 5% or less at any wavelength in the visible light region (wavelength 400 nm to 800 nm), and in the same range of wavelengths, The transmittance was 0.5% or less.
  • Spectral reflectance is the spectral reflectance when specularly reflected light from the substrate is captured using a device (Minolta Camera, CM-2002) equipped with an illumination / light-receiving optical system that conforms to JIS Z 8722 Condition C. was measured.
  • a substrate 1 having an outer shape of 76 mm in length, 26 mm in width, and 2.5 mm in thickness was produced by injection molding.
  • the substrate 1 is provided with twelve elliptical concave reaction portions 2 having a major axis of 4.8 mm, a minor axis of 2.40 mm, and a depth of 1.5 mm, and each reaction unit has a diameter of 0.1 mm and a height of 0. 96 projections 19 of 12 mm were provided.
  • the volume of the reaction part of the substrate was about 13.5 ⁇ L. Further, the variation in the height of the upper surface of the convex portion was 3 ⁇ m or less. Moreover, the pitch of the convex part was 0.18 mm.
  • the substrate 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 carboxy groups on the substrate surface.
  • an elliptical groove 3 having a rectangular cross section of 0.5 mm in width and 0.5 mm in depth was formed on the outer periphery of each reaction part by cutting. This was used as the substrate A (FIGS. 18C and 18D). Moreover, the board
  • a selective binding substance was immobilized on each of the substrate A and the substrate B1 by the following method.
  • a selective binding substance capture probe
  • the nucleotide sequence SEQ ID NO: 1 described in the literature (J. Clin. Microbiol, 1995. p. 901-905) reported in the research on type discrimination of human papillomaviruses.
  • This oligonucleotide was dissolved in pure water to a concentration of 0.3 nmol / ⁇ L to obtain a stock solution.
  • PBS 8 g NaCl, 2.9 g Na 2 HPO 4 ⁇ 12H 2 O, 0.2 g KCl, and 0.2 g KH 2 PO 4 were combined. And then diluted to 1 L, adjusted to pH 5.5 by adding hydrochloric acid) to 10-fold dilution to a final concentration of probe DNA of 0.03 nmol / ⁇ L.
  • the molded product was removed from the mold, and molded articles of cover A and cover B1 having outer dimensions of 76 mm in length, 26 mm in width, and 2 mm in thickness were obtained.
  • the groove of the cover A was set on the substrate A, it was confirmed that the groove was located at the same location.
  • the contact angle of water on the surfaces of the cover A and the cover B1 was 90 °.
  • Example 1 an analysis chip (illustrated in FIG. 21A) composed of a substrate A in which a groove is formed on the outer periphery of the reaction part and a cover B1 in which no groove is formed is used.
  • the hybridization operation was carried out 20 times as described above.
  • test substance solution As a test substance, a recombinant plasmid (pHPV16 (16,600 base pairs in total length)) cloned from human papillomavirus genomic DNA purchased from the Human Science Research Resource Bank was fragmented by ultrasound.
  • MY11 SEQ ID NO: 2; complementary to the 50th to 69th base sequence on the 5 'end side with reference to the base position of the 5' end when the test substance binds to the capture probe
  • GP5 SEQ ID NO: 3; similar to MY11, complementary sequence to the 10th to 34th nucleotide sequence on the 5 ′ end side
  • GP6 SEQ ID NO: 4; when the test substance binds to the capture probe
  • MY09 SEQ ID NO: 5; similar to GP6, the 3 ′ end side 340-359 th position
  • the sequences complementary to the base sequence) were synthesized with biotin-labeled 3 ′ end and 5 ′ end, respectively. These were diluted with sterilized water to a concentration of 100 fmol to obtain each detection probe
  • Hybridization operation In order to confirm the presence or absence of liquid leakage and contamination in the adjacent reaction part due to the hybridization operation, the following operation was performed.
  • the detection probe solution was added to and mixed with the solution containing the test substance, 10 ⁇ L was dropped onto 6 reaction parts of the substrate A, the cover B1 was set, and the reaction part was covered.
  • the mixture was set in a stirrer (rotation radius: 2 mm, rotation speed: 2000 rpm) installed in an oven adjusted to a temperature of 32 ° C. and stirred for 2 hours.
  • liquid leakage when the cover was removed and the presence or absence of the solution in the adjacent reaction part were evaluated by the following operation.
  • the liquid leakage is a state in which the solution leaks to the outside from the reaction portion at a predetermined position, but remains on the contact surface of the substrate, and the mixing refers to a solution that leaks to the outside from the reaction portion at a position other than the predetermined position.
  • the state which flowed into the reaction part which adjoins each is pointed out. According to 20 evaluations, “good” indicates that neither liquid leakage nor mixing occurred once, “possible” indicates that liquid leakage occurred but never mixed, and “when mixed” occurs even once.
  • the signal intensity (fluorescence intensity) of each reaction spot was measured using a high-resolution fluorescence detector (Toray Industries, Inc .; “3D-Gene (registered trademark) Scanner”).
  • the signal intensity of the spot (20 spots) where the selective binding substance is immobilized, for a total of 12 places including 6 reaction parts into which the solution containing the test substance was injected and 6 reaction parts into which the test substance was not injected.
  • Each blank signal intensity was read, and each average value and S / N ratio were calculated.
  • the results are shown in Table 1.
  • the S / N ratio in the reaction part injected with the solution containing the test substance was 5.2, while the S / N ratio in the reaction part not injected with the solution containing the test substance was 1.0. Therefore, it was confirmed that the test substance was correctly detected.
  • Example 2 Hybridization operations were performed 20 times using an analysis chip (illustrated in FIG. 21B) composed of a substrate A having grooves formed on the outer periphery of the reaction part and a cover A having grooves.
  • Example 2 The hybridization operation was carried out 20 times in the same manner as in Example 1, and the liquid leakage and the presence / absence of contamination during cover removal were evaluated. As a result, as shown in Table 1, liquid leakage occurred three times, but no mixing occurred.
  • Example 3 Hybridization operations were performed 20 times using an analysis chip (illustrated in FIG. 21 (c)) composed of the substrate B1 in which no groove was formed on the outer periphery of the reaction part and the cover A in which the groove was formed. .
  • the hybridization operation was carried out 20 times in the same manner as in Example 1, and the liquid leakage and the presence / absence of contamination during cover removal were evaluated. As a result, as shown in Table 1, although liquid leakage occurred twice, no mixing occurred.
  • Example 4 On the contact surface of the cover B1, nitoflon no. 903UL (Nitto Denko) was attached to give a water repellent surface. At this time, the contact angle of the water repellent surface was 110 °.
  • This cover is referred to as a cover B2.
  • Hybridization operations were performed 20 times using an analysis chip (illustrated in FIG. 21 (d)) composed of a substrate A having a groove formed on the outer periphery of the reaction portion and a cover B2.
  • Example 2 The hybridization operation was carried out 20 times in the same manner as in Example 1, and the liquid leakage and the presence / absence of contamination during cover removal were evaluated. As a result, as shown in Table 1, neither liquid leakage nor mixing occurred.
  • Example 5 Nittofuron No. which cut out the part corresponding to the reaction part. 903UL was affixed on the contact surface of the substrate B1 to give a water repellent surface. At this time, the contact angle of the water repellent surface was 110 °.
  • This substrate is referred to as a substrate B2.
  • Hybridization operations were performed 20 times using an analysis chip (illustrated in FIG. 21 (e)) composed of the substrate B2 and the cover A having grooves.
  • Example 2 The hybridization operation was carried out 20 times in the same manner as in Example 1, and the liquid leakage and the presence / absence of contamination during cover removal were evaluated. As a result, as shown in Table 1, neither liquid leakage nor mixing occurred.
  • Comparative Example 1 Using an analysis chip (illustrated in FIG. 17A) composed of a substrate B1 in which no groove was formed on the outer periphery of the reaction part and a cover B1 in which no groove was formed, the hybridization operation was performed 20 times. Carried out.
  • the hybridization operation was carried out 20 times in the same manner as in Example 1, and the liquid leakage and the presence / absence of contamination during cover removal were evaluated. As a result, as shown in Table 1, liquid leakage and contamination occurred in all 20 operations.
  • Example 1 For one example in which contamination occurred, the operation of reading a fluorescent label and a fluorescent signal was carried out in the same manner as in Example 1. As a result, as shown in Table 1, the S / N ratio in the reaction part into which the solution containing the test substance was injected was 4.1, while the reaction part in which the solution containing the test substance was not injected. The S / N ratio was 2.0, indicating that the test substance was erroneously detected.
  • Comparative Example 2 Hybridization using an analysis chip (illustrated in FIG. 17 (b)) composed of a substrate B2 provided with water repellency without forming grooves on the outer periphery of the reaction part and a cover B1 without grooves. The operation was performed 20 times.
  • the hybridization operation was carried out 20 times in the same manner as in Example 1, and the liquid leakage and the presence / absence of contamination during cover removal were evaluated. As a result, as shown in Table 1, liquid leakage occurred 20 times out of 20 operations, and mixing occurred 15 times.
  • Example 1 For one example in which contamination occurred, the operation of reading a fluorescent label and a fluorescent signal was carried out in the same manner as in Example 1. As a result, as shown in Table 1, the S / N ratio in the reaction part into which the solution containing the test substance was injected was 4.0, while in the reaction part into which the solution containing the test substance was not injected. The S / N ratio was 2.3, indicating that the test substance was erroneously detected.
  • Comparative Example 3 Hybridization using an analysis chip (illustrated in FIG. 17 (c)) composed of a substrate B1 in which no groove is formed on the outer periphery of the reaction part and a cover B2 which has no groove and is provided with water repellency The operation was performed 20 times.
  • the hybridization operation was carried out 20 times in the same manner as in Example 1, and the liquid leakage and the presence / absence of contamination during cover removal were evaluated. As a result, as shown in Table 1, liquid leakage and contamination occurred in all 20 operations.
  • Example 1 For one example in which contamination occurred, the operation of reading a fluorescent label and a fluorescent signal was carried out in the same manner as in Example 1. As a result, as shown in Table 1, the S / N ratio in the reaction part into which the solution containing the test substance was injected was 4.0, while in the reaction part into which the solution containing the test substance was not injected. The S / N ratio was 2.4, indicating that the test substance was erroneously detected.
  • Example 6 Preparation of analysis chip substrate
  • a substrate X (FIGS. 19A and 19B) in which a groove was provided in a shape surrounding the concave reaction portion shown in FIG. 19 and a substrate Y in which no groove was provided in the outer peripheral portion were produced. .
  • a silicone rubber substrate 1 having an outer shape of 76 mm in length, 26 mm in width, and 2 mm in thickness was produced by liquid silicone rubber injection molding (LIM).
  • the substrate was provided with 24 reaction portions 2 each having a concave side with a rectangular bottom and a long side of 7.8 mm, a short side of 3.3 mm, and a depth of 0.5 mm.
  • a substrate having a groove formed on the outer periphery of the reaction portion by forming a convex structure having a width of 0.4 mm and a height of 0.4 mm surrounding the outer periphery of each reaction portion was designated as substrate X.
  • FIG. 19A shows a top view of the substrate X
  • FIG. 19B shows a top view and a cross-sectional view of the concave reaction portion of the substrate X.
  • substrate Y a substrate in which no groove was formed on the outer periphery of the concave reaction portion
  • a cover Y1 (FIGS. 20A and 20B) having a recess formed on the cover surface, having a protrusion on the recess plane, and having no groove on the outer periphery of the recess is produced as follows. did.
  • a black PMMA cover 5 having a length of 76 mm, a width of 26 mm, and a thickness of 1 mm was produced by injection molding in the same manner as the analysis chip substrate of Example 1. Twenty-four rectangular recesses 20 having a long side of 7.2 mm, a short side of 2.7 mm, and a depth of 0.12 mm are provided, and a convex part 19 having a diameter of 0.1 mm and a height of 0.12 mm is formed on the plane of each recess 20. 300 places at a pitch of 0.17 mm. The variation in the height of the upper surface of the convex portion was 3 ⁇ m or less.
  • a cover Y1 was prepared by spotting a selective binding substance (capture probe) on a convex portion and immobilizing it in the same manner as in Example 1.
  • FIG. 20A shows a top view of the cover Y1
  • FIG. 20B shows a top view and a sectional view of the concave portion of the cover Y1.
  • a water repellent (FS-1060C (Fluoro Technology) was applied to the contact surface of the cover Y1 so as to surround each concave portion of the cover Y1, thereby providing a water repellent surface.
  • This cover is referred to as a cover Y2.
  • the water contact angle of the water repellent surface of the cover Y2 was 115 °.
  • Example 6 an analysis chip comprising a substrate X having a groove formed on the outer periphery of the reaction portion and a cover Y1 not formed with a groove and not subjected to water repellency treatment (FIG. 22A) And the hybridization operation was performed 20 times.
  • Example 2 The hybridization operation was carried out 20 times in the same manner as in Example 1, and the liquid leakage and the presence / absence of contamination during cover removal were evaluated. As a result, as shown in Table 1, liquid leakage occurred once, but no mixing occurred.
  • Example 7 Hybridization using an analysis chip (illustrated in FIG. 22 (b)) composed of a substrate X having grooves formed on the outer periphery of the reaction part and a cover Y2 having no grooves and water-repellent treatment. The operation was performed 20 times.
  • Example 2 The hybridization operation was carried out 20 times in the same manner as in Example 1, and the liquid leakage and the presence / absence of contamination during cover removal were evaluated. As a result, as shown in Table 1, neither liquid leakage nor mixing occurred.
  • Comparative Example 4 Using an analysis chip (illustrated in FIG. 17D) composed of a substrate Y in which no groove is formed on the outer periphery of the reaction part and a cover Y1 that is not formed with a groove and is not subjected to water repellent treatment. The hybridization operation was performed 20 times.
  • the hybridization operation was carried out 20 times in the same manner as in Example 1, and the liquid leakage and the presence / absence of contamination during cover removal were evaluated. As a result, as shown in Table 1, liquid leakage and contamination occurred in all 20 operations.
  • Example 1 For one example in which contamination occurred, the operation of reading a fluorescent label and a fluorescent signal was carried out in the same manner as in Example 1. As a result, as shown in Table 1, the S / N ratio in the reaction part into which the solution containing the test substance was injected was 4.2, whereas in the reaction part into which the solution containing the test substance was not injected, The S / N ratio was 2.2, indicating that the test substance was erroneously detected because contamination occurred.
  • the analysis chip of the present invention can accurately detect or quantify a test substance. Therefore, the present invention enables diagnosis and diagnosis of diseases by measuring markers of genes, proteins, etc. at clinical sites and inspection centers, and can obtain highly reliable data especially in measurement using an automated device. Very useful.

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Abstract

　L'invention est motivée par le fait qu'avec une puce analytique conventionnelle ayant des parties de réaction multiples, il existe un problème en ce que, lorsque le couvercle est enlevé du substrat après la réaction, la solution contenant l'analyte peut s'écouler dans des parties de réaction adjacentes, conduisant à un mélange. En particulier, lorsque des puces analytiques sont utilisées dans des applications de test/diagnostic, il existe un risque d'obtenir des résultats de test/diagnostic significativement affectés, tels que l'occurrence de faux positifs et similaire. Selon la présente invention, le substrat et/ou le couvercle qui constituent la puce analytique sont pourvus de rainures qui, lorsque la pluralité de parties de réaction dans lesquelles une solution contenant un analyte a été introduite sont scellées par le couvercle de façon à produire un espace étanche, forment des espacements ayant une forme entourant l'extérieur des ouvertures des parties de réaction, de manière à éviter la fuite ou le mélange entre des parties de réaction adjacentes, ce qui se produit fréquemment lorsque le couvercle est retiré du substrat.
PCT/JP2015/058967 2014-03-26 2015-03-24 Puce analytique WO2015147004A1 (fr)

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JP2018014961A (ja) * 2016-07-29 2018-02-01 川崎重工業株式会社 薬液交換装置
WO2021162028A1 (fr) * 2020-02-14 2021-08-19 東レ株式会社 Procédé de production de biopuces
US11513076B2 (en) 2016-06-15 2022-11-29 Ludwig-Maximilians-Universität München Single molecule detection or quantification using DNA nanotechnology

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Publication number Priority date Publication date Assignee Title
US11513076B2 (en) 2016-06-15 2022-11-29 Ludwig-Maximilians-Universität München Single molecule detection or quantification using DNA nanotechnology
JP2018014961A (ja) * 2016-07-29 2018-02-01 川崎重工業株式会社 薬液交換装置
WO2021162028A1 (fr) * 2020-02-14 2021-08-19 東レ株式会社 Procédé de production de biopuces

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