WO2018034109A1 - Inspection method and inspection device - Google Patents

Inspection method and inspection device Download PDF

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Publication number
WO2018034109A1
WO2018034109A1 PCT/JP2017/026705 JP2017026705W WO2018034109A1 WO 2018034109 A1 WO2018034109 A1 WO 2018034109A1 JP 2017026705 W JP2017026705 W JP 2017026705W WO 2018034109 A1 WO2018034109 A1 WO 2018034109A1
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WIPO (PCT)
Prior art keywords
inspection
chip
pores
test
solution
Prior art date
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PCT/JP2017/026705
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French (fr)
Japanese (ja)
Inventor
孝明 伊藤
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富士フイルム株式会社
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Publication of WO2018034109A1 publication Critical patent/WO2018034109A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals

Definitions

  • the present invention relates to a test method and a test apparatus for detecting an antigen as a test substance, an antibody, deoxyribonucleic acid and the like.
  • the binding phenomenon between a test substance and a capture substance is optically detected as one of the methods for examining a specific binding reaction such as an enzymatic reaction, nucleic acid hybridization, or an antigen-antibody reaction.
  • Methods for detecting are known.
  • a test substance is bound to a capture substance fixed at a predetermined position, a label which emits fluorescence upon receiving excitation light, or a label which catalyzes a reaction of a substrate to generate color, fluorescence or chemiluminescence, etc. Is applied to the test substance to detect light generated due to such a label.
  • a method of attaching a fluorescent label to a binding substance such as an antibody that specifically binds to a test substance and detecting fluorescence generated from the fluorescent label, or a binding such as an antibody that specifically binds to a test substance
  • a binding substance such as an antibody that specifically binds to a test substance
  • Methods for labeling substances with enzymes and reacting them using these enzymes as catalysts, chromogenic substrates, fluorescent substrates, methods for detecting color development, fluorescence, or chemiluminescence generated from chemiluminescent substrates, etc. are known. Identification is possible.
  • Japanese Patent Application Publication No. 9-504864 describes that a means for providing fluid flow in the pores of a biochip comprising a porous substrate and a detection means for detecting a binding reaction are provided.
  • the specific device configuration is not sufficiently disclosed.
  • the capture substance is immobilized in the pores of the porous substrate, and the sample fluid containing the test substance is pumped up through the through holes and circulated from the back side to the front side of the porous substrate.
  • a method is proposed in U.S. Pat. No. 7,470,056 in which the liquid is brought into effective contact with the capture substance to bind the analyte to the capture substance. By this method, the measurement time can be significantly shortened.
  • U.S. Pat. No. 7,470,056 as a specific configuration, a pipette whose tip is inserted into a sample liquid is connected to the lower surface of a porous substrate, a diaphragm pump is provided on the upper surface, and pressure control is performed.
  • a method has been proposed in which the liquid is supplied into the pores and the liquid level is controlled.
  • JP 2001-521148 A proposes a capillary assay method in which an assay is performed in a capillary of a device provided with a plurality of capillaries and light is detected from the longitudinal direction of the capillary.
  • the method of supplying the sample liquid into the capillary is not mentioned, and the state of the liquid level at the time of light detection is not described.
  • a method of controlling the distance between the detector and an optical waveguide tip provided between the detector and the specimen for light measurement Specifically, there is disclosed a sample storage unit provided with a through hole for storing a sample at the bottom of a mortar-like opening, and the sample supplied from the top is lowered by the weight of the sample itself at the opening. It is stated that the liquid level can be managed at a constant level because it stops at a substantially constant place.
  • the same assay may be performed on a plurality of micropores to average the signals from a plurality of micropores.
  • the distance between the sample containers is at least It is necessary to be in the order of mm, and it is difficult to increase the density of sample storage sections.
  • An object of the present invention is to provide an inspection method and an inspection apparatus which can suppress signal variation among a plurality of pores in an inspection chip and enable signal detection with higher accuracy in view of the above-mentioned circumstances.
  • the inspection method of the present invention is a plate-like inspection chip having a plurality of pores penetrating at a constant pore diameter from one surface to the other surface, and specifically binding to a specific substance on the inner wall surface of the pores
  • a test chip having a shape in which the capture substance is immobilized and the pore is filled in the pore by capillary action under atmospheric pressure
  • the sample solution to be tested is supplied to the pores of the test chip to bind a specific substance in the sample fluid to the capture substance, and the labeled substance that specifically binds to the specific substance is bound to the specific substance rear
  • the test chip is horizontally supported in a container storing the test solution with one side facing vertically upward,
  • the solution for inspection is supplied to the pores from the other side which is the lower surface of the inspection chip by pressurizing the inside of the container or by reducing the pressure on the space on the inspection chip, Then, open the pressurized container or the depressurized space to atmospheric pressure, With the test solution held in the pores of the test chip
  • the inspection chip is rotated so that the top and bottom of the inspection chip are reversed, the other surface is vertically oriented, and arranged horizontally, and emitted from the inspection chip
  • the light to be detected is detected from the other side.
  • the shape of each of the plurality of pores of the inspection chip is the surface tension of the solution for inspection, T, the contact angle of the solution for inspection with the inner wall surface of the pores, and the gravitational acceleration.
  • T the surface tension of the solution for inspection
  • g the density of the test solution as ⁇ Peripheral length of pore ⁇ T ⁇ Cos ⁇ > Hollow volume of pore ⁇ g ⁇ ⁇ It is preferable that the
  • the inspection chip is preferably made of one or more of Si, SiO 2 , Al, Al 2 O 3 , stainless steel and resin material.
  • the inspection chip preferably has an equivalent circle diameter of 1 ⁇ m to 100 ⁇ m in the opening area on one side of the pore.
  • the inspection chip preferably has a thickness of 100 ⁇ m to 2000 ⁇ m.
  • the capture substance immobilized on the inner wall surface of the pore of the test chip is preferably an antigen, an antibody or deoxyribonucleic acid (DNA).
  • a substance containing an enzyme label is used as a labeling substance, and a solution containing a substrate that is catalyzed and reacted by the enzyme label is used as a test solution, and light emitted from the test solution is used. It is preferred to detect the light produced by the substrate being catalyzed by the enzyme label.
  • a chromogenic substrate, a fluorescent substrate, a chemiluminescent substrate, etc. are mentioned, These substrates are suitably selected according to the kind of enzyme label
  • the light emitted from the test chip is different, and the light to be detected is light absorption (coloring), fluorescence or chemiluminescence.
  • the inspection apparatus of the present invention is a plate-like inspection chip having a plurality of pores penetrating at a constant pore diameter from one surface to the other surface, and specifically binds to a specific substance on the inner wall surface of the pores
  • a test chip having a shape capable of holding the capture solution immobilized therein and having the pores filled in the pores by capillary action under atmospheric pressure;
  • a container for storing a test solution;
  • a support member for supporting the inspection chip in the container;
  • a liquid supply unit for supplying an inspection container to the pores of the inspection chip;
  • a photodetector for detecting light emitted from the inspection chip from vertically above the inspection chip disposed horizontally.
  • the inspection method of the present invention is a plate-like inspection chip having a plurality of pores penetrating at a constant pore diameter from one surface to the other surface, which is specific to a specific substance on the inner wall surface of the pores Using a test chip that is shaped so as to be able to hold the capture solution bound to the inside of the pore by means of capillary action under atmospheric pressure, using pressure or pressure reduction. After supplying the test solution to the chip, the test solution can be retained in the pores of the test chip even when the pressure is returned to atmospheric pressure. When the test chip is separated from the test solution in the container, the surface position of the test solution in the pores of the test chip is substantially the same among the plurality of pores.
  • the surface position of the test solution is almost the same among the many pores, the light emitted from the test chip is detected from the vertical upper side of the test chip, so that the light signal measurement is caused by the dispersion of the liquid level. Highly accurate signal detection without signal variation is possible.
  • FIG. 1 is a view showing a schematic configuration of the inspection apparatus 1.
  • the inspection apparatus 1 of the present embodiment has a plurality of pores 11 penetrating at a constant pore diameter from one surface 10a to the other surface 10b, and is trapped specifically binding to a specific substance on the inner wall surface of the pores 11
  • the inspection chip 10 is horizontally supported with one surface 10a vertically upward in the plate-like inspection chip 10 on which the substance is immobilized, the container 12 for storing liquid, and the container 12
  • a support member 14 for holding liquid on one surface 10a is provided.
  • the support member 14 in this example also serves as a liquid holding portion that holds liquid on one surface 10 a of the inspection chip 10, but the liquid holding portion may be separate from the support member.
  • supporting horizontally refers to supporting the test chip 10 so that the lower surface of the test chip 10 is parallel to the surface of the liquid, but it becomes the surface of the liquid and the lower surface of the test chip 10 It is assumed that the angle formed by the surface 10b is in the range of ⁇ 10 °.
  • the vertical direction corresponds to the vertical direction, and the vertically upward surface of the inspection chip 10 in use in which the inspection chip 10 is horizontally disposed is the upper surface, and the vertically downward surface is the lower surface.
  • the inspection apparatus 1 further functions as a liquid supply unit for supplying liquid to the pore 11 from the other surface 10 b which is the lower surface when the inspection chip 10 is horizontally disposed at the time of liquid supply to the pore of the inspection chip 10.
  • a space 16 enclosed by one surface 10 a of the inspection chip 10 and the support member 14 is closed, and a pump 16 is provided to reduce the pressure of the space 15.
  • the inspection apparatus 1 includes an optical signal measurement unit 22 configured of a light detector 22 a disposed in the dark room 23 and a lens 22 b for condensing light from the inspection chip 10.
  • the inspection chip 10 is disposed below the light detector 22 a, and the light detector 22 a detects light emitted from the inspection chip 10 from vertically above the inspection chip 10.
  • the inspection chip 10 is a plate-like base material in which a plurality of pores 11 are two-dimensionally arranged.
  • the inspection chip 10 is made of one or more of Si (silicon), SiO 2 (silicon oxide), Al (aluminum), Al 2 O 3 (alumina), stainless steel and a resin material. Is preferred.
  • the thickness of the inspection chip 10 is not particularly limited, but preferably about 100 ⁇ m to 2000 ⁇ m.
  • the opening shape of the pores 11 of the inspection chip 10 is not particularly limited, and may be circular, oval or polygonal.
  • the pores 11 are columnar, the cross-sectional shape of which does not change, and the pore diameter is constant.
  • the equivalent circle diameter of the opening on at least one surface of the pore 11 is preferably about 1 ⁇ m to 100 ⁇ m. More preferably, it is 3 ⁇ m to 50 ⁇ m, and particularly preferably 5 ⁇ m to 30 ⁇ m.
  • the equivalent circle diameter refers to the diameter of a circle having an area equal to the area of the opening region.
  • pores 11 having the same shape are periodically arranged in a two-dimensional manner in one inspection chip, and the ratio d / ⁇ of the distance d between the nearest neighboring pores 11 to the aperture diameter ⁇ is 5 It is preferable that it is the following. More preferably, d / ⁇ is 3 or less, and particularly preferably 1 or less. The smaller the ratio d / ⁇ , the larger the pore density in the test chip.
  • inspection chip 10 has a shape which can hold
  • the shape of the pore 11 of the inspection chip 10 is the surface tension of the solution for inspection T, the contact angle ⁇ with the inner wall surface of the pore of the solution for inspection, the gravitational acceleration g, and the solution for inspection
  • the hollow volume of the pore ⁇ g ⁇ ⁇ is satisfied.
  • the outer peripheral length of the pore is the outer peripheral length of the opening on one surface of the inspection chip, and for example, if the opening of the pore is a circle of diameter ⁇ , it is represented by ⁇ .
  • FIG. 2 An enlarged view of the area A of the inspection chip 10 set in the light signal measurement unit 22 of FIG. 1 is shown in FIG.
  • the pores 11 can hold the test solution 50 inside.
  • the test solution 50 is held in the pores 11, and the surface position of the test solution 50 in the pores 11 is aligned with the upper surface position of the test chip 10 horizontally disposed, and is substantially the same in all the pores 11. It becomes a position.
  • the planar shape of the inspection chip 10 is not particularly limited, but a square, a rectangle such as a rectangle, or a circle is preferable.
  • FIG. 3 is a perspective view of the support member 14 and the inspection chip 10 shown in FIG.
  • the support member 14 includes an inspection chip receiving portion 14 a corresponding to the outer shape of the inspection chip 10.
  • the inspection chip receiving portion 14a of the support member 14 and the inspection chip 10 may be provided with an engagement portion for engaging both or a fitting portion for fitting each other.
  • the support member 14 holds the liquid on the upper surface 10 a of the test chip 10, and the flange 14 c is locked to a part of the container 12 to support the test chip 10 in the container 12. Is equipped.
  • the support member 14 can be fixed to the container 12 by locking the collar portion 14 c to a part of the upper surface of the container 12.
  • the form of the support member 14 is not limited to this example.
  • the liquid holding portion may be configured separately from the support member.
  • a pedestal for mounting the inspection chip on a part of the container 12 is provided and the pedestal is used as the inspection chip.
  • a cylindrical member capable of holding a liquid and installed so as to be pressed against the upper surface of the inspection chip may be provided as a liquid holding portion.
  • FIG. 4 and 5 are schematic cross-sectional views of other configuration examples of the inspection chip and the support member.
  • the support member 114 may be connected to one surface having the opening of the pore 11 of the inspection chip 10, or as shown in FIG. Instead of being connected, the inspection chip 10 may be holdable from the center of the end of the support member 214.
  • the support member and the inspection chip may be detachable, and the support member may be reused, or after the support member and the inspection chip are once connected and used, they may be discarded as they are.
  • the support member and the inspection chip may be integrally formed.
  • At least the liquid holding portion of the support member has light transparency for confirmation of the liquid level position.
  • the test substance (target molecule) to be tested in this test method is mainly a biological molecule, and proteins such as antigens and antibodies, saccharides, peptides, DNA, ribonucleic acid (RNA), peptide nucleic acids (Peptide nucleic acid: PNA) and the like.
  • the capture substance fixed to the inner wall surface of the pore 11 and specifically binding to a specific substance is a substance that specifically binds to these test substances.
  • the supply and discharge of the liquid to the pores 11 of the inspection chip 10 is performed by the pump 16.
  • the space 15 is depressurized by the pump 16 with the test chip 10 horizontally supported so that the other surface 10 b serving as the lower surface is in contact with the liquid in the container 12, the other surface 10 b side of the test chip 10
  • the liquid is drawn into the pores 11 from the Further, when the space 15 is pressurized in a state where the liquid is contained in the pore 11, the liquid is pushed downward and discharged from the pore 11 of the inspection chip 10.
  • supply (suction) and discharge of the liquid into the pore 11 are performed.
  • the pressure reducing operation of the space 15 by the pump 16 corresponds to the liquid supplying operation
  • the pressurizing operation of the space 15 corresponds to the liquid discharging operation.
  • supply and discharge of various liquids to the pores 11 of the inspection chip 10 are performed by the pressure reducing operation of the pump 16.
  • one surface 10a of the inspection chip 10 may be referred to as the upper surface 10a
  • the other surface 10b may be referred to as the lower surface 10b.
  • FIG. 6 is a diagram showing a test flow
  • FIG. 7 is a diagram schematically showing reactions in the first to third reaction processes of the test flow shown in FIG.
  • the first reaction liquid in the first reaction process is a sample liquid to be subjected to a test.
  • Specific examples of the sample fluid include plasma or serum.
  • the sample liquid is placed in the container 12, and the test chip 10 is set so that the lower surface 10b is immersed in the surface of the sample liquid in the container 12. Thereafter, the space 15 is depressurized by the pump 16 to supply the sample liquid into the pore 11. The sample liquid is sucked into the pores 11 by the pressure reduction of the space 15, and the liquid level gradually rises to the upper surface 10 a side of the test chip 10.
  • the pressure reducing operation by the pump 16 is stopped and the space 15 is returned to the atmospheric pressure.
  • the sample liquid remains in the pore 11 even if the space 15 is returned to the atmospheric pressure.
  • the sample liquid is held in the pore 11 for 30 minutes.
  • the space 15 is pressurized by the pump 16 and the sample liquid in the pore 11 is discharged (S2).
  • the steps S1-S2 may be repeated multiple times. Here, for example, it is repeated three times to promote a specific binding reaction between a specific substance in the sample solution and the capture substance immobilized on the inner wall in the pore 11.
  • a first cleaning process is performed (S3).
  • the cleaning solution is placed in the container 12, and the inspection chip 10 is set so that the lower surface 10b is immersed in the surface of the cleaning solution in the container 12.
  • the space 15 is depressurized by the pump 16 to supply the cleaning liquid into the pores 11.
  • the reduced pressure in the space 15 sucks the cleaning solution into the pores 11, and the liquid level gradually rises to the upper surface 10 a side of the inspection chip 10.
  • the pressure reducing operation by the pump 16 is switched to the pressurizing operation to open the space 15 Pressurize to discharge the cleaning solution in the pores 11.
  • the washing liquid is supplied to the upper side of the position where the sample liquid is supplied, so that sufficient washing can be performed.
  • Step S3 may be repeated multiple times. Here, for example, the washing effect is enhanced three times repeatedly.
  • the second reaction solution in the second reaction process is a labeling solution containing a labeling substance.
  • the labeling substance is one in which a substance that specifically binds to the test substance is labeled.
  • the labeling solution is placed in the container 12, and the test chip 10 is set so that the lower surface 10b is immersed in the surface of the labeling solution in the container 12.
  • the space 15 is depressurized by the pump 16 to supply the labeling solution into the pore 11.
  • the label solution is sucked into the pores 11 by the pressure reduction of the space 15, and the liquid level gradually rises to the upper surface 10 a side of the test chip 10.
  • the pressure reducing operation by the pump 16 is stopped to return the space 15 to the atmospheric pressure. Then, the labeling solution is held in the pores 11 for 30 minutes. Thereafter, the space 15 is pressurized by the pump 16 to discharge the labeling solution in the pores 11 (S5).
  • the steps S4 to S5 may be repeated multiple times.
  • the binding reaction of the labeled substance to the specifically bound test substance with the capture substance fixed on the inner wall in the pore 11 is repeated three times.
  • the second cleaning process is similar to the first cleaning process.
  • the third reaction process is carried out (S7).
  • the third reaction solution in the third reaction process is a test solution.
  • the test solution is placed in the container 12, and the test chip 10 is set so that the lower surface 10b is immersed in the surface of the test solution in the container 12.
  • the space 15 is depressurized by the pump 16 to supply the test solution into the pore 11.
  • the reduced pressure of the space 15 sucks the test solution into the pores 11 and the liquid level gradually rises to the upper surface 10 a side of the test chip 10.
  • the pump operation is stopped and the space 15 is returned to the atmospheric pressure.
  • the sample liquid remains in the pore 11 even if the space 15 is returned to the atmospheric pressure.
  • the test chip 10 While holding the test solution in the pores 11 of the test chip 10, the test chip 10 is removed from the container 12, and the lower surface 10b thereof is separated from the test solution (S8).
  • the inspection chip 10 is rotated to turn over the upper and lower surfaces so that the other surface 10b of the inspection chip 10, which was the lower surface at the time of supplying the solution for inspection, is the upper surface (S9).
  • the inspection chip 10 is disposed horizontally below the light detector 22a of the light signal measurement unit 22 so that the other surface 10b is vertically upward and faces the light detector 22a (S10).
  • the light emitted from the inspection chip 10 is detected by the light detector 22a in a state where the inspection solution is retained in the pores 11 of the inspection chip 10 under atmospheric pressure (S11), and the inspection process is completed.
  • the pore 11 of the inspection chip 10 has a shape capable of holding the inspection solution in the pore 11 in the atmosphere, as described above, the inspection solution sucked from the lower surface is kept in the pore 11 It is possible to move to the light signal measurement unit 22 without performing pressure control. Further, in the plurality of pores 11 of the inspection chip 10, the liquid surface position on the upper surface side of the inspection chip 10 is aligned at a substantially constant position by the balance of the liquid's own weight and surface tension (see FIG. 2). The inspection accuracy from each of the eleven pores 11 can be improved.
  • the upper surface and the lower surface of the inspection chip 10 are inverted to provide light to one surface 10 b of the inspection chip 10
  • the other members such as the support member 14 are not located between the detector 22a and the like.
  • FIG. 7 is a view schematically showing a reaction in each of the above reaction processes.
  • a capture substance 30 such as an allergen is fixed to the inner wall surface 11a of the pore 11 of the test chip 10 (S0).
  • the analyte fluid is supplied to the pores 11, and the analyte (for example, a specific IgE antibody that specifically binds to the above-mentioned allergen) 32 contained in the analyte fluid is captured 30 specifically bind.
  • the analyte for example, a specific IgE antibody that specifically binds to the above-mentioned allergen
  • the labeled substance obtained by applying the label F to the substance 33 (for example, secondary antibody) that specifically binds to the test substance 32 The labeling solution containing 35 is supplied to the pore 11, and the labeling substance 35 contained in the labeling solution is bound to the test substance 32.
  • the label F is an enzyme label that functions as a catalyst for chemiluminescent substrates such as, for example, luminol, lophine, lucigenin and oxalate.
  • a test solution containing a luminescent substrate that emits light as a catalyst with the label F is supplied into the pore 11 to label the luminescent substrate.
  • the light emission reaction is performed using F as a catalyst.
  • the light signal due to the light emission reaction is detected by the light detector 22a.
  • the test solution 32 or the like which is nonspecifically adsorbed in the pores 11 is washed by washing the residual solution of the sample solution or the labeling solution. Since the labeling substance is removed, noise in the measurement signal can be suppressed.
  • ALP alkaline phosphatase enzyme is a reaction solution (luminol reaction solution) containing a luminol-based chemiluminescent substrate in which HRP functions as a catalyst.
  • reaction solution containing a dioxetane-based chemiluminescent substrate.
  • the luminol reaction solution contains at least a luminol substrate and a hydrogen peroxide solution.
  • the enzyme label is one that catalyzes the oxidation of luminol in the presence of hydrogen peroxide water.
  • the reaction solution preferably contains a sensitizer for sensitizing chemiluminescence.
  • a color reaction (light absorption) reaction or fluorescence may be detected using a reaction solution containing not only the above-mentioned chemiluminescent substrate but also a luminescent substrate or a fluorescent substrate.
  • FIG. 8 is a schematic block diagram of the inspection apparatus 2 according to the second embodiment of the present invention.
  • differences from the inspection apparatus 1 according to the first embodiment will be mainly described, and the same reference numerals will be given to the common components and the detailed description will be omitted.
  • the inspection device 2 of the present embodiment differs from the configuration of the inspection device 1 in that the shape of the container 24 for holding liquid and the pump 16 as the liquid supply unit are connected to the container 24.
  • the pump 16 supplies and discharges the liquid to and from the pores 11 of the inspection chip 10 by pressurizing and depressurizing the space 25 of the container 24.
  • the container 24 has a shape that can form a closed space 25 inside the container 24 by the inspection chip 10 and the container 24.
  • the flange portion 14 c of the support member 14 can be locked to a part of the upper surface of the container 24 to horizontally support the inspection chip 10 in the container 24.
  • the space 25 When the space 25 is pressurized by the pump 16 while the inspection chip 10 is horizontally supported so that the other surface 10 b is in contact with the liquid in the container 24, the pores 11 from the surface 10 b side of the inspection chip 10 The liquid is pushed up inside.
  • the space 25 When the space 25 is depressurized with the liquid contained in the pores 11, the liquid is sucked downward from the pores 11 of the inspection chip 10 and discharged.
  • the operation of the pump 16 supplies and discharges the liquid into the pores 11.
  • the pressurizing operation of the space 25 by the pump 16 corresponds to the liquid supplying operation
  • the depressurizing operation of the space 25 corresponds to the liquid discharging operation.
  • the inspection method is the same as the inspection method described above except that the operation of supplying and discharging the liquid into the pore 11 is either pressure reduction or pressure reduction.
  • the upper and lower surfaces of the inspection chip 10 are reversed after the inspection solution is supplied to the inspection chip 10.
  • the optical signal measurement unit 22 of FIG. It may be installed in the light signal measuring unit 22 without changing the upper and lower surfaces of the chip 10 and in the vertical relation at the time of supply, and may be used for light detection.
  • it is more preferable to go through the reversing step since the noise due to the test solution remaining in the liquid holding portion 14b can be suppressed by reversing the upper and lower surfaces.
  • inspection apparatus 10 inspection chip 10a one surface of an inspection chip 10b the other surface of an inspection chip 11 pore 11a inner wall surface 12 container 14, 114, 214 support member (inspection chip support portion) 14a Test chip receiving portion 14b Liquid holding portion 14c Edge portion 15 Space 16 Pump (liquid supply portion) 22 light signal measurement unit 22a light detector 22b lens 23 dark room 24 container 25 space in container 30 capture substance 32 test substance 33 substance 35 labeled substance 50 test solution F label

Abstract

Through use of a plate-shaped inspection chip 10 having a plurality of pores 11 penetrating from one surface 10a to another surface 10b and having a constant pore diameter, a capture substance for specifically binding to a specific substance being immobilized on inside wall surfaces of the pores 11, and the pores 11 being shaped so as to be capable of retaining, by capillary action at atmospheric pressure, an inspection solution with which the pores 11 are filled, the other surface 10b is brought into contact with the inspection solution and the inspection solution is supplied into the pores 11, after which the inspection chip 10 is separated from the inspections solution stored in a container while the inspection solution remains retained in the pores 11 of the inspection chip 10, and light emitted from the inspection chip 10 is detected from vertically above the inspection chip 10.

Description

検査方法および検査装置Inspection method and inspection device
　本発明は、被検物質である抗原、抗体もしくはデオキシリボ核酸などを検出するための検査方法および検査装置に関する。 The present invention relates to a test method and a test apparatus for detecting an antigen as a test substance, an antibody, deoxyribonucleic acid and the like.
　生化学的な反応、例えば、酵素反応、核酸ハイブリダイゼーション、抗原－抗体反応などの特異的結合反応を検査する方法の一つとして、被検物質と捕捉物質との間の結合現象を光学的に検出する方法が知られている。この方法は、所定位置に固定されている捕捉物質に被検物質を結合させ、励起光を受けて蛍光を発する標識、あるいは基質の反応を触媒して発色、蛍光もしくは化学発光を生じさせる標識などをその被検物質に付与し、かかる標識に起因して生じる光を検出するものである。より具体的には、被検物質に特異的に結合する抗体などの結合物質に蛍光標識を付与し、蛍光標識から生じる蛍光を検出する方法、被検物質に特異的に結合する抗体などの結合物質に酵素を標識し、この酵素を触媒として反応する発色基質、蛍光基質、あるいは化学発光基質から生じる発色や蛍光、化学発光を検出する方法等が知られており、これらにより、被検物質の特定が可能となる。 The binding phenomenon between a test substance and a capture substance is optically detected as one of the methods for examining a specific binding reaction such as an enzymatic reaction, nucleic acid hybridization, or an antigen-antibody reaction. Methods for detecting are known. In this method, a test substance is bound to a capture substance fixed at a predetermined position, a label which emits fluorescence upon receiving excitation light, or a label which catalyzes a reaction of a substrate to generate color, fluorescence or chemiluminescence, etc. Is applied to the test substance to detect light generated due to such a label. More specifically, a method of attaching a fluorescent label to a binding substance such as an antibody that specifically binds to a test substance and detecting fluorescence generated from the fluorescent label, or a binding such as an antibody that specifically binds to a test substance Methods for labeling substances with enzymes and reacting them using these enzymes as catalysts, chromogenic substrates, fluorescent substrates, methods for detecting color development, fluorescence, or chemiluminescence generated from chemiluminescent substrates, etc. are known. Identification is possible.
　このような検査に用いられるバイオチップとしては、支持体に多数の貫通孔（細孔）が整列配置されてなる多孔性基板からなるデバイスの検討が進められている。 As a biochip used for such an inspection, examination of a device comprising a porous substrate in which a large number of through holes (pores) are aligned in a support has been advanced.
　特表平９－５０４８６４号公報には、多孔性基板からなるバイオチップの細孔中に流体流動を供する手段や、結合反応を検出するための検出手段を備える旨は記載されている。しかしながら、具体的な装置構成は十分に開示されていない。 Japanese Patent Application Publication No. 9-504864 describes that a means for providing fluid flow in the pores of a biochip comprising a porous substrate and a detection means for detecting a binding reaction are provided. However, the specific device configuration is not sufficiently disclosed.
　一方、多孔性基板の細孔内に捕捉物質を固定化し、被検物質を含有する検体液を多孔性基板の裏面側から表面へと貫通孔を介してポンプで汲み上げ、循環させることにより、検体液が捕捉物質に効率的に接触されて、被検物質を捕捉物質に結合させる方法が米国特許第７４７０５６号明細書に提案されている。この方法により、測定時間の大幅な短縮化を図ることができる。米国特許第７４７０５６号明細書には、具体的な構成として、多孔性基板の下面に検体液中に先端が挿入されたピペットを接続し、上面にダイアフラムポンプを備え、圧力制御を行うことで検体液を細孔中に供給し、その液面を制御する方法が提案されている。 On the other hand, the capture substance is immobilized in the pores of the porous substrate, and the sample fluid containing the test substance is pumped up through the through holes and circulated from the back side to the front side of the porous substrate. A method is proposed in U.S. Pat. No. 7,470,056 in which the liquid is brought into effective contact with the capture substance to bind the analyte to the capture substance. By this method, the measurement time can be significantly shortened. In U.S. Pat. No. 7,470,056, as a specific configuration, a pipette whose tip is inserted into a sample liquid is connected to the lower surface of a porous substrate, a diaphragm pump is provided on the upper surface, and pressure control is performed. A method has been proposed in which the liquid is supplied into the pores and the liquid level is controlled.
　特表２００１－５２１１４８号公報には、複数の毛細管を備えたデバイスの毛細管内でアッセイを行い、毛細管の縦方向から光を検出する毛細管アッセイ方法が提案されている。しかしながら、毛細管中への検体液の供給方法については言及されておらず、光検出時における液面の状態についても記載がない。 JP 2001-521148 A proposes a capillary assay method in which an assay is performed in a capillary of a device provided with a plurality of capillaries and light is detected from the longitudinal direction of the capillary. However, the method of supplying the sample liquid into the capillary is not mentioned, and the state of the liquid level at the time of light detection is not described.
　他方、国際公開第２００６/０１３８３２号には、検体の光情報を検出する光情報認識装置において、光測定の精度を高めるために、光検出時における検体収容部に収容されている検体の液面と光測定のために検出器と検体との間に備えられる光導波先端との距離を制御する方法が開示されている。具体的には、すり鉢状の開口部の底部に検体を収容する貫通穴が設けられた検体収容部が開示されており、上部から供給された検体は開口部にある検体自体の重みによって下降してゆき、ほぼ一定の場所で止まるため、液面を一定に管理することができる旨記載されている。 On the other hand, according to WO2006 / 013832, in the optical information recognition apparatus for detecting optical information of a specimen, the liquid level of the specimen stored in the specimen storage unit at the time of light detection in order to enhance the accuracy of light measurement. And a method of controlling the distance between the detector and an optical waveguide tip provided between the detector and the specimen for light measurement. Specifically, there is disclosed a sample storage unit provided with a through hole for storing a sample at the bottom of a mortar-like opening, and the sample supplied from the top is lowered by the weight of the sample itself at the opening. It is stated that the liquid level can be managed at a constant level because it stops at a substantially constant place.
　米国特許第７４７０５６号明細書においては、ピペット毎にダイアフラムを配置する必要があり、また、ポンプの圧力を制御してチップ内に反応液を留めたまま、チップの下方から光検出を行うため、系が煩雑である。加えて、検査チップの孔の径や反応液の表面張力が違うと細孔中に吸い上げられる液面レベルが変わってしまい、光取り出し側の液表面の状態が不安定であり定量的な測定は困難である。このような系において、定量的な測定を行うためには、チップの設計や反応液の表面張力が変わるたびにダイアフラムの設定値を変更する必要があり、管理が煩雑なものとなる。 In U.S. Pat. No. 7,470,056, it is necessary to arrange a diaphragm for each pipette, and in order to control the pressure of the pump and perform the light detection from below the tip while keeping the reaction liquid in the tip, The system is complicated. In addition, if the diameter of the hole of the inspection chip or the surface tension of the reaction liquid is different, the level of the liquid absorbed into the pore will change, the state of the liquid surface on the light extraction side is unstable, and quantitative measurement Have difficulty. In such a system, in order to perform quantitative measurement, it is necessary to change the set value of the diaphragm every time the design of the chip or the surface tension of the reaction liquid changes, which makes management complicated.
　多数の微細孔を備えた検査チップにおいて、微細孔からの低出力を精度よく測定するためには、複数の微細孔で同一のアッセイを行い、複数の微細孔からの信号を平均化することが好ましい。しかしながら、国際公開第２００６/０１３８３２号のすり鉢状の開口部の底部に検体を収容する貫通穴が設けられた検体収容部では、検体収容部を複数設ける場合に、検体収容部同士の間隔は少なくともｍｍオーダー必要であり、検体収容部密度を高めることが難しい。 In an inspection chip provided with a large number of micropores, in order to accurately measure the low output from the micropores, the same assay may be performed on a plurality of micropores to average the signals from a plurality of micropores. preferable. However, in a sample container provided with a through hole for containing a sample at the bottom of the mortar-like opening of WO2006 / 013832, when a plurality of sample containers are provided, the distance between the sample containers is at least It is necessary to be in the order of mm, and it is difficult to increase the density of sample storage sections.
　本発明は、上記事情に鑑み、検査チップ内の複数の細孔間での信号バラツキを抑制し、より高い精度の信号検出を可能とする検査方法および検査装置を提供することを目的とする。 An object of the present invention is to provide an inspection method and an inspection apparatus which can suppress signal variation among a plurality of pores in an inspection chip and enable signal detection with higher accuracy in view of the above-mentioned circumstances.
　本発明の検査方法は、一方の面から他方の面まで一定の孔径で貫通した複数の細孔を有する板状の検査チップであって、細孔の内壁面に特定の物質と特異的に結合する捕捉物質が固定化されてなり、細孔が細孔内に充填された検査用溶液を、大気圧下において毛細管現象により保持できる形状である検査チップを用い、
　検査チップの細孔に、検査に供する検体液を供給して、検体液中の特定の物質を捕捉物質に結合させ、特定の物質と特異的に結合する標識物質を特定の物質に結合させた後、
　検査チップを、検査用溶液が貯留された容器内に、一方の面を鉛直上向きとして水平に支持し、
　容器内を加圧することにより、あるいは検査チップ上の空間を減圧することにより、検査用溶液を、検査チップの下面である他方の面側から細孔に供給し、
　その後、加圧された容器内、あるいは減圧された空間を大気圧に開放し、
　検査チップの細孔内に検査用溶液を保持させたまま、検査チップを容器に貯留された検査用溶液から離間させ、
　検査チップから出射される光を、検査チップの鉛直上方から検出する検査方法である。 The inspection method of the present invention is a plate-like inspection chip having a plurality of pores penetrating at a constant pore diameter from one surface to the other surface, and specifically binding to a specific substance on the inner wall surface of the pores Using a test chip having a shape in which the capture substance is immobilized and the pore is filled in the pore by capillary action under atmospheric pressure,
The sample solution to be tested is supplied to the pores of the test chip to bind a specific substance in the sample fluid to the capture substance, and the labeled substance that specifically binds to the specific substance is bound to the specific substance rear,
The test chip is horizontally supported in a container storing the test solution with one side facing vertically upward,
The solution for inspection is supplied to the pores from the other side which is the lower surface of the inspection chip by pressurizing the inside of the container or by reducing the pressure on the space on the inspection chip,
Then, open the pressurized container or the depressurized space to atmospheric pressure,
With the test solution held in the pores of the test chip, the test chip is separated from the test solution stored in the container,
It is an inspection method which detects light emitted from an inspection chip from the perpendicular upper part of an inspection chip.
　本発明の検査方法においては、検査チップを検査用溶液から離間させた後に、検査チップの上下が逆になるように回転させて他方の面を鉛直上向きとして、水平に配置し、検査チップから出射される光を他方の面側から検出することが好ましい。 In the inspection method of the present invention, after separating the inspection chip from the solution for inspection, the inspection chip is rotated so that the top and bottom of the inspection chip are reversed, the other surface is vertically oriented, and arranged horizontally, and emitted from the inspection chip Preferably, the light to be detected is detected from the other side.
　本発明の検査方法においては、検査チップが、複数の細孔の各細孔の形状が、検査用溶液の表面張力をＴ、検査用溶液の細孔内壁面に対する接触角をθ、重力加速度をｇ、および検査用溶液の密度をρとしたとき、
細孔の外周長×Ｔ×Cosθ＞細孔の中空部体積×g×ρ
を満たすものであることが好ましい。 In the inspection method of the present invention, the shape of each of the plurality of pores of the inspection chip is the surface tension of the solution for inspection, T, the contact angle of the solution for inspection with the inner wall surface of the pores, and the gravitational acceleration. g and the density of the test solution as ρ
Peripheral length of pore × T × Cos θ> Hollow volume of pore × g × ρ
It is preferable that the
　本発明の検査方法においては、検査チップが、Ｓｉ、ＳｉＯ２、Ａｌ、Ａｌ２Ｏ３、ステンレス鋼および樹脂材料のうちの１つまたは２つ以上の材料からなることが好ましい。
　検査チップは、細孔の一方の面における開口領域の円相当直径が１μｍ～１００μｍであることが好ましい。
　また、検査チップは、厚さが１００μｍ～２０００μｍであることが好ましい。 In the inspection method of the present invention, the inspection chip is preferably made of one or more of Si, SiO 2 , Al, Al 2 O 3 , stainless steel and resin material.
The inspection chip preferably has an equivalent circle diameter of 1 μm to 100 μm in the opening area on one side of the pore.
The inspection chip preferably has a thickness of 100 μm to 2000 μm.
　本発明の検査方法においては、検査チップの細孔の内壁面に固定化されている捕捉物質が、抗原、抗体またはデオキシリボ核酸（deoxyribonucleic acid：ＤＮＡ）であることが好ましい。 In the test method of the present invention, the capture substance immobilized on the inner wall surface of the pore of the test chip is preferably an antigen, an antibody or deoxyribonucleic acid (DNA).
　本発明の検査方法においては、標識物質として酵素標識を含む物質を用い、検査用溶液として、酵素標識により触媒されて反応する基質を含む溶液を用い、出射される光として、検査用溶液中の基質が酵素標識により触媒されて生じる光を検出することが好ましい。
　なお、上記基質としては、発色基質、蛍光基質および化学発光基質などが挙げられ、これらの基質は酵素標識の種類に応じて適宜選択される。また、この基質に応じて、検査チップから出射される光は異なり、検出される光は、吸光（呈色）、蛍光または化学発光である。 In the test method of the present invention, a substance containing an enzyme label is used as a labeling substance, and a solution containing a substrate that is catalyzed and reacted by the enzyme label is used as a test solution, and light emitted from the test solution is used. It is preferred to detect the light produced by the substrate being catalyzed by the enzyme label.
In addition, as said substrate, a chromogenic substrate, a fluorescent substrate, a chemiluminescent substrate, etc. are mentioned, These substrates are suitably selected according to the kind of enzyme label | marker. Also, depending on the substrate, the light emitted from the test chip is different, and the light to be detected is light absorption (coloring), fluorescence or chemiluminescence.
　本発明の検査装置は、一方の面から他方の面まで一定の孔径で貫通した細孔を複数有する板状の検査チップであって、細孔の内壁面に特定の物質と特異的に結合する捕捉物質が固定化されてなり、細孔が細孔内に充填された検査用溶液を、大気圧下において毛細管現象により保持できる形状である検査チップと、
　検査用溶液を貯留する容器と、
　検査チップを、容器内において支持する支持部材と、
　検査チップの細孔に検査用容器を供給する液体供給部と、
　検査チップから出射される光を、水平に配置された検査チップの鉛直上方から検出する光検出器とを備えている。 The inspection apparatus of the present invention is a plate-like inspection chip having a plurality of pores penetrating at a constant pore diameter from one surface to the other surface, and specifically binds to a specific substance on the inner wall surface of the pores A test chip having a shape capable of holding the capture solution immobilized therein and having the pores filled in the pores by capillary action under atmospheric pressure;
A container for storing a test solution;
A support member for supporting the inspection chip in the container;
A liquid supply unit for supplying an inspection container to the pores of the inspection chip;
And a photodetector for detecting light emitted from the inspection chip from vertically above the inspection chip disposed horizontally.
　本発明の検査方法によれば、一方の面から他方の面まで一定の孔径で貫通した複数の細孔を有する板状の検査チップであって、細孔の内壁面に特定の物質と特異的に結合する捕捉物質が固定化されてなり、細孔が細孔内に充填された検査用溶液を、大気圧下において毛細管現象により保持できる形状である検査チップを用い、加圧もしくは減圧により検査チップに検査用溶液を供給した後、大気圧に戻しても検査チップの細孔内に検査用溶液を留まらせることができる。検査チップを容器中の検査用溶液から離間した状態において、検査チップの細孔内の検査用溶液の表面位置は複数の細孔間でほぼ同一となる。多数の細孔間で検査用溶液の表面位置がほぼ一致しているので、検査チップから出射される光を、検査チップの鉛直上方から検出することにより、光信号測定において、液面のバラツキによる信号ばらつきのない、高精度な信号検出が可能である。 According to the inspection method of the present invention, it is a plate-like inspection chip having a plurality of pores penetrating at a constant pore diameter from one surface to the other surface, which is specific to a specific substance on the inner wall surface of the pores Using a test chip that is shaped so as to be able to hold the capture solution bound to the inside of the pore by means of capillary action under atmospheric pressure, using pressure or pressure reduction. After supplying the test solution to the chip, the test solution can be retained in the pores of the test chip even when the pressure is returned to atmospheric pressure. When the test chip is separated from the test solution in the container, the surface position of the test solution in the pores of the test chip is substantially the same among the plurality of pores. Since the surface position of the test solution is almost the same among the many pores, the light emitted from the test chip is detected from the vertical upper side of the test chip, so that the light signal measurement is caused by the dispersion of the liquid level. Highly accurate signal detection without signal variation is possible.
本発明の検査方法の第１の実施形態を実施するための検査装置の概略構成図である。It is a schematic block diagram of the inspection apparatus for enforcing 1st Embodiment of the inspection method of this invention. 検査チップの細孔中に保持されている検査用溶液の状態を示す図である。It is a figure which shows the state of the solution for test | inspection hold | maintained in the pore of a test | inspection chip. 検査チップおよび支持部材の斜視図である。It is a perspective view of an inspection chip and a support member. 検査チップおよび支持部材の組合せ例を示す断面模式図である。It is a cross-sectional schematic diagram which shows the example of a combination of a test | inspection chip and a support member. 検査チップおよび支持部材の組合せの他の例を示す断面模式図である。It is a cross-sectional schematic diagram which shows the other example of the combination of a test | inspection chip and a support member. 検査工程のフローを示す図である。It is a figure which shows the flow of an inspection process. 反応プロセスにおける各種結合反応の過程を示す模式図である。It is a schematic diagram which shows the process of various coupling reaction in a reaction process. 本発明の検査方法の第２の実施形態を実施するための検査装置の概略構成図である。It is a schematic block diagram of the inspection apparatus for enforcing 2nd Embodiment of the inspection method of this invention.
　以下、図面を参照して本発明の実施形態を詳細に説明する。なお、本明細書において「～」とは、その前後に記載される数値を下限値および上限値として含む意味で使用される。 Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. In the present specification, “to” is used in the meaning including the numerical values described before and after it as the lower limit value and the upper limit value.
　本発明の第１の実施形態にかかる検査方法および検査装置を説明する。図１は検査装置１の概略構成を示す図である。
　本実施形態の検査装置１は、一方の面１０ａから他方の面１０ｂまで一定の孔径で貫通した細孔１１を複数有し、細孔１１の内壁面に特定の物質と特異的に結合する捕捉物質が固定化されてなる板状の検査チップ１０と、液体を貯留する容器１２と、容器１２中において、検査チップ１０を一方の面１０ａを鉛直上向きとして水平に支持すると共に、検査チップ１０の一方の面１０ａ上に液体を保持する支持部材１４とを備えている。本例における支持部材１４は、検査チップ１０の一方の面１０ａ上に液体を保持する液体保持部を兼ねているが、液体保持部は支持部材とは別体としてもよい。 An inspection method and an inspection apparatus according to a first embodiment of the present invention will be described. FIG. 1 is a view showing a schematic configuration of the inspection apparatus 1.
The inspection apparatus 1 of the present embodiment has a plurality of pores 11 penetrating at a constant pore diameter from one surface 10a to the other surface 10b, and is trapped specifically binding to a specific substance on the inner wall surface of the pores 11 In the inspection chip 10, the inspection chip 10 is horizontally supported with one surface 10a vertically upward in the plate-like inspection chip 10 on which the substance is immobilized, the container 12 for storing liquid, and the container 12 A support member 14 for holding liquid on one surface 10a is provided. The support member 14 in this example also serves as a liquid holding portion that holds liquid on one surface 10 a of the inspection chip 10, but the liquid holding portion may be separate from the support member.
　なお、ここで「水平に支持する」とは、検査チップ１０の下面が液体の表面と平行となるように検査チップ１０を支持することをいうが、液体の表面と検査チップ１０の下面となる面１０ｂのなす角度が±１０°の範囲であれば許容されるものとする。
　また、本明細書中において上下方向は鉛直方向に一致し、検査チップ１０が水平配置される使用時における検査チップ１０の鉛直上向きの面を上面、鉛直下向きの面を下面とする。 Here, "supporting horizontally" refers to supporting the test chip 10 so that the lower surface of the test chip 10 is parallel to the surface of the liquid, but it becomes the surface of the liquid and the lower surface of the test chip 10 It is assumed that the angle formed by the surface 10b is in the range of ± 10 °.
In the present specification, the vertical direction corresponds to the vertical direction, and the vertically upward surface of the inspection chip 10 in use in which the inspection chip 10 is horizontally disposed is the upper surface, and the vertically downward surface is the lower surface.
　本検査装置１は、さらに、検査チップ１０の細孔への液体供給時において検査チップ１０を水平配置した際の下面となる他方の面１０ｂから細孔１１に液体を供給する液体供給部として、検査チップ１０の一方の面１０ａおよび支持部材１４で囲われた空間１５を閉じて、その空間１５を減加圧するポンプ１６を備えている。 The inspection apparatus 1 further functions as a liquid supply unit for supplying liquid to the pore 11 from the other surface 10 b which is the lower surface when the inspection chip 10 is horizontally disposed at the time of liquid supply to the pore of the inspection chip 10. A space 16 enclosed by one surface 10 a of the inspection chip 10 and the support member 14 is closed, and a pump 16 is provided to reduce the pressure of the space 15.
　さらに、本検査装置１は、暗室２３内に配置された光検出器２２ａおよび検査チップ１０からの光を集光するレンズ２２ｂから構成される光信号測定部２２を備えている。光信号測定部２２において、検査チップ１０は光検出器２２ａの下方に配置され、光検出器２２ａは検査チップ１０から出射される光を、検査チップ１０の鉛直上方から検出する。 Furthermore, the inspection apparatus 1 includes an optical signal measurement unit 22 configured of a light detector 22 a disposed in the dark room 23 and a lens 22 b for condensing light from the inspection chip 10. In the light signal measurement unit 22, the inspection chip 10 is disposed below the light detector 22 a, and the light detector 22 a detects light emitted from the inspection chip 10 from vertically above the inspection chip 10.
　本実施形態において、検査チップ１０は、複数の細孔１１が二次元状に配列されてなる板状基材である。検査チップ１０は、Ｓｉ（シリコン）、ＳｉＯ２（シリコン酸化物）、Ａｌ（アルミニウム）、Ａｌ２Ｏ３（アルミナ）、ステンレス鋼および樹脂材料のいずれか１種もしくは２種以上の材料から構成されていることが好ましい。 In the present embodiment, the inspection chip 10 is a plate-like base material in which a plurality of pores 11 are two-dimensionally arranged. The inspection chip 10 is made of one or more of Si (silicon), SiO 2 (silicon oxide), Al (aluminum), Al 2 O 3 (alumina), stainless steel and a resin material. Is preferred.
　検査チップ１０の厚さには、特に制限はないが、１００μｍ～２０００μｍ程度が好ましい。 The thickness of the inspection chip 10 is not particularly limited, but preferably about 100 μm to 2000 μm.
　検査チップ１０の細孔１１の開口形状は、特に制限されず、円形、楕円形あるいは多角形であってもよい。細孔１１は、柱状で断面形状が変化せず、孔径が一定のものである。なお、細孔１１の少なくとも一方の面における開口の円相当直径が１μｍ～１００μｍ程度であることが好ましい。より好ましくは３μｍ～５０μｍであり、特に好ましくは５μｍ～３０μｍである。なお、円相当直径とは、開口領域の面積と同等の面積を有する円の直径をいう。
　１つの検査チップには基本的に同一形状の細孔１１が二次元状に周期的に配列されており、最隣接の細孔１１同士の距離ｄと開口直径φとの比ｄ／φが５以下であることが好ましい。より好ましくはｄ／φが３以下であり、特に好ましくは１以下である。比ｄ／φが小さいほど検査チップにおける細孔密度が大きくなる。 The opening shape of the pores 11 of the inspection chip 10 is not particularly limited, and may be circular, oval or polygonal. The pores 11 are columnar, the cross-sectional shape of which does not change, and the pore diameter is constant. The equivalent circle diameter of the opening on at least one surface of the pore 11 is preferably about 1 μm to 100 μm. More preferably, it is 3 μm to 50 μm, and particularly preferably 5 μm to 30 μm. The equivalent circle diameter refers to the diameter of a circle having an area equal to the area of the opening region.
Basically, pores 11 having the same shape are periodically arranged in a two-dimensional manner in one inspection chip, and the ratio d / φ of the distance d between the nearest neighboring pores 11 to the aperture diameter φ is 5 It is preferable that it is the following. More preferably, d / φ is 3 or less, and particularly preferably 1 or less. The smaller the ratio d / φ, the larger the pore density in the test chip.
　検査チップ１０は、細孔１１内に充填された検査用溶液５０を、大気圧下において毛細管現象により保持できる形状を有している。具体的には、検査チップ１０は、細孔１１の形状が、検査用溶液の表面張力をＴ、検査用溶液の細孔の内壁面に対する接触角をθ、重力加速度をｇ、および検査用溶液の密度をρとしたとき、細孔の外周長×Ｔ×Cosθ＞細孔の中空部体積×g×ρを満たす。
　なお、細孔の外周長は、検査チップの一面における開口の外周長であり、例えば、細孔の開口が直径φの円であればπφで表される。 The test | inspection chip 10 has a shape which can hold | maintain the test solution 50 with which it filled in the pore 11 by a capillary phenomenon under atmospheric pressure. Specifically, the shape of the pore 11 of the inspection chip 10 is the surface tension of the solution for inspection T, the contact angle θ with the inner wall surface of the pore of the solution for inspection, the gravitational acceleration g, and the solution for inspection When the density of is 、, the peripheral length of the pore × T × Cos θ> The hollow volume of the pore × g × × is satisfied.
The outer peripheral length of the pore is the outer peripheral length of the opening on one surface of the inspection chip, and for example, if the opening of the pore is a circle of diameter φ, it is represented by πφ.
　図１の光信号測定部２２にセットされた検査チップ１０の領域Ａの拡大図を図２に示す。図２に示すように、大気圧下において、細孔１１は検査用溶液５０を内部に保持することができる。検査用溶液５０は細孔１１内に保持され、その検査用溶液５０の細孔１１内における表面位置は、水平配置された検査チップ１０の上面位置に揃い、すべての細孔１１においてほぼ同一の位置となる。 An enlarged view of the area A of the inspection chip 10 set in the light signal measurement unit 22 of FIG. 1 is shown in FIG. As shown in FIG. 2, under atmospheric pressure, the pores 11 can hold the test solution 50 inside. The test solution 50 is held in the pores 11, and the surface position of the test solution 50 in the pores 11 is aligned with the upper surface position of the test chip 10 horizontally disposed, and is substantially the same in all the pores 11. It becomes a position.
　検査チップ１０の平面形状は特に限定されないが、正方形、長方形などの矩形あるいは円形が好ましい。 The planar shape of the inspection chip 10 is not particularly limited, but a square, a rectangle such as a rectangle, or a circle is preferable.
　図３は図１に示す支持部材１４および検査チップ１０の斜視図である。
　支持部材１４は、検査チップ１０の外形に応じた検査チップ受容部１４ａを備えている。支持部材１４の検査チップ受容部１４ａおよび検査チップ１０に、両者を係合するための係合部あるいは互いに嵌め合せるための嵌合部等を備えていればよい。
　さらに、支持部材１４は検査チップ１０の上面１０ａ上に液体を保持する液体保持部１４ｂと、検査チップ１０を容器１２中において支持するために、容器１２の一部に係止される鍔部１４ｃを備えている。この鍔部１４ｃを容器１２の上面の一部に係止することによって支持部材１４を容器１２に対して固定することができる。 FIG. 3 is a perspective view of the support member 14 and the inspection chip 10 shown in FIG.
The support member 14 includes an inspection chip receiving portion 14 a corresponding to the outer shape of the inspection chip 10. The inspection chip receiving portion 14a of the support member 14 and the inspection chip 10 may be provided with an engagement portion for engaging both or a fitting portion for fitting each other.
Furthermore, the support member 14 holds the liquid on the upper surface 10 a of the test chip 10, and the flange 14 c is locked to a part of the container 12 to support the test chip 10 in the container 12. Is equipped. The support member 14 can be fixed to the container 12 by locking the collar portion 14 c to a part of the upper surface of the container 12.
　支持部材１４の形態は本例に限るものではない。既述の通り、液体保持部は支持部材とは別体として構成されていてもよく、例えば、容器１２の一部に検査チップを載置するための台座を設けてその台座を、検査チップを支持する支持部材とし、検査チップの上面に対して押し付けられるようにして設置される、液体を保持可能な筒状部材を液体保持部として設けてもよい。 The form of the support member 14 is not limited to this example. As described above, the liquid holding portion may be configured separately from the support member. For example, a pedestal for mounting the inspection chip on a part of the container 12 is provided and the pedestal is used as the inspection chip. As a supporting member to support, a cylindrical member capable of holding a liquid and installed so as to be pressed against the upper surface of the inspection chip may be provided as a liquid holding portion.
　図４、図５は、検査チップと支持部材の他の構成例の断面模式図である。
　図４に示すように、検査チップ１０の細孔１１の開口を有する一面に接続される支持部材１１４であってもよいし、図５に示すように、検査チップ１０は支持部材の端部に接続されるのではなく、支持部材２１４の端部より中央よりに検査チップ１０を保持可能にされていてもよい。
　なお、支持部材と検査チップは、脱着自在として、支持部材を再利用するようにしてもよいし、支持部材と検査チップを一旦接続して使用した後は、そのまま破棄するようにしてもよい。また、支持部材と検査チップは一体的に形成されていてもよい。 4 and 5 are schematic cross-sectional views of other configuration examples of the inspection chip and the support member.
As shown in FIG. 4, the support member 114 may be connected to one surface having the opening of the pore 11 of the inspection chip 10, or as shown in FIG. Instead of being connected, the inspection chip 10 may be holdable from the center of the end of the support member 214.
The support member and the inspection chip may be detachable, and the support member may be reused, or after the support member and the inspection chip are once connected and used, they may be discarded as they are. In addition, the support member and the inspection chip may be integrally formed.
　なお、支持部材の少なくとも液体保持部は、液面位置の確認のため、光透過性を有するものであることが好ましい。 In addition, it is preferable that at least the liquid holding portion of the support member has light transparency for confirmation of the liquid level position.
　本検査方法において検査対象とされる被検物質（標的分子）は、主として、生体由来分子であり、抗原および抗体などのタンパク質、糖類、ペプチド、ＤＮＡ、リボ核酸（ribonucleic acid：ＲＮＡ）、ペプチド核酸（peptide nucleic acid：ＰＮＡ）などである。そして、細孔１１の内壁面に固定されている、特定の物質と特異的に結合する捕捉物質としては、これらの被検物質と特異的に結合する物質である。 The test substance (target molecule) to be tested in this test method is mainly a biological molecule, and proteins such as antigens and antibodies, saccharides, peptides, DNA, ribonucleic acid (RNA), peptide nucleic acids (Peptide nucleic acid: PNA) and the like. The capture substance fixed to the inner wall surface of the pore 11 and specifically binding to a specific substance is a substance that specifically binds to these test substances.
　本実施形態の検査方法において、検査チップ１０の細孔１１への液体の供給排出はポンプ１６により行う。検査チップ１０を、容器１２中の液体に下面となる他方の面１０ｂが接触するように水平に支持した状態で、ポンプ１６により空間１５が減圧されると、検査チップ１０の他方の面１０ｂ側から細孔１１中に液体が吸引される。また、細孔１１中に液体が入っている状態で、空間１５が加圧されると、検査チップ１０の細孔１１中から液体が下方に押し出され排出される。このポンプ１６の動作により、細孔１１内への液体の供給（吸引）および排出が行われる。本例では、ポンプ１６による空間１５の減圧動作が液体の供給動作に相当し、空間１５の加圧動作が液体の排出動作に相当する。
　以下において、検査チップ１０の細孔１１への各種液体の供給および排出は上記ポンプ１６の減加圧動作によって行う。なお、検査チップ１０への液体の供給排出を伴う工程における以下の説明では検査チップ１０の一方の面１０ａを上面１０ａ、他方の面１０ｂを下面１０ｂという場合がある。 In the inspection method of the present embodiment, the supply and discharge of the liquid to the pores 11 of the inspection chip 10 is performed by the pump 16. When the space 15 is depressurized by the pump 16 with the test chip 10 horizontally supported so that the other surface 10 b serving as the lower surface is in contact with the liquid in the container 12, the other surface 10 b side of the test chip 10 The liquid is drawn into the pores 11 from the Further, when the space 15 is pressurized in a state where the liquid is contained in the pore 11, the liquid is pushed downward and discharged from the pore 11 of the inspection chip 10. By the operation of the pump 16, supply (suction) and discharge of the liquid into the pore 11 are performed. In this example, the pressure reducing operation of the space 15 by the pump 16 corresponds to the liquid supplying operation, and the pressurizing operation of the space 15 corresponds to the liquid discharging operation.
In the following, supply and discharge of various liquids to the pores 11 of the inspection chip 10 are performed by the pressure reducing operation of the pump 16. In the following description of the process involving the supply and discharge of liquid to the inspection chip 10, one surface 10a of the inspection chip 10 may be referred to as the upper surface 10a, and the other surface 10b may be referred to as the lower surface 10b.
　以下、図６および図７を参照して、図１に示した第１の実施形態の検査装置１を用いた、本発明の第１の実施形態の検査方法を説明する。図６は検査フローを示す図であり、図７は図６に示す検査フローの第１～第３の反応プロセスにおける反応を模式的に示す図である。 The inspection method of the first embodiment of the present invention using the inspection device 1 of the first embodiment shown in FIG. 1 will be described below with reference to FIGS. 6 and 7. FIG. 6 is a diagram showing a test flow, and FIG. 7 is a diagram schematically showing reactions in the first to third reaction processes of the test flow shown in FIG.
　まず、第１の反応プロセスを実施する（Ｓ１）。第１の反応プロセスにおける第１の反応液は、検査に供する検体液である。検体液の具体例としては、血漿、または血清が挙げられる。
　検体液を容器１２に入れ、検査チップ１０を容器１２中の検体液の表面に下面１０ｂが浸されるようにセットする。その後、ポンプ１６により空間１５を減圧して、細孔１１内に検体液を供給する。空間１５の減圧により検体液が細孔１１中に吸い上げられ、その液面は、徐々に検査チップ１０の上面１０ａ側に上昇する。このとき、検体液の液面が検査チップ１０の上面１０ａよりも上方に予め設定された反応液基準位置まで到達したら、ポンプ１６による減圧動作を停止して、空間１５を大気圧に戻す。空間１５を大気圧に戻しても検体液は細孔１１内に留まる。検体液を細孔１１中に留めた状態で３０分間保持する。その後、ポンプ１６により空間１５を加圧して細孔１１中の検体液を排出する（Ｓ２）。
　Ｓ１－Ｓ２の工程は、複数回繰り返してもよい。ここでは、例えば、３回繰り返し、検体液中の特定の物質と細孔１１中の内壁に固定されている捕捉物質との特異的な結合反応を促進させる。 First, the first reaction process is carried out (S1). The first reaction liquid in the first reaction process is a sample liquid to be subjected to a test. Specific examples of the sample fluid include plasma or serum.
The sample liquid is placed in the container 12, and the test chip 10 is set so that the lower surface 10b is immersed in the surface of the sample liquid in the container 12. Thereafter, the space 15 is depressurized by the pump 16 to supply the sample liquid into the pore 11. The sample liquid is sucked into the pores 11 by the pressure reduction of the space 15, and the liquid level gradually rises to the upper surface 10 a side of the test chip 10. At this time, when the liquid level of the sample liquid reaches the reaction liquid reference position preset above the upper surface 10a of the inspection chip 10, the pressure reducing operation by the pump 16 is stopped and the space 15 is returned to the atmospheric pressure. The sample liquid remains in the pore 11 even if the space 15 is returned to the atmospheric pressure. The sample liquid is held in the pore 11 for 30 minutes. Thereafter, the space 15 is pressurized by the pump 16 and the sample liquid in the pore 11 is discharged (S2).
The steps S1-S2 may be repeated multiple times. Here, for example, it is repeated three times to promote a specific binding reaction between a specific substance in the sample solution and the capture substance immobilized on the inner wall in the pore 11.
　次に、第１の洗浄プロセスを実施する（Ｓ３）。洗浄液を容器１２に入れ、検査チップ１０を容器１２中の洗浄液の表面に下面１０ｂが浸されるようにセットする。その後、ポンプ１６により空間１５を減圧して、細孔１１内に洗浄液を供給する。空間１５の減圧により洗浄液が細孔１１中に吸い上げられ、その液面は、徐々に検査チップ１０の上面１０ａ側に上昇する。洗浄液の液面が少なくとも検査チップ１０の上面１０ａを超え、反応液基準位置よりも上方に予め設定された洗浄液基準位置まで到達したら、ポンプ１６による減圧動作を加圧動作に切り換えて、空間１５を加圧して細孔１１中の洗浄液を排出する。洗浄液は検体液が供給された位置よりも上方まで供給されるので、十分洗浄を行うことができる。
　工程Ｓ３は、複数回繰り返してもよい。ここでは、例えば、３回繰り返して洗浄効果を高める。 Next, a first cleaning process is performed (S3). The cleaning solution is placed in the container 12, and the inspection chip 10 is set so that the lower surface 10b is immersed in the surface of the cleaning solution in the container 12. Thereafter, the space 15 is depressurized by the pump 16 to supply the cleaning liquid into the pores 11. The reduced pressure in the space 15 sucks the cleaning solution into the pores 11, and the liquid level gradually rises to the upper surface 10 a side of the inspection chip 10. When the liquid level of the cleaning liquid exceeds at least the upper surface 10a of the inspection chip 10 and reaches the cleaning liquid reference position preset above the reaction liquid reference position, the pressure reducing operation by the pump 16 is switched to the pressurizing operation to open the space 15 Pressurize to discharge the cleaning solution in the pores 11. The washing liquid is supplied to the upper side of the position where the sample liquid is supplied, so that sufficient washing can be performed.
Step S3 may be repeated multiple times. Here, for example, the washing effect is enhanced three times repeatedly.
　次に、第２の反応プロセスを実施する（Ｓ４）。第２の反応プロセスにおける第２の反応液は、標識物質を含む標識溶液である。標識物質は、被検物質と特的に結合する物質に標識が付与されたものである。
　標識溶液を容器１２に入れ、検査チップ１０を容器１２中の標識溶液の表面に下面１０ｂが浸されるようにセットする。その後、ポンプ１６により空間１５を減圧して、細孔１１内に標識溶液を供給する。空間１５の減圧により標識溶液が細孔１１中に吸い上げられ、その液面は、徐々に検査チップ１０の上面１０ａ側に上昇する。このとき、標識溶液が反応液基準位置まで到達したら、ポンプ１６による減圧動作を停止して、空間１５を大気圧に戻す。そして、標識溶液を細孔１１中に留めた状態で３０分間保持する。その後、ポンプ１６により空間１５を加圧して細孔１１中の標識溶液を排出する（Ｓ５）。
　Ｓ４－Ｓ５の工程は、複数回繰り返してもよい。ここでは、例えば、３回繰り返し、細孔１１中の内壁に固定されている捕捉物質との特異的な結合した被検物質への標識物質の結合反応を促進させる。 Next, a second reaction process is performed (S4). The second reaction solution in the second reaction process is a labeling solution containing a labeling substance. The labeling substance is one in which a substance that specifically binds to the test substance is labeled.
The labeling solution is placed in the container 12, and the test chip 10 is set so that the lower surface 10b is immersed in the surface of the labeling solution in the container 12. Thereafter, the space 15 is depressurized by the pump 16 to supply the labeling solution into the pore 11. The label solution is sucked into the pores 11 by the pressure reduction of the space 15, and the liquid level gradually rises to the upper surface 10 a side of the test chip 10. At this time, when the labeling solution reaches the reaction liquid reference position, the pressure reducing operation by the pump 16 is stopped to return the space 15 to the atmospheric pressure. Then, the labeling solution is held in the pores 11 for 30 minutes. Thereafter, the space 15 is pressurized by the pump 16 to discharge the labeling solution in the pores 11 (S5).
The steps S4 to S5 may be repeated multiple times. Here, for example, the binding reaction of the labeled substance to the specifically bound test substance with the capture substance fixed on the inner wall in the pore 11 is repeated three times.
　次に、第２の洗浄プロセスを実施する（Ｓ６）。第２の洗浄プロセスは第１の洗浄プロセスと同様である。 Next, a second cleaning process is performed (S6). The second cleaning process is similar to the first cleaning process.
　続いて、第３の反応プロセスを実施する（Ｓ７）。第３の反応プロセスにおける第３の反応液は、検査用溶液である。
　検査用溶液を容器１２に入れ、検査チップ１０を容器１２中の検査用溶液の表面に下面１０ｂが浸されるようにセットする。その後、ポンプ１６により空間１５を減圧して、検査用溶液を細孔１１内に供給する。空間１５の減圧により検査用溶液が細孔１１中に吸い上げられ、その液面は、徐々に検査チップ１０の上面１０ａ側に上昇する。検査用溶液の液面が検査チップ１０の上面１０ａを超えて反応液基準位置に到達したら、ポンプ動作を停止して、空間１５を大気圧に戻す。空間１５を大気圧に戻しても検体液は細孔１１内に留まる。 Subsequently, the third reaction process is carried out (S7). The third reaction solution in the third reaction process is a test solution.
The test solution is placed in the container 12, and the test chip 10 is set so that the lower surface 10b is immersed in the surface of the test solution in the container 12. Thereafter, the space 15 is depressurized by the pump 16 to supply the test solution into the pore 11. The reduced pressure of the space 15 sucks the test solution into the pores 11 and the liquid level gradually rises to the upper surface 10 a side of the test chip 10. When the liquid level of the test solution exceeds the upper surface 10 a of the test chip 10 and reaches the reaction liquid reference position, the pump operation is stopped and the space 15 is returned to the atmospheric pressure. The sample liquid remains in the pore 11 even if the space 15 is returned to the atmospheric pressure.
　検査チップ１０の細孔１１中に検査用溶液を保持させたまま、検査チップ１０を容器１２から取り外し、その下面１０ｂを検査用溶液から離間させる（Ｓ８）。ここで、検査用溶液供給時に下面であった検査チップ１０の他方の面１０ｂを上面となるように、検査チップ１０を回転させて上下面を反転させる（Ｓ９）。 While holding the test solution in the pores 11 of the test chip 10, the test chip 10 is removed from the container 12, and the lower surface 10b thereof is separated from the test solution (S8). Here, the inspection chip 10 is rotated to turn over the upper and lower surfaces so that the other surface 10b of the inspection chip 10, which was the lower surface at the time of supplying the solution for inspection, is the upper surface (S9).
　その後、検査チップ１０を、光信号測定部２２の光検出器２２ａの下方に、他方の面１０ｂが鉛直上方となり、光検出器２２ａと対向するように水平に配置する（Ｓ１０）。
　大気圧下において、検査チップ１０の細孔１１中に検査用溶液を留めた状態で、光検出器２２ａにより検査チップ１０から出射される光を検出し（Ｓ１１）、検査工程は終了となる。 Thereafter, the inspection chip 10 is disposed horizontally below the light detector 22a of the light signal measurement unit 22 so that the other surface 10b is vertically upward and faces the light detector 22a (S10).
The light emitted from the inspection chip 10 is detected by the light detector 22a in a state where the inspection solution is retained in the pores 11 of the inspection chip 10 under atmospheric pressure (S11), and the inspection process is completed.
　検査チップ１０の細孔１１が、大気中において細孔１１内に検査用溶液を保持できる形状であることから、上記のように、下面から吸い上げた検査用溶液を細孔１１中に留めたまま、圧力制御をすることなく光信号測定部２２へ移動することが可能である。また、検査チップ１０の複数の細孔１１中において、検査チップ１０上面側の液体表面位置が、液体の自重と表面張力のバランスによりほぼ一定位置に揃うため（図２参照）、複数の細孔１１の各細孔１１からの検査精度を向上させることができる。特に、本実施形態のように、支持部材１４の一端に検査チップ１０が支持される構成の場合には、検査チップ１０の上下面を反転させることにより、検査チップ１０の一方の面１０ｂと光検出器２２ａとの間に支持部材１４等の他の部材が位置しない状態となる。これにより、支持部材１４の側壁などに残留する検査用溶液によるノイズを抑制することができ、さらなる精度向上が可能となる。 Since the pore 11 of the inspection chip 10 has a shape capable of holding the inspection solution in the pore 11 in the atmosphere, as described above, the inspection solution sucked from the lower surface is kept in the pore 11 It is possible to move to the light signal measurement unit 22 without performing pressure control. Further, in the plurality of pores 11 of the inspection chip 10, the liquid surface position on the upper surface side of the inspection chip 10 is aligned at a substantially constant position by the balance of the liquid's own weight and surface tension (see FIG. 2). The inspection accuracy from each of the eleven pores 11 can be improved. In particular, in the case where the inspection chip 10 is supported by one end of the support member 14 as in the present embodiment, the upper surface and the lower surface of the inspection chip 10 are inverted to provide light to one surface 10 b of the inspection chip 10 The other members such as the support member 14 are not located between the detector 22a and the like. As a result, noise due to the test solution remaining on the side wall or the like of the support member 14 can be suppressed, and the accuracy can be further improved.
　図７は上記各反応プロセスにおける反応を模式的に示す図である。
　検査チップ１０の細孔１１の内壁面１１ａにはアレルゲンなどの捕捉物質３０が固定されている（Ｓ０）。第１の反応プロセス（Ｓ１）では、細孔１１に検体液が供給され、検体液に含まれている被検物質（例えば、上記アレルゲンと特異的に結合する特異的ＩｇＥ抗体）３２を捕捉物質３０に特異的に結合させる。 FIG. 7 is a view schematically showing a reaction in each of the above reaction processes.
A capture substance 30 such as an allergen is fixed to the inner wall surface 11a of the pore 11 of the test chip 10 (S0). In the first reaction process (S1), the analyte fluid is supplied to the pores 11, and the analyte (for example, a specific IgE antibody that specifically binds to the above-mentioned allergen) 32 contained in the analyte fluid is captured 30 specifically bind.
　そして、検体液を排出し洗浄した後、第２の反応プロセス（Ｓ４）では、被検物質３２と特異的に結合する物質３３（例えば、二次抗体）に標識Ｆが付与されてなる標識物質３５を含む標識溶液を細孔１１が供給され、標識溶液に含まれている標識物質３５を被検物質３２に結合させる。
　標識Ｆは、例えば、ルミノール、ロフィン、ルシゲニンおよびシュウ酸エステルなどの化学発光基質に対して触媒として機能する酵素標識である。 Then, after the sample liquid is discharged and washed, in the second reaction process (S4), the labeled substance obtained by applying the label F to the substance 33 (for example, secondary antibody) that specifically binds to the test substance 32 The labeling solution containing 35 is supplied to the pore 11, and the labeling substance 35 contained in the labeling solution is bound to the test substance 32.
The label F is an enzyme label that functions as a catalyst for chemiluminescent substrates such as, for example, luminol, lophine, lucigenin and oxalate.
　さらに、標識溶液を排出し洗浄した後、第３の反応プロセス（Ｓ７）では、細孔１１内に標識Ｆを触媒として発光反応する発光基質を含む検査用溶液を供給し、発光基質を、標識Ｆを触媒として発光反応させる。光信号測定部２２においては、この発光反応による光信号を光検出器２２ａにより検出する。 Furthermore, after the labeling solution is discharged and washed, in the third reaction process (S7), a test solution containing a luminescent substrate that emits light as a catalyst with the label F is supplied into the pore 11 to label the luminescent substrate. The light emission reaction is performed using F as a catalyst. In the light signal measurement unit 22, the light signal due to the light emission reaction is detected by the light detector 22a.
　なお、第１の反応プロセスおよび第２の反応プロセスの後に実施される洗浄プロセスでは、検体液あるいは標識溶液の残液を洗浄し、細孔１１中に非特異吸着している被検物質３２や標識物質を除去するので、測定信号におけるノイズを抑制することができる。 In the washing process performed after the first reaction process and the second reaction process, the test solution 32 or the like which is nonspecifically adsorbed in the pores 11 is washed by washing the residual solution of the sample solution or the labeling solution. Since the labeling substance is removed, noise in the measurement signal can be suppressed.
　上記において、ＨＲＰ（西洋わさびペルオキシダーゼ）酵素が標識として用いられる場合には、ＨＲＰが触媒として機能するルミノール系の化学発光基質を含有する反応液（ルミノール反応液）を、ＡＬＰ（アルカリホスファターゼ）酵素が用いられる場合にはジオキセタン系化学発光基質を含有する反応液を用いることが好ましい。 In the above, when HRP (horseradish peroxidase) enzyme is used as a label, ALP (alkaline phosphatase) enzyme is a reaction solution (luminol reaction solution) containing a luminol-based chemiluminescent substrate in which HRP functions as a catalyst. When used, it is preferable to use a reaction solution containing a dioxetane-based chemiluminescent substrate.
　ルミノール反応液には、少なくともルミノール基質と過酸化水素水が含まれる。酵素標識は、過酸化水素水存在下において、ルミノールの酸化を触媒するものである。なお、反応液中には、化学発光を増感する増感剤を含むことが好ましい。 The luminol reaction solution contains at least a luminol substrate and a hydrogen peroxide solution. The enzyme label is one that catalyzes the oxidation of luminol in the presence of hydrogen peroxide water. The reaction solution preferably contains a sensitizer for sensitizing chemiluminescence.
　なお、酵素標識を用いた光検出においては、上記の化学発光基質のみならず、発光基質あるいは蛍光基質を含む反応液を用い、呈色（吸光）反応や、蛍光を検出してもよい。 In the light detection using an enzyme label, a color reaction (light absorption) reaction or fluorescence may be detected using a reaction solution containing not only the above-mentioned chemiluminescent substrate but also a luminescent substrate or a fluorescent substrate.
　図８は、本発明の第２の実施形態にかかる検査装置２の概略構成図である。以下においては、第１の実施形態の検査装置１と異なる点を主に説明し、共通する構成については同一の符号を付して詳細な説明を省略する。 FIG. 8 is a schematic block diagram of the inspection apparatus 2 according to the second embodiment of the present invention. In the following, differences from the inspection apparatus 1 according to the first embodiment will be mainly described, and the same reference numerals will be given to the common components and the detailed description will be omitted.
　本実施形態の検査装置２は、液体を保持する容器２４の形状、および液体供給部であるポンプ１６が容器２４に接続されている点で、上記検査装置１の構成と異なる。本検査装置２においては、ポンプ１６は容器２４の空間２５を加減圧することにより検査チップ１０の細孔１１への液体の供給及び細孔１１からの液体の排出を行う。 The inspection device 2 of the present embodiment differs from the configuration of the inspection device 1 in that the shape of the container 24 for holding liquid and the pump 16 as the liquid supply unit are connected to the container 24. In the inspection apparatus 2, the pump 16 supplies and discharges the liquid to and from the pores 11 of the inspection chip 10 by pressurizing and depressurizing the space 25 of the container 24.
　容器２４は、容器２４内部に検査チップ１０と容器２４により閉じた空間２５を構成できる形状を有している。支持部材１４の鍔部１４ｃは容器２４の上面の一部に係止可能とされて、容器２４中に検査チップ１０を水平に支持することができる。 The container 24 has a shape that can form a closed space 25 inside the container 24 by the inspection chip 10 and the container 24. The flange portion 14 c of the support member 14 can be locked to a part of the upper surface of the container 24 to horizontally support the inspection chip 10 in the container 24.
　検査チップ１０が容器２４中の液体に他方の面１０ｂが接触するように水平に支持された状態で、ポンプ１６により空間２５が加圧されると、検査チップ１０の面１０ｂ側から細孔１１中に液体が押し上げられる。また、細孔１１中に液体が入っている状態で、空間２５が減圧されると、検査チップ１０の細孔１１中から液体が下方に吸引されて排出される。このポンプ１６の動作により、細孔１１内への液体の供給および排出が行われる。本例では、ポンプ１６による空間２５の加圧動作が液体の供給動作に相当し、空間２５の減圧動作が液体の排出動作に相当する。 When the space 25 is pressurized by the pump 16 while the inspection chip 10 is horizontally supported so that the other surface 10 b is in contact with the liquid in the container 24, the pores 11 from the surface 10 b side of the inspection chip 10 The liquid is pushed up inside. When the space 25 is depressurized with the liquid contained in the pores 11, the liquid is sucked downward from the pores 11 of the inspection chip 10 and discharged. The operation of the pump 16 supplies and discharges the liquid into the pores 11. In this example, the pressurizing operation of the space 25 by the pump 16 corresponds to the liquid supplying operation, and the depressurizing operation of the space 25 corresponds to the liquid discharging operation.
　検査方法は、細孔１１内への液体の供給および排出の動作が減加圧であるか加減圧であるかが違うだけで、先に説明した検査方法と同様である。なお、上記で説明した検査方法においては、検査チップ１０に検査溶液を供給した後、検査チップ１０の上下面を反転させることとしたが、図８の光信号測定部２２において示すように、検査チップ１０の上下面の反転することなく、供給時の上下関係のまま光信号測定部２２に設置されて、光検出に供されても構わない。但し、既述の通り、上下面を反転させることにより、液体保持部１４ｂに残留する検査溶液によるノイズを抑制することができるため、反転工程を経ることがより好ましい。 The inspection method is the same as the inspection method described above except that the operation of supplying and discharging the liquid into the pore 11 is either pressure reduction or pressure reduction. In the inspection method described above, the upper and lower surfaces of the inspection chip 10 are reversed after the inspection solution is supplied to the inspection chip 10. However, as shown in the optical signal measurement unit 22 of FIG. It may be installed in the light signal measuring unit 22 without changing the upper and lower surfaces of the chip 10 and in the vertical relation at the time of supply, and may be used for light detection. However, as described above, it is more preferable to go through the reversing step, since the noise due to the test solution remaining in the liquid holding portion 14b can be suppressed by reversing the upper and lower surfaces.
　１、２　検査装置
　１０　検査チップ
　１０ａ　検査チップの一方の面
　１０ｂ　検査チップの他方の面
　１１　細孔
　１１ａ　内壁面
　１２　容器
　１４、１１４、２１４　支持部材（検査チップ支持部）
　１４ａ　検査チップ受容部
　１４ｂ　液体保持部
　１４ｃ　鍔部
　１５　空間
　１６　ポンプ（液体供給部）
　２２　光信号測定部
　２２ａ　光検出器
　２２ｂ　レンズ
　２３　暗室
　２４　容器
　２５　容器内の空間
　３０　捕捉物質
　３２　被検物質
　３３　物質
　３５　標識物質
　５０　検査用溶液
　Ｆ　　標識 1, 2 inspection apparatus 10 inspection chip 10a one surface of an inspection chip 10b the other surface of an inspection chip 11 pore 11a inner wall surface 12 container 14, 114, 214 support member (inspection chip support portion)
14a Test chip receiving portion 14b Liquid holding portion 14c Edge portion 15 Space 16 Pump (liquid supply portion)
22 light signal measurement unit 22a light detector 22b lens 23 dark room 24 container 25 space in container 30 capture substance 32 test substance 33 substance 35 labeled substance 50 test solution F label

Claims (9)

  1. 　一方の面から他方の面まで一定の孔径で貫通した複数の細孔を有する板状の検査チップであって、前記細孔の内壁面に特定の物質と特異的に結合する捕捉物質が固定化されてなり、前記細孔が該細孔内に充填された検査用溶液を、大気圧下において毛細管現象により保持できる形状である検査チップを用い、
    　該検査チップの前記細孔に、検査に供する検体液を供給して、前記検体液中の特定の物質を前記捕捉物質に結合させ、前記特定の物質と特異的に結合する標識物質を前記特定の物質に結合させた後、
    　前記検査チップを、前記検査用溶液が貯留された容器内に、一方の面を鉛直上向きとして水平に支持し、
    　前記容器内を加圧することにより、あるいは前記検査チップ上の空間を減圧することにより、前記検査用溶液を、前記検査チップの下面である前記他方の面側から前記細孔に供給し、
    　その後、前記加圧された前記容器内、あるいは前記減圧された前記空間を大気圧に開放し、
    　前記検査チップの前記細孔内に前記検査用溶液を保持させたまま、前記検査チップを前記容器に貯留された前記検査用溶液から離間させ、
    　前記検査チップから出射される光を、前記検査チップの鉛直上方から検出する検査方法。     A plate-like inspection chip having a plurality of pores penetrating at a constant pore diameter from one surface to the other surface, wherein a capture substance that specifically binds to a specific substance is immobilized on the inner wall surface of the pores Using a test chip having a shape capable of holding the test solution having the pores filled in the pores by capillary action under atmospheric pressure,
    A sample fluid to be tested is supplied to the pores of the test chip to bind a specific substance in the sample fluid to the capture substance, and a labeled substance which specifically binds to the specific substance is identified. After binding to the substance of
    The test chip is horizontally supported in a container in which the test solution is stored, with one surface facing vertically upward,
    The solution for inspection is supplied to the pores from the other surface, which is the lower surface of the inspection chip, by pressurizing the inside of the container or by reducing the pressure on the space above the inspection chip.
    Thereafter, the pressurized container or the depressurized space is opened to atmospheric pressure,
    With the test solution held in the pores of the test chip, the test chip is separated from the test solution stored in the container;
    The inspection method which detects the light emitted from the inspection chip from the perpendicular upper part of the inspection chip.
  2. 　前記検査チップを前記検査用溶液から離間させた後に、前記検査チップの上下が逆になるように回転させて前記他方の面を鉛直上向きとして、水平に配置し、
    　前記光を前記他方の面側から検出する請求項１記載の検査方法。     After separating the test chip from the test solution, the test chip is rotated so that the top and bottom of the test chip are reversed so that the other surface is vertically oriented and horizontally arranged.
    The inspection method according to claim 1, wherein the light is detected from the other surface side.
  3. 　前記検査チップが、前記複数の細孔の各細孔の形状が、前記検査用溶液の表面張力をＴ、前記検査用溶液の前記細孔の内壁面に対する接触角をθ、重力加速度をｇ、および前記検査用溶液の密度をρとしたとき、
    　前記細孔の外周長×Ｔ×Cosθ＞前記細孔の中空部体積×g×ρ
    を満たす、請求項１または２に記載の検査方法。     The shape of each of the plurality of pores of the inspection chip is the surface tension of the solution for inspection, T, the contact angle of the solution for inspection with the inner wall surface of the pores, and the gravitational acceleration g. And the density of the test solution is ρ
    Outer peripheral length of the pore × T × Cos θ> hollow volume of the pore × g × ρ
    The inspection method according to claim 1 or 2, wherein
  4. 　前記検査チップが、Ｓｉ、ＳｉＯ２、Ａｌ、Ａｌ２Ｏ３、ステンレス鋼および樹脂材料のうちの１つまたは２つ以上の材料からなる請求項１から３いずれか１項に記載の検査方法。 The test chip, Si, SiO 2, Al, Al 2 O 3, 1 or 2 or more inspection method according to claim 1 comprising a material 3 any one of stainless steel and a resin material.
  5. 　前記検査チップは、前記細孔の前記一方の面における開口領域の円相当直径が１μｍ～１００μｍである請求項１から４いずれか１項に記載の検査方法。 5. The inspection method according to any one of claims 1 to 4, wherein an equivalent circle diameter of an opening area on the one surface of the pore of the inspection chip is 1 μm to 100 μm.
  6. 　前記検査チップは、厚さが１００μｍ～２０００μｍである請求項１から５いずれか１項に記載の検査方法。 The inspection method according to any one of claims 1 to 5, wherein the inspection chip has a thickness of 100 μm to 2000 μm.
  7. 　前記捕捉物質が、抗原、抗体またはデオキシリボ核酸である請求項１から６いずれか１項に記載の検査方法。 The test method according to any one of claims 1 to 6, wherein the capture substance is an antigen, an antibody or deoxyribonucleic acid.
  8. 　前記標識物質として酵素標識を含む物質を用い、
    　前記検査用溶液として、前記酵素標識により触媒されて反応する基質を含む溶液を用い、
    　前記出射される光として、前記検査用溶液中の前記基質が前記酵素標識により触媒されて生じる光を検出する請求項１から７いずれか１項に記載の検査方法。     A substance containing an enzyme label is used as the labeling substance,
    As the test solution, a solution containing a substrate which is catalyzed and reacted by the enzyme label is used.
    The inspection method according to any one of claims 1 to 7, wherein as the emitted light, light generated by being catalyzed by the enzyme label by the substrate in the test solution is detected.
  9. 　一方の面から他方の面まで一定の孔径で貫通した細孔を複数有する板状の検査チップであって、前記細孔の内壁面に特定の物質と特異的に結合する捕捉物質が固定化されてなり、前記細孔が該細孔内に充填された検査用溶液を、大気圧下において毛細管現象により保持できる形状である検査チップと、
    　前記検査用溶液を貯留する容器と、
    　前記検査チップを、前記容器内において支持する支持部材と、
    　前記検査チップの前記細孔に前記検査用溶液を供給する液体供給部と、
    　前記検査チップから出射される光を、水平に配置された前記検査チップの鉛直上方から検出する光検出器とを備えた検査装置。     A plate-like inspection chip having a plurality of pores penetrating at a constant pore diameter from one surface to the other surface, wherein a capture substance that specifically binds to a specific substance is immobilized on the inner wall surface of the pores An inspection chip having a shape capable of holding the inspection solution in which the pores are filled in the pores by capillary action under atmospheric pressure;
    A container for storing the test solution;
    A support member for supporting the inspection chip in the container;
    A liquid supply unit for supplying the test solution to the pores of the test chip;
    An inspection apparatus comprising: a light detector that detects light emitted from the inspection chip from vertically above the inspection chip arranged horizontally.
PCT/JP2017/026705 2016-08-16 2017-07-24 Inspection method and inspection device WO2018034109A1 (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0627111A (en) * 1992-07-09 1994-02-04 Olympus Optical Co Ltd Reaction vessel
JP2005148048A (en) * 2003-04-25 2005-06-09 Jsr Corp Biochip, biochip kit, and method for manufacturing and using the same
JP2006153889A (en) * 2001-03-21 2006-06-15 Olympus Corp Biochemical test method

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0627111A (en) * 1992-07-09 1994-02-04 Olympus Optical Co Ltd Reaction vessel
JP2006153889A (en) * 2001-03-21 2006-06-15 Olympus Corp Biochemical test method
JP2005148048A (en) * 2003-04-25 2005-06-09 Jsr Corp Biochip, biochip kit, and method for manufacturing and using the same

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