WO2017169716A1 - Inspection device, inspection apparatus, and inspection method - Google Patents

Inspection device, inspection apparatus, and inspection method Download PDF

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Publication number
WO2017169716A1
WO2017169716A1 PCT/JP2017/010099 JP2017010099W WO2017169716A1 WO 2017169716 A1 WO2017169716 A1 WO 2017169716A1 JP 2017010099 W JP2017010099 W JP 2017010099W WO 2017169716 A1 WO2017169716 A1 WO 2017169716A1
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Prior art keywords
inspection
pores
substance
inspection device
test
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PCT/JP2017/010099
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French (fr)
Japanese (ja)
Inventor
弘隆 渡野
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富士フイルム株式会社
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Publication of WO2017169716A1 publication Critical patent/WO2017169716A1/en

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    • 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
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • 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
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
    • 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
    • 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
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N37/00—Details not covered by any other group of this subclass

Definitions

  • the present invention relates to an inspection device for detecting a test substance that is an antibody or an antigen, an inspection apparatus provided with the inspection device, and an inspection method.
  • the binding phenomenon between a test substance and a capture substance is optically determined.
  • a method of detecting is known. In this method, a test substance is bound to a capture substance fixed at a predetermined position, and a label that emits fluorescence upon receiving excitation light, or a label that catalyzes a substrate reaction to generate color, fluorescence, or chemiluminescence. Or the like is applied to the test substance, and light generated due to the label is detected.
  • a method of detecting a fluorescence generated from a fluorescent label by attaching a fluorescent label to a binding substance such as an antibody that specifically binds to the test substance, a binding of an antibody that specifically binds to the test substance
  • a binding substance such as an antibody that specifically binds to the test substance
  • a binding of an antibody that specifically binds to the test substance There are known methods for detecting color development, fluorescence, chemiluminescence, etc. generated from a chromogenic substrate, fluorescent substrate, or chemiluminescent substrate that reacts with this enzyme as a catalyst. Identification becomes possible.
  • a multi-item simultaneous detection chip in which biologically related molecules are regularly arranged on a two-dimensional substrate has been conventionally used.
  • a device made of a porous substrate in which a large number of through-holes (pores) are arranged on a support has been studied.
  • Patent Document 1 discloses a method in which a solution containing a trapping substance is impregnated into a thread or a membrane, and the trapping substance is fixed to the thread by drying. And a method of immobilizing a capture substance inside.
  • Patent Document 2 proposes a device in which silicon having a large number of pores is thermally oxidized to form locally transparent silicon oxide (SiO 2 ), and an organic silicon compound as a linker is used as a DNA.
  • a technique is disclosed in which a capturing substance such as a protein or a ligand is immobilized in a pore by a covalent bond.
  • JP 2001-515735 A Japanese Patent No. 4125244
  • Patent Document 1 as a specific method for immobilizing a capture substance, only a method for immobilizing the capture substance on a thread or a membrane is cited, and the capture substance in the pores of a device made of a porous substrate is mentioned. The immobilization of is not described.
  • Patent Document 2 discloses a method of covalently binding a capture substance to a linker made of an organosilicon compound. However, since covalent bond strongly binds only to a specific substance, a plurality of different types of capture substances are different in one device. It is not suitable for fixing.
  • the inspection device of the present invention is an inspection device having a plurality of pores penetrating from one side of a plate-like substrate made of an inorganic material to the other side, A hydrophobic polymer is bonded to the inner wall surface of at least two or more pores,
  • This is a test device in which different capture substances consisting of an antigen or an antibody that specifically bind to a test substance are physically adsorbed to hydrophobic polymers of different pores.
  • the hydrophobic polymer means a polymer having a contact angle of 80 ° or more measured by an inspection method of JIS (Japanese Industrial Standards) R3257.
  • the hydrophobic polymer and the plate-like substrate are R—O— via an oxygen atom O, where R is a hydrophobic polymer and M is an inorganic substance constituting the plate-like substrate. It is preferable that it is covalently bonded by M bond.
  • the hydrophobic polymer is preferably polystyrene or polypropylene.
  • the inorganic material is preferably one or more of Si, SiO 2 , Al, and Al 2 O 3 .
  • the inspection device of the present invention includes a plurality of inspection regions including a plurality of pores to which the trapping substance is adsorbed when viewed from one surface, and between the inspection regions arranged adjacent to each other among the plurality of inspection regions. , Separated by a non-inspection region having a width wider than the pore spacing in the adjacent inspection regions, and the same type of trapping substance is physically adsorbed in the pores in one inspection region, It is preferable that different trapping substances are physically adsorbed in the pores of one inspection region and the other inspection region among the inspection regions arranged adjacent to each other.
  • the non-inspection region can be a region in which non-inspection pores in which a trapping substance is absent from a plurality of pores are arranged.
  • the inspection device of the present invention may include a cavity that extends in the depth direction of the pores and is non-penetrating on at least the other surface in the non-inspection region.
  • the non-inspection region may be a portion of a plate-like base material that does not have pores.
  • one side of the inspection region in the alignment direction of the non-inspection region and the inspection region is longer than the width of the non-inspection region.
  • one inspection region and the other inspection region are periodically arranged.
  • differently captured substances may include a crude purified allergen and at least one allergen component contained in the crude purified allergen.
  • the inspection apparatus of the present invention includes the inspection device of the present invention, A solution supply unit for supplying an inspection solution into the pores of the inspection device;
  • the inspection apparatus includes a photodetector that is disposed on one surface side or the other surface side of the inspection device and detects light emitted from the inspection device.
  • a specimen liquid containing a test substance is supplied to at least some of the pores of the test device of the present invention, and the test substance is bound to a capture substance, A labeling substance that specifically binds to the test substance is bound to the test substance,
  • the inspection method detects light emitted from the inspection device in a state where the inspection solution is supplied to the pores and the inspection solution is retained in the pores.
  • a substance containing an enzyme label is used as the labeling substance
  • a reaction solution containing a substrate that reacts catalyzed by the enzyme label is used as the inspection solution
  • light emitted from the inspection device is used.
  • light generated by catalyzing a substrate in the reaction solution by an enzyme label may be detected.
  • a substance containing a fluorescent label is used as the labeling substance, the inspection device is irradiated with excitation light that excites the fluorescent label, and the light emitted from the inspection device is irradiated with excitation light. Fluorescence generated from the labeling substance may be detected.
  • An inspection device of the present invention is an inspection device having a plurality of pores penetrating from one surface of a plate-like substrate made of an inorganic material to the other surface, and is hydrophobic on the inner wall surface of at least two or more pores. Since different types of capture substances can be easily immobilized in a single device, different capture molecules can be captured by different hydrophobic polymers composed of antigens or antibodies. Since the substance is physically adsorbed, simultaneous inspection of multiple items is possible.
  • FIG. 1 is a perspective view of an inspection device according to a first embodiment of the present invention. It is a top view of the inspection device shown in FIG. It is sectional drawing of the test
  • FIG. 1 is a perspective view of an inspection device according to a first embodiment of the present invention
  • FIG. 2 is a plan view of the inspection device of FIG. 1
  • FIG. 3 is a cross-sectional view taken along line III-III of the inspection device of FIG. .
  • the plan view shown in FIG. 2 is a view of the inspection device 1 as seen from one surface 11.
  • the inspection device 1 of this embodiment includes a plurality of pores 14 penetrating from one surface 11 to the other surface 12 in a plate-like substrate 10 made of an inorganic material.
  • a hydrophobic polymer is bonded to the inner wall surface 14a of the pore 14 of the test device 1, and different capture substances 30A, 30B, 30C made of antigens or antibodies are attached to the hydrophobic polymer of the different pores 14 ... Is physically adsorbed.
  • the part where the species of the trapping substance needs to be distinguished is simply indicated as the trapping substance 30, and the branch code such as the trapping substance 30A, the trapping substance 30B,. It is set as the notation which added A, B ....
  • this inspection device 1 includes a plurality of pores 14 and different capture substances 30A, 30B, 30C,... Are physically adsorbed between the pores 14, by using this inspection device 1, a plurality of analytes can be detected. Inspection can be performed at the same time.
  • the external shape of the inspection device 1 is configured by the external shape of the plate-like base material 10
  • one surface 11 and the other surface 12 of the plate-like base material 10 are hereinafter referred to as one of the inspection devices 1.
  • the surface and the other surface are hereinafter referred to as the surface and the other surface.
  • both surfaces may be named generically and may be called the surface of the plate-shaped base material 10 (surface of a test
  • the plate-like substrate 10 may be composed of one or more materials of Si (silicon), SiO 2 (silicon oxide), Al (aluminum), and Al 2 O 3 (alumina). preferable.
  • the plate-like substrate 10 may be opaque made of Si or Al, or may be transparent made of SiO 2 or Al 2 O 3 .
  • the plate-like substrate 10 may be composed of any single material, or may include, for example, a Si portion and a SiO 2 portion by partially oxidizing the Si substrate,
  • the Al base material may be partially oxidized to include an Al portion and an Al 2 O 3 portion.
  • a hydrophobic polymer is bonded to the inner wall surface 14 a of the pore 14 of the inspection device 1.
  • the hydrophobic polymer is a polymer having a contact angle of 80 ° or more, and examples thereof include polystyrene, polypropylene, polyethylene, polytetrafluoroethylene, and silicon rubber. Of these, polystyrene and polypropylene are preferred.
  • FIG. 4A schematically shows a step of bonding the hydrophobic polymer pores 14 to the inner wall surface 14a.
  • FIG. 4A only the cross section of one pore 14 of the inspection device 1 is shown, but the binding treatment of the hydrophobic polymer to all the pores 14 in the inspection device 1 can be performed simultaneously.
  • a hydrophobic polymer may be simultaneously applied to the pores 14 provided in the non-inspection region described later.
  • the surface of the plate-like substrate 10 made of an inorganic material is cleaned by ultraviolet irradiation (A1).
  • the pores 14 are filled with a solution 45 containing a hydrophobic polymer having a hydroxyl group (OH group) (A2).
  • the inner wall surface 14a of the pores 14 and the hydrophobic polymer are covalently bonded by heat treatment and dehydration condensation (A3).
  • the hydrophobic polymer that has not been bonded is removed with a solvent to form a chemically modified layer (hydrophobic polymer layer) 46 in which the hydrophobic polymer is bonded to the inner wall surface 14 a of the pore 14. .
  • the plate-like substrate 10 having the pores 14 in which the hydrophobic polymer is bonded to the inner wall surface 14a can be obtained (A4).
  • FIG. 4B is a conceptual diagram schematically showing the binding state between the hydrophobic polymer and the plate-like substrate and the adsorption state of the trapping substance.
  • a case where polystyrene is bonded as a hydrophobic polymer to the pore inner wall surface 14a of the substrate 10 made of Si will be described.
  • Heat treatment is performed in a state where a solution containing polystyrene having an OH group is in contact with the Si surface (inner wall surface 14a). Thereby, the OH group of polystyrene and the OH group naturally formed on the Si surface are dehydrated and condensed.
  • the antigen or antibody that is the capture substance 30 is a protein.
  • a protein molecule is a polymer in which an amino acid having a hydrophilic group and an amino acid having a hydrophobic group are bonded.
  • the surface of a protein molecule has a mosaic structure composed of a hydrophilic part and a hydrophobic part. The hydrophobic part of the protein surface is easily adsorbed to the hydrophobic polymer layer.
  • the capture substance 30 made of a protein molecule such as an antigen or an antibody has a hydrophilic part and a hydrophobic part, so that it is easily adsorbed to the hydrophobic polymer.
  • the hydrophobic polymer is covalently bonded to each inner wall surface 14a of the plurality of pores 14, different capture substances 30A, 30B, 30C,. Is easy.
  • the capture substance on the inner wall surface with a covalent bond only a specific molecule capable of covalent bond can be immobilized, and the desired capture substance may not be immobilized. Physical adsorption is preferable because it can be applied to almost all protein molecules.
  • the thickness of the plate-like substrate 10 is not particularly limited, but is preferably about 100 ⁇ m to 2000 ⁇ m.
  • the pores 14 provided in the plate-like substrate 10 are preferably arranged in an aligned manner as shown in the present embodiment, but may be arranged randomly.
  • 49 pores of 7 rows ⁇ 7 columns are provided for easy visual recognition, but the inspection device 1 includes, for example, 100 pores of 10 rows ⁇ 10 columns.
  • n ⁇ m pores of n rows ⁇ m columns may be used as one inspection region, and a plurality of inspection regions may be provided (n and m are positive integers).
  • the opening and cross-sectional shape of the pore 14 are square in this embodiment, but are not limited to a rectangle such as a square or a rectangle, but may be a circle, an ellipse, a triangle, or a polygon that is a pentagon or more. Note that the corners of the polygon may be rounded due to manufacturing reasons.
  • the pores 14 are generally columnar and the cross-sectional shape does not change, but the cross-sectional shape may partially change or the cross-sectional size may change.
  • the equivalent circle diameter of the opening in at least one surface 11 of the pore 14 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 the same area as the area of the opening region.
  • the test substance (target molecule) to be tested in the present testing device 1 is mainly a biological molecule, and is an antibody or an antigen.
  • the capture substance 30 made of an antigen or antibody fixed to the inner wall surface 14a of the pore 14 a substance that specifically binds to a desired test substance can be selected.
  • the test device 1 is suitable for an allergy test provided with an allergen that is a kind of antigen as a capture substance.
  • the inspection device 1 preferably includes an inspection region provided with the same kind of trapping substance in common with the plurality of pores 14.
  • 5A and 5B are schematic plan views of inspection devices 1A and 1B of a design change example each having a plurality of inspection regions.
  • the inspection device 1A includes an inspection area 20A and an inspection area 20B.
  • 9 ⁇ 3 ⁇ 9 pores 14 are provided as shown in the enlarged views.
  • the capture substance 30A is physically adsorbed in the pores 14 in the inspection region 20A, and the capture material 30B of a type different from the capture material 30A is provided in the pores 14 in the inspection region 20B.
  • the inspection device 1B includes inspection areas 20A, 20B, 20C, and 20D. Each inspection region 20A, 20B, 20C and 20D is provided with a plurality of pores 14 as shown in an enlarged view in FIG. 5A. Further, in the inspection region 20A, the trapping substance 30A is in the pore 14, the trapping material 30B is in the pore 14 in the inspection region 20B, and the trapping substance 30C is in the pore 14 in the inspection region 20C. The trapping substance 30D is physically adsorbed to the pores 14 in 20D. In the inspection device 1B, four sets are arranged with these four regions as one set.
  • the inspection accuracy can be improved by providing an inspection region containing a plurality of pores including the same type of capture substance. Furthermore, by periodically arranging a plurality of inspection regions having the same type of capturing substance, it is possible to make the accuracy variation uniform on the inspection device 1 and further improve the inspection accuracy.
  • allergen components are called allergen components, and allergen components include allergen components that are strongly associated with clinical symptoms and other allergen components. For this reason, if it is not possible to determine the presence or absence of allergy by measuring crudely purified allergen, it is desirable to perform IgE measurement on allergen components that are highly related to clinical symptoms.
  • a test region in which the crude purified allergen is provided as a capture substance and a test pore in which an allergen component that is strongly associated with clinical symptoms in the crude purified allergen is provided as a capture substance. It is preferable to prepare and enable inspection at the same time.
  • a crudely purified allergen containing a plurality of allergen components is also regarded as one kind of trapping substance.
  • egg white is used as the capturing substance 30A as the inspection area 20A
  • ovomucoid which is an egg white allergen component
  • wheat is used as the capturing substance 30C as the inspection area 20C
  • wheat ⁇ -5 gliadin which is an allergen component
  • the inspection device when viewed from one surface, the inspection device includes a plurality of inspection regions including a plurality of pores to which the trapping substance is physically adsorbed, and is disposed adjacent to each other among the plurality of inspection regions.
  • the gap is preferably separated by a non-inspection region having a width wider than the pore interval in the adjacent inspection regions.
  • FIG. 6 is a plan view of the inspection device 2 according to the second embodiment.
  • the trapping substance 30 ⁇ / b> A is physically adsorbed in the pores 14 indicated by the diagonal lines rising to the right
  • the trapping substance 30 ⁇ / b> B is physically adsorbed in the pores 14 indicated by the diagonal lines rising to the left.
  • the pores 14 not hatched are non-inspection pores to which no capture substance is applied and no capture substance is present (absent).
  • the inspection region 20 provided with the capture substance 30A corresponds to the inspection region 20A in FIGS. 5A and 5B
  • the inspection region 20 provided with the capture material 30B corresponds to the inspection region 20B in FIGS. 5A and 5B.
  • the inspection area 20 is simply indicated.
  • the four pores 14 in one inspection region 20 are provided with the same kind of capture substance, and the region provided with the capture material 30A and the region provided with the capture material 30B are not inspected.
  • the regions 21 are arranged alternately.
  • the width b of the non-inspection region 21 between the inspection regions 20 arranged adjacent to each other is wider than the pore interval c in the inspection region 20 arranged with the width b therebetween.
  • the width b of the non-inspection area 21 is the shortest distance between the inspection areas 20 in the alignment direction of the inspection area 20 and the non-inspection area 21.
  • the pore interval c is the shortest distance between re-adjacent pores arranged along the direction of arrangement.
  • the length “a” of one side of the inspection region 20 along the arrangement direction is longer than the width “b” of the non-inspection region 21. That is, it is preferable that the side a of the inspection region 20, the width b of the non-inspection region 21, and the pore interval c have a relationship of a> b> c.
  • the inspection area 20 for the capture substance 30A and the inspection area 20 for the capture substance 30B are provided, the inspection for the capture substances 30A and 30B can be performed simultaneously. Further, by separating the inspection regions 20 by the non-inspection region 21, crosstalk between the inspection regions 20 can be suppressed, and a more accurate inspection can be performed.
  • FIG. 7 shows a plan view of the inspection device 3 of the third embodiment.
  • the same four inspection regions 20 as the inspection device 2 of the second embodiment are provided.
  • the inspection device 3 of the present embodiment is different from the inspection device 2 of the second embodiment in that the non-inspection region 21 that separates the inspection regions 20 is not provided with the pores 14.
  • the non-inspection region 21 does not need to be provided with the pores 14, and the same effect as the inspection device 2 of the second embodiment can be obtained.
  • the side a of the inspection region 20, the width b of the non-inspection region 21, and the pore interval c have a relationship of a>b> c.
  • FIG. 8 shows a plan view of the inspection device 4 of the fourth embodiment.
  • inspection regions 20 including four pores 14 are arranged 6 ⁇ 6 vertically and horizontally.
  • the trapping substance 30 ⁇ / b> A is physically adsorbed in the pores 14 indicated by the diagonal lines rising to the right
  • the trapping substance 30 ⁇ / b> B is physically adsorbed in the pores 14 indicated by the diagonal lines rising to the left.
  • the trapping substance 30C is physically adsorbed in the pores 14 indicated by the horizontal stripes
  • the trapping substance 30D is physically adsorbed in the pores 14 indicated by the vertical stripes.
  • 3 ⁇ 3 sets are arranged vertically and horizontally in one inspection device 4 with one region including each of the capture substances 30A, 30B, 30C, and 30D as one set.
  • the present inspection device 4 it is possible to simultaneously perform the inspection on the four capture substances 30A, 30B, 30C, and 30D.
  • by providing a plurality of sets in one device it is possible to suppress inspection variations and increase detection sensitivity.
  • the non-inspection region in the inspection device 4 of the present embodiment has pores 14 that do not include the trapping substance 30.
  • the relationship between the width of the inspection region 20, the width of the non-inspection region, and the pore interval is the same as that of the inspection device 2.
  • FIG. 9 shows a cross-sectional view of the inspection device 5 of the fifth embodiment.
  • the plan view seen from one surface 11 of the inspection device 5 of the present embodiment is the same as the plan view of the inspection device of the second embodiment shown in FIG.
  • the non-inspection region 21 is provided with a hollow portion 16 extending in the depth direction of the pore 14 and not penetrating at least on the other surface 12 instead of the pore.
  • the cavity 16 is a space where at least the other surface 12 is closed.
  • the hollow portion 16 is formed to extend from one surface 11 of the plate-like base material 10 to the front of the other surface 12, and an opening connected to the hollow portion 16 is provided on the other surface 12 of the plate-like base material 10. It is not done.
  • formed to extend to the front means that it extends toward the other surface 12 but does not reach the other surface 12.
  • the length of the cavity 16 extending from the one surface 11 to the other surface 12 side, that is, the depth along the depth direction of the pores 14 is at least half the thickness of the plate-like substrate 10. Is more preferable, and more preferably 3/4 or more.
  • the cavity portion 17 is provided with a through hole 40 penetrating from one surface 11 of the plate-like substrate 10 to the other surface 12.
  • the opening on the other surface 12 side may be formed so as to be closed with a closing member 42 made of a material different from that of the plate-like substrate 10.
  • the hollow portion 17 may have its opening on one surface 11 side closed by a second closing member 44 made of a material different from that of the plate-like substrate 10. That is, the hollow portion 17 may be a closed space.
  • the inside of the cavity may be a vacuum, or a gas having a refractive index of about 1 such as air may be enclosed.
  • the material constituting the closing member 42 and the second closing member 44 constituting the cavity portion 17 is not particularly limited, and is configured as a closing member by being solidified or cured after being dropped by an ink jet apparatus such as a metal or a resin material. Suitable materials are preferred.
  • the inspection region 20 having the plurality of pores 14 is provided as in the inspection devices 2 to 6 of the second to sixth embodiments, at least the inspection region 20 has the plate-like substrate 10 having light transmittance It is preferable that Since the aperture of a plurality of pores is gathered from the aperture of light from one aperture 14 to make the entire inspection region 20 an optical aperture, the optical aperture ratio increases, so that the light emitted to the surface of the device is extracted. The amount of light can be increased.
  • having light transmittance means that the transmittance of detection light described later is 50% or more. Specifically, the average transmittance in the visible light wavelength range of 400 to 700 nm is 50% or more. If there is, it will be regarded as having light transparency.
  • the entire region surrounded by the cavities 16 and 17 has light transmittance.
  • FIG. 11 is a schematic view showing a part of the inspection device 5 in an enlarged manner.
  • a hydrophobic polymer is bonded to the inner wall surface 14a of the pore 14, and a trapping substance (not shown in FIG. 11) is immobilized on the hydrophobic polymer by physical adsorption.
  • Detect the presence or absence of the test substance by binding the test substance to this capture substance, binding the label substance labeled with an enzyme to the test substance, and detecting chemiluminescence using the label substance as a catalyst, or It is used in a test method for detecting the presence and amount of a test substance.
  • the detection of the optical signal from the present inspection device 1 is performed in a state where the pore 14 is filled with an inspection solution 61 such as a buffer solution.
  • an inspection solution 61 such as a buffer solution.
  • the solution 61 may not enter into the cavity part 16, and the cavity part 16 comprises an air layer.
  • the refractive index of the light-transmitting plate-like substrate 10 and the refractive index of the test solution are equivalent in the range of about 1.3 to 1.5, whereas the refractive index of the air layer is about 1. It is. That is, since the refractive index difference between the plate-like substrate 10 and the air layer is very large, a large reflection occurs at the interface.
  • the plate-like substrate 10 since the plate-like substrate 10 has a higher refractive index than the air layer, light incident on the interface C 1 at an incident angle ⁇ 1 greater than the critical angle from the plate-like substrate 10 side toward the air layer is totally reflected. Then, it goes to the surface side of the plate-like substrate 10. Even when the incident light is incident on the interface C 1 from the plate-like substrate 10 side at an incident angle ⁇ 2 smaller than the critical angle, a part of the incident light is specularly reflected at the interface C 1 and It will go to the surface side.
  • the light transmitted through the interface C 1 is incident on the plate-like base material 10 constituting the cavity 16 from the air layer, and at least a part of the light is regularly reflected at the interface C 2 , and the surface of the plate-like base material 10 is again formed. Head to the side.
  • the function of condensing light generated in the pores 14 toward the surface of the plate-like substrate 10 is given. As a result, the amount of light emitted from the surface of the inspection device 1 is increased, and the light extraction efficiency is improved.
  • the amount of light can be reflected at the interface between the plate-like base material and the air layer with almost no attenuation, and the light can be condensed on the surface side of the device.
  • the same effect can be obtained for the inspection device 6 of the sixth embodiment.
  • a plate-like substrate made of SiO 2 having a plurality of pores can be produced as follows. First, a plate-like substrate made of Si is prepared, and pores are formed from one surface side by wet etching or dry etching. For example, a microporous Si manufacturing process known in MEMS (Micro Electro Mechanical System) technology can be used.
  • MEMS Micro Electro Mechanical System
  • the mask when it is set as the structure provided with the cavity part 16 in the non-inspection area
  • the cavity can be formed by etching from one side and stopping the etching before the other side.
  • the whole base material can be made transparent or partially transparent.
  • thermal oxidation treatment is performed at a temperature of 1100 ° C. for a predetermined time.
  • the thermal oxidation treatment time may be appropriately adjusted according to the size and shape of the plate-like substrate and the desired oxidation treatment region.
  • the through hole 40 and the blocking members 42 and 44 are formed as in the hollow portion 17 shown in FIG. 10, first, the through hole 40 constituting a part of the hollow portion is etched simultaneously with the pore 14. Form. Thereafter, the Si base material is changed to SiO 2 by the above thermal oxidation treatment, and then the opening on the other surface side of the through hole 40 constituting a part of the cavity is closed with a closing member 42 made of a material different from the base material. . Similarly, the opening on one surface side of the through hole 40 is closed with the second closing member 44. Thereby, the hollow part 17 used as the closed space can be formed.
  • the blocking member 42 only needs to be able to block the opening on the other surface side of the through hole 40, and may be formed so as to partially enter the through hole 40.
  • the closing member 42 can be formed, for example, by dripping a metal or a resin material so as to close the opening on the other surface side of the through hole using an ink jet device, and solidifying or curing.
  • a plate-like substrate made of Al having a plurality of pores can be produced by forming pores in an Al plate-like substrate by a mechanical method such as a drill, or by etching or the like. Can also be produced. Further, by performing the anodic oxidation process to the plate-shaped substrates made of Al, it is possible to obtain a plate-shaped substrate made of Al 2 O 3 having a plurality of pores.
  • the production methods of the plate-like substrate 10 having pores are not limited to these, and various known methods for forming a porous substrate can be used.
  • Immobilizing the capture substance which is a specific binding substance that specifically binds to the test substance, on the inner wall surface of the pores of the plate-like substrate having a plurality of pores produced as described above
  • an inspection device can be manufactured.
  • the trapping substance is fixed to the inner wall surface of the pore by forming a chemically modified layer with a hydrophobic polymer on the inner wall surface of the pore, and then by physical adsorption of the trapping substance on the hydrophobic polymer. Can be implemented.
  • a polystyrene solution in which polystyrene having a molecular weight of about 1000 to 10,000 is dissolved in a toluene solvent at 1% by mass is applied by dip coating, etc. This fills the pores 14 with the polystyrene solution, which is 160 to 180 ° C. Then, heat treatment is performed for several hours to 3 days to dehydrate and condense, and the inner wall surface 14a of the pores 14 and polystyrene are bonded to each other, and then polystyrene not bonded to the inner wall surface 14a is removed with toluene to 10 nm.
  • the following chemically modified layer 46 can be formed.
  • a method for physically adsorbing allergen on polystyrene as a capture substance is as follows.
  • the allergen extract is diluted with PBS (Phosphate buffered saline) so as to be 10 ⁇ g / mL, and dispensed into predetermined pores 14 on which the allergen is physically adsorbed by a spotter.
  • PBS Phosphate buffered saline
  • BSA Bovine serum albumin
  • an inspection device in which blocking is applied can be obtained. Note that different allergens can be physically adsorbed in the same procedure for each pore of the inspection device or for each inspection region including a plurality of pores.
  • FIG. 12 is a diagram schematically showing the configuration of the inspection apparatus 50 according to an embodiment of the present invention.
  • the inspection apparatus 50 according to the present embodiment is disposed on the inspection device 5, the solution supply unit 60 that supplies the inspection solution into the pores 14 of the inspection device 5, and the one surface 11 side of the inspection device 5.
  • a photodetector 70 a photodetector 70.
  • the light detector 70 detects light emitted from the inspection device 5.
  • the light detector 70 is disposed on the one surface 11 side of the inspection device 5, but is disposed on the other surface 12 side. It may be.
  • the inspection apparatus 50 is not limited to the inspection device 5 and may include any of the inspection devices described above.
  • FIG. 13 is a diagram illustrating a schematic configuration of the solution supply unit 60.
  • the solution supply unit 60 stores the inspection solution 61 installed on the other surface 12 side of the inspection device 5, and the inspection installed in the upper part of the storage unit 62 and stored in the storage unit 62.
  • the pipette part 64 that sucks the solution 61 for use, and the pressure of the depressurizing space part 66 and the depressurizing space part 66 arranged on the one surface 11 side of the inspection device 5 installed on the pipette part 64 are reduced.
  • a pump 68 for pressurization is provided.
  • the reduced pressure space 66 is decompressed by the pump 68 so that the inspection solution 61 passes through the pipette part 64 and the pores 14 of the inspection device 5. Supplied in. Note that the inspection solution 61 is supplied to the pores 14 of the inspection device 5 and the inspection solution 61 is discharged from the pores 14 by the pressure reduction and pressurization of the reduced pressure space 66 by the pump 68. it can.
  • the test solution 61 is a solution supplied into the pores 14 at the time of light detection, but the solution supply unit 60 includes a sample solution, a label solution, a cleaning solution, and the like to be supplied into the pores 14 in the test process. It is also used for supply. That is, the solution supply unit 60 supplies a necessary solution to the pores 14 for each inspection process.
  • the cavity 16 is not open on the other surface 12 side, so that the supplied solution does not enter the cavity 16 from the other surface 12 side.
  • the solution supply unit 60 supplies the solution so that the solution does not enter the cavity from one surface 11 where the cavity 16 is opened. Control. As shown in FIG.
  • the solution supply unit 60 was provided with a lid portion 67 that covers the opening of the cavity portion 16 in a part of the depressurizing space portion 66, and overflowed from the opening of the pores 14 on one surface 11.
  • the configuration may be such that the solution does not enter the cavity 16.
  • the light emitted from the inspection device and detected by the photodetector is, for example, fluorescence generated when the label attached to the test substance is excited, or luminescence generated by the label acting as a substrate of the reaction solution It is an optical signal resulting from a label, such as luminescence by reaction (hereinafter referred to as “chemiluminescence”).
  • chemiluminescence an optical signal resulting from a label, such as luminescence by reaction
  • the optical signal includes absorbance (colorimetric) in addition to fluorescence and chemiluminescence light.
  • FIG. 14 is a diagram schematically showing the inspection process.
  • a hydrophobic polymer is bonded to the inner wall surface 14a of the pore 14 of the inspection device 5, and a capture substance 30 (30A or 30B) such as an allergen is fixed to the hydrophobic polymer by physical adsorption (S1). .
  • a specimen liquid containing a test substance (for example, a specific IgE antibody that specifically binds to the allergen) is supplied to the pores 14 of the test device 5, and the test substance 32 is bound to the capture substance 30 (S2). ).
  • the above-described solution supply unit 60 is used for supplying the sample liquid.
  • the sample liquid is efficiently brought into contact with the capture substance 30 in the pores 14 by repeatedly pumping and discharging the sample liquid by the pipette unit 64 while the sample liquid is stored in the storage unit 62. Can do.
  • a labeled solution containing a labeled substance 35 in which a label F is added to a substance 33 (for example, a secondary antibody) that specifically binds to the test substance 32 is supplied to the pores 14.
  • a substance 33 for example, a secondary antibody
  • the labeling substance 35 is bound to the test substance 32 (S3).
  • the label solution is supplied to the pores 14 by the solution supply unit 60 in the same manner as the sample solution.
  • the labeling substance 35 is bound to the test substance 32 after the test substance 32 is bound to the capture substance 30.
  • the sample liquid and the labeling solution are mixed in advance outside the test apparatus 50.
  • the test substance 32 and the labeling substance 35 may be combined to supply the mixed liquid into the pores 14.
  • the test substance 32 to which the labeling substance 35 is bound can be bound to the capture substance 30 fixed in the pores 14.
  • the cleaning solution is supplied to the pores 14 using the solution supply unit 60 in the same manner as the supply of the sample solution, and nonspecific adsorption is performed in the pores 14.
  • the test substance 32 and the labeling substance 35 are removed.
  • the cleaning solution is discharged, and light generated from a luminescence reaction that acts using the label F as a substrate is detected by the photodetector 70 in a state where the inspection solution such as a buffer solution is filled in the pores 14 (S4). ).
  • the inspection solution such as a buffer solution is filled in the pores 14 (S4).
  • the fluorescence measurement is performed in a state where the buffer solution is supplied into the pore 14 as a test solution and the pore 14 is filled with the buffer solution. I do.
  • the inspection device is irradiated with light having a wavelength that excites the fluorescent label as excitation light, and fluorescence from the label excited by the excitation light is detected.
  • the photodetector 70 used for detecting the fluorescent label is provided with an excitation light irradiation unit.
  • the label F is an enzyme label that catalyzes a chemiluminescent substrate such as luminol
  • a reaction including a chemiluminescent substrate that promotes a chemical reaction using the enzyme label as a catalyst after the washing treatment The liquid is supplied into the pores 14 as a test solution.
  • the photodetector 70 by supplying this reaction solution to the pores 14, the enzyme label imparted to the test substance 32 captured by the capture material 30 in the pores 14 becomes the substance in the reaction solution.
  • Luminescence generated by catalyzing a chemical reaction is detected. In this case, luminescence detection is performed in a state where the pores 14 are filled with the reaction solution.
  • enzyme labels that generate chemiluminescence include enzymes that react with chemiluminescent substrates such as luminol, lophine, lucigenin and oxalate.
  • HRP horseradish peroxidase
  • a reaction liquid luminol reaction liquid
  • HRP functions as a catalyst an enzyme for converting HRP to alkaline phosphatase. It is preferable to use a reaction solution containing a dioxetane chemiluminescent substrate.
  • the luminol reaction solution contains at least a luminol substrate and a hydrogen peroxide solution.
  • the enzyme label catalyzes the oxidation of luminol in the presence of hydrogen peroxide.
  • the reaction solution preferably contains a sensitizer that sensitizes chemiluminescence.
  • chemiluminescent substrate not only the chemiluminescent substrate described above but also a reaction solution containing a luminescent substrate or a fluorescent substrate may be used to detect a color reaction (absorption) or fluorescence.
  • the inspection device of the present invention by using the inspection device of the present invention, the light generated in the pores is efficiently emitted from the surface of the inspection device, and the light extraction efficiency is high. Is possible.

Abstract

[Problem] To provide: an inspection device having a configuration that allows captured substances of multiple different types to easily be immobilized in a single device; an inspection apparatus equipped with the inspection device; and an inspection method. [Solution] An inspection device (1) having a plurality of pores (14) that penetrate from one face (11) to the other face (12) of a plate-like base material (10) made from an inorganic substance. A hydrophobic polymer is bonded to the inner wall faces (14a) of at least two pores (14), and different captured substances (30A, 30B, 30C, etc.) comprising antigens or antibodies are physisorbed to the hydrophobic polymers of different pores (14).

Description

検査デバイス、検査装置および検査方法Inspection device, inspection apparatus, and inspection method
　本発明は、抗体または抗原である被検物質を検出するための検査デバイス、その検査デバイスを備えた検査装置および検査方法に関する。 The present invention relates to an inspection device for detecting a test substance that is an antibody or an antigen, an inspection apparatus provided with the inspection device, and an inspection method.
　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。 As one of the methods for examining biochemical reactions, for example, specific binding reactions such as enzyme reactions, nucleic acid hybridizations, and antigen-antibody reactions, the binding phenomenon between a test substance and a capture substance is optically determined. A method of detecting is known. In this method, a test substance is bound to a capture substance fixed at a predetermined position, and a label that emits fluorescence upon receiving excitation light, or a label that catalyzes a substrate reaction to generate color, fluorescence, or chemiluminescence. Or the like is applied to the test substance, and light generated due to the label is detected. More specifically, a method of detecting a fluorescence generated from a fluorescent label by attaching a fluorescent label to a binding substance such as an antibody that specifically binds to the test substance, a binding of an antibody that specifically binds to the test substance There are known methods for detecting color development, fluorescence, chemiluminescence, etc. generated from a chromogenic substrate, fluorescent substrate, or chemiluminescent substrate that reacts with this enzyme as a catalyst. Identification becomes possible.
　このような検査に用いられるバイオチップとしては、二次元基板上に生体関連分子が規則的に配列された多項目同時検出のためのチップが従来用いられている。近年では、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。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ことができ、測定時間の大幅な短縮化を図ることができる。 As a biochip used for such an inspection, a multi-item simultaneous detection chip in which biologically related molecules are regularly arranged on a two-dimensional substrate has been conventionally used. In recent years, a device made of a porous substrate in which a large number of through-holes (pores) are arranged on a support has been studied. By immobilizing the capture substance in the pores, pumping the sample liquid containing the test substance from the back side to the surface of the porous substrate through the through-hole and circulating it, the sample liquid becomes efficient as the capture substance The test substance can be bonded to the capture substance and the measurement time can be greatly shortened.
　バイオチップにおいて、細孔に捕捉物質を固定するための種々の固定化方法が提案されている。例えば、特許文献１には、糸やメンブレンに捕捉物質を含む溶液をしみ込ませ、乾燥させることにより糸に捕捉物質を固定化する方法や、管の内側を捕捉物質の水溶液でコーティングすることによって管の内側に捕捉物質を固定化する方法などが挙げられている。 In the biochip, various immobilization methods have been proposed for immobilizing a capture substance in pores. For example, Patent Document 1 discloses a method in which a solution containing a trapping substance is impregnated into a thread or a membrane, and the trapping substance is fixed to the thread by drying. And a method of immobilizing a capture substance inside.
　また、特許文献２には、多数の細孔を有するシリコンを熱酸化処理して局部的に透明な酸化シリコン（ＳｉＯ２）化してなるデバイスが提案されており、有機シリコン化合物をリンカーとして、ＤＮＡ、たんぱく質あるいはリガンドなどの捕捉物質を共有結合により細孔内に固定する手法が開示されている。 Patent Document 2 proposes a device in which silicon having a large number of pores is thermally oxidized to form locally transparent silicon oxide (SiO 2 ), and an organic silicon compound as a linker is used as a DNA. In addition, a technique is disclosed in which a capturing substance such as a protein or a ligand is immobilized in a pore by a covalent bond.
特表２００１－５１５７３５号公報JP 2001-515735 A 特許第４１２５２４４号公報Japanese Patent No. 4125244
　しかしながら、特許文献１において具体的な捕捉物質の固定化方法としては、糸あるいはメンブレンに捕捉物質を固定化する方法しか挙げられておらず、多孔性基板からなるデバイスの細孔内への捕捉物質の固定化については記載されていない。 However, in Patent Document 1, as a specific method for immobilizing a capture substance, only a method for immobilizing the capture substance on a thread or a membrane is cited, and the capture substance in the pores of a device made of a porous substrate is mentioned. The immobilization of is not described.
　特許文献２では、捕捉物質を有機シリコン化合物からなるリンカーに共有結合する方法が開示されているが、共有結合では、特定の物質とのみ強く結合するため、１つのデバイスにおいて異なる複数種類の捕捉物質を固定化するのには適さない。 Patent Document 2 discloses a method of covalently binding a capture substance to a linker made of an organosilicon compound. However, since covalent bond strongly binds only to a specific substance, a plurality of different types of capture substances are different in one device. It is not suitable for fixing.
　本発明は、上記事情に鑑み、１つのデバイス中において異なる複数種類の捕捉物質を容易に固定化可能な構成の検査デバイスを提供することを目的とする。また、本発明は多項目同時検査を可能とした検査デバイスを備えた検査装置および検査方法を提供することを目的とする。 In view of the above circumstances, an object of the present invention is to provide an inspection device having a configuration capable of easily immobilizing a plurality of different types of capture substances in one device. Another object of the present invention is to provide an inspection apparatus and an inspection method provided with an inspection device that enables simultaneous inspection of multiple items.
　本発明の検査デバイスは、無機物からなる板状基材の一方の面から他方の面に貫通する複数の細孔を備えた検査デバイスであって、
　少なくとも２つ以上の細孔の内壁面に疎水性高分子が結合しており、
　互いに異なる細孔の疎水性高分子に、被検物質と特異的に結合する、抗原または抗体からなる互いに異なる捕捉物質が物理吸着されている検査デバイスである。 The inspection device of the present invention is an inspection device having a plurality of pores penetrating from one side of a plate-like substrate made of an inorganic material to the other side,
A hydrophobic polymer is bonded to the inner wall surface of at least two or more pores,
This is a test device in which different capture substances consisting of an antigen or an antibody that specifically bind to a test substance are physically adsorbed to hydrophobic polymers of different pores.
　ここで、疎水性高分子とは、ＪＩＳ（Japanese Industrial Standards）　Ｒ３２５７の検査方法により測定される接触角が８０°以上である高分子をいう。 Here, the hydrophobic polymer means a polymer having a contact angle of 80 ° or more measured by an inspection method of JIS (Japanese Industrial Standards) R3257.
　本発明の検査デバイスにおいて、疎水性高分子と板状基材は、疎水性高分子をＲ、板状基材を構成する無機物をＭとしたとき、酸素原子Ｏを介して、Ｒ－Ｏ－Ｍ結合で共有結合していることが好ましい。 In the inspection device of the present invention, the hydrophobic polymer and the plate-like substrate are R—O— via an oxygen atom O, where R is a hydrophobic polymer and M is an inorganic substance constituting the plate-like substrate. It is preferable that it is covalently bonded by M bond.
　本発明の検査デバイスにおいて、疎水性高分子はポリスチレンまたはポリプロピレンであることが好ましい。 In the inspection device of the present invention, the hydrophobic polymer is preferably polystyrene or polypropylene.
　本発明の検査デバイスにおいて、無機物はＳｉ、ＳｉＯ２、ＡｌおよびＡｌ２Ｏ３のうちの１種もしくは２種以上であることが好ましい。 In the inspection device of the present invention, the inorganic material is preferably one or more of Si, SiO 2 , Al, and Al 2 O 3 .
　本発明の検査デバイスは、一方の面から見た場合において、捕捉物質が吸着された複数の細孔を含む検査領域を複数備え、複数の検査領域のうち隣り合って配置された検査領域間は、隣り合って配置された検査領域中における細孔間隔よりも広い幅の非検査領域によって分離されており、１つの検査領域中の細孔には同一種の捕捉物質が物理吸着されてなり、隣り合って配置された検査領域のうち一方の検査領域の細孔と他方の検査領域の細孔には互いに異なる捕捉物質が物理吸着されていることが好ましい。 The inspection device of the present invention includes a plurality of inspection regions including a plurality of pores to which the trapping substance is adsorbed when viewed from one surface, and between the inspection regions arranged adjacent to each other among the plurality of inspection regions. , Separated by a non-inspection region having a width wider than the pore spacing in the adjacent inspection regions, and the same type of trapping substance is physically adsorbed in the pores in one inspection region, It is preferable that different trapping substances are physically adsorbed in the pores of one inspection region and the other inspection region among the inspection regions arranged adjacent to each other.
　本発明の検査デバイスは、非検査領域を、複数の細孔のうちの捕捉物質が不存在である非検査用細孔が配置された領域とすることができる。 In the inspection device of the present invention, the non-inspection region can be a region in which non-inspection pores in which a trapping substance is absent from a plurality of pores are arranged.
　本発明の検査デバイスは、非検査領域に、細孔の深さ方向に延び、少なくとも他方の面に非貫通である空洞部を備えていてもよい。 The inspection device of the present invention may include a cavity that extends in the depth direction of the pores and is non-penetrating on at least the other surface in the non-inspection region.
　本発明の検査デバイスは、非検査領域が、細孔を備えていない板状基材の部分であってもよい。 In the inspection device of the present invention, the non-inspection region may be a portion of a plate-like base material that does not have pores.
　本発明の検査デバイスは、一方の面において、非検査領域と検査領域の並び方向における検査領域の一辺が、非検査領域の幅よりも長いことが好ましい。 In the inspection device of the present invention, on one side, it is preferable that one side of the inspection region in the alignment direction of the non-inspection region and the inspection region is longer than the width of the non-inspection region.
　本発明の検査デバイスにおいては、一方の検査領域と他方の検査領域とが周期的に配置されていることが好ましい。 In the inspection device of the present invention, it is preferable that one inspection region and the other inspection region are periodically arranged.
　本発明の検査デバイスにおいては、互いに異なる捕捉物質として、粗精製アレルゲンと、粗精製アレルゲン中に含まれる少なくとも１つのアレルゲンコンポーネントとを含むものとすることができる。 In the test device of the present invention, differently captured substances may include a crude purified allergen and at least one allergen component contained in the crude purified allergen.
　本発明の検査装置は、本発明の検査デバイスと、
　検査デバイスの細孔中に検査用溶液を供給する溶液供給部と、
　検査デバイスの一方の面側または他方の面側に配置され、検査デバイスから出射される光を検出する光検出器とを備えた検査装置である。 The inspection apparatus of the present invention includes the inspection device of the present invention,
A solution supply unit for supplying an inspection solution into the pores of the inspection device;
The inspection apparatus includes a photodetector that is disposed on one surface side or the other surface side of the inspection device and detects light emitted from the inspection device.
　本発明の検査方法は、本発明の検査デバイスの少なくとも一部の細孔に、被検物質を含有する検体液を供給して、被検物質を捕捉物質に結合させ、
　被検物質と特異的に結合する標識物質をその被検物質に結合させ、
　細孔に検査用溶液を供給して細孔に検査用溶液を留めた状態で、検査デバイスから出射される光を検出する検査方法である。 In the inspection method of the present invention, a specimen liquid containing a test substance is supplied to at least some of the pores of the test device of the present invention, and the test substance is bound to a capture substance,
A labeling substance that specifically binds to the test substance is bound to the test substance,
The inspection method detects light emitted from the inspection device in a state where the inspection solution is supplied to the pores and the inspection solution is retained in the pores.
　本発明の検査方法においては、上記標識物質として酵素標識を含む物質を用い、上記検査用溶液として、酵素標識により触媒されて反応する基質を含む反応液を用い、上記検査デバイスから出射される光として、反応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してもよい。 In the inspection method of the present invention, a substance containing an enzyme label is used as the labeling substance, a reaction solution containing a substrate that reacts catalyzed by the enzyme label is used as the inspection solution, and light emitted from the inspection device is used. Alternatively, light generated by catalyzing a substrate in the reaction solution by an enzyme label may be detected.
　本発明の検査方法においては、上記標識物質として蛍光標識を含む物質を用い、上記蛍光標識を励起させる励起光を検査デバイスに照射し、上記検査デバイスから出射される光として、励起光の照射により標識物質から生じる蛍光を検出してもよい。 In the inspection method of the present invention, a substance containing a fluorescent label is used as the labeling substance, the inspection device is irradiated with excitation light that excites the fluorescent label, and the light emitted from the inspection device is irradiated with excitation light. Fluorescence generated from the labeling substance may be detected.
　本発明の検査デバイスは、無機物からなる板状基材の一方の面から他方の面に貫通する複数の細孔を備えた検査デバイスであって、少なくとも２つ以上の細孔の内壁面に疎水性高分子が結合しているので、１つのデバイス中において異なる複数種類の捕捉物質を容易に固定化することができ、抗原または抗体からなる互いに異なる細孔の疎水性高分子に、互いに異なる捕捉物質が物理吸着されているので、多項目同時検査が可能である。 An inspection device of the present invention is an inspection device having a plurality of pores penetrating from one surface of a plate-like substrate made of an inorganic material to the other surface, and is hydrophobic on the inner wall surface of at least two or more pores. Since different types of capture substances can be easily immobilized in a single device, different capture molecules can be captured by different hydrophobic polymers composed of antigens or antibodies. Since the substance is physically adsorbed, simultaneous inspection of multiple items is possible.
本発明の第１の実施形態にかかる検査デバイスの斜視図である。1 is a perspective view of an inspection device according to a first embodiment of the present invention. 図１に示す検査デバイスの平面図である。It is a top view of the inspection device shown in FIG. 図１に示す検査デバイスの断面図である。It is sectional drawing of the test | inspection device shown in FIG. 疎水性高分子の細孔内壁面への結合工程を模式的に示す図である。It is a figure which shows typically the coupling | bonding process to the pore inner wall face of hydrophobic polymer. 疎水性高分子と細孔内壁面との結合状態および捕捉物質の吸着状態を示す概念図である。It is a conceptual diagram which shows the coupling | bonding state of a hydrophobic polymer and a pore inner wall surface, and the adsorption state of a capture | acquisition substance. 設計変更例１の検査デバイスの平面模式図である。It is a plane schematic diagram of the inspection device of design change example 1. 設計変更例２の検査デバイスの平面模式図である。It is a plane schematic diagram of the inspection device of the design change example 2. 本発明の第２の実施形態にかかる検査デバイスの平面図である。It is a top view of the inspection device concerning a 2nd embodiment of the present invention. 本発明の第３の実施形態にかかる検査デバイスの平面図である。It is a top view of the inspection device concerning a 3rd embodiment of the present invention. 本発明の第４の実施形態にかかる検査デバイスの平面図である。It is a top view of the inspection device concerning a 4th embodiment of the present invention. 本発明の第５の実施形態にかかる検査デバイスの断面図である。It is sectional drawing of the test | inspection device concerning the 5th Embodiment of this invention. 本発明の第６の実施形態にかかる検査デバイスの断面図である。It is sectional drawing of the test | inspection device concerning the 6th Embodiment of this invention. 検査デバイスの効果を説明するための模式図である。It is a schematic diagram for demonstrating the effect of a test | inspection device. 本発明の検査デバイスを備えた一実施形態の検査装置の概略構成を示す図である。It is a figure which shows schematic structure of the test | inspection apparatus of one Embodiment provided with the test | inspection device of this invention. 検査装置における溶液供給部を説明するための模式図である。It is a schematic diagram for demonstrating the solution supply part in a test | inspection apparatus. 本発明の検査デバイスを用いた検査工程を示す模式図である。It is a schematic diagram which shows the test | inspection process using the test | inspection device 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 to mean that the numerical values described before and after it are included as a lower limit value and an upper limit value.
　図１は本発明の第１の実施形態にかかる検査デバイスの斜視図であり、図２は図１の検査デバイスの平面図、図３は図２の検査デバイスのIII-III線断面図である。図２に示す平面図は、検査デバイス１を一方の面１１から見た図である。 1 is a perspective view of an inspection device according to a first embodiment of the present invention, FIG. 2 is a plan view of the inspection device of FIG. 1, and FIG. 3 is a cross-sectional view taken along line III-III of the inspection device of FIG. . The plan view shown in FIG. 2 is a view of the inspection device 1 as seen from one surface 11.
　本実施形態の検査デバイス１は、無機物からなる板状基材１０に一方の面１１から他方の面１２に貫通する複数の細孔１４を備えてなる。検査デバイス１の細孔１４の内壁面１４ａには疎水性高分子が結合しており、互いに異なる細孔１４の疎水性高分子に、抗原または抗体からなる互いに異なる捕捉物質３０Ａ、３０Ｂ、３０Ｃ…が物理吸着されている。なお、以下の説明において、捕捉物質の種を区別する必要の箇所においては単に捕捉物質３０と表記し、種を区別して説明する必要がある箇所においてのみ捕捉物質３０Ａ、捕捉物質３０Ｂ…など枝符号Ａ，Ｂ…を付与した表記とする。 The inspection device 1 of this embodiment includes a plurality of pores 14 penetrating from one surface 11 to the other surface 12 in a plate-like substrate 10 made of an inorganic material. A hydrophobic polymer is bonded to the inner wall surface 14a of the pore 14 of the test device 1, and different capture substances 30A, 30B, 30C made of antigens or antibodies are attached to the hydrophobic polymer of the different pores 14 ... Is physically adsorbed. In the following description, the part where the species of the trapping substance needs to be distinguished is simply indicated as the trapping substance 30, and the branch code such as the trapping substance 30A, the trapping substance 30B,. It is set as the notation which added A, B ....
　本検査デバイス１は、複数の細孔１４を備え、細孔１４間で異なる捕捉物質３０Ａ，３０Ｂ，３０Ｃ…が物理吸着されているので、本検査デバイス１を用いることにより複数の被検物質に対する検査を同時に実施可能である。 Since this inspection device 1 includes a plurality of pores 14 and different capture substances 30A, 30B, 30C,... Are physically adsorbed between the pores 14, by using this inspection device 1, a plurality of analytes can be detected. Inspection can be performed at the same time.
　なお、検査デバイス１の外形は板状基材１０の外形により構成されるものであるため、以下において、板状基材１０の一方の面１１および他方の面１２を、検査デバイス１の一方の面および他方の面と称する場合がある。また、板状基材１０の一方の面、他方の面を区別しない場合には、両方の面を総称して板状基材１０の表面（検査デバイスの表面）と称する場合がある。 In addition, since the external shape of the inspection device 1 is configured by the external shape of the plate-like base material 10, one surface 11 and the other surface 12 of the plate-like base material 10 are hereinafter referred to as one of the inspection devices 1. Sometimes referred to as the surface and the other surface. Moreover, when not distinguishing one surface and the other surface of the plate-shaped base material 10, both surfaces may be named generically and may be called the surface of the plate-shaped base material 10 (surface of a test | inspection device).
　板状基材１０は、Ｓｉ（シリコン）、ＳｉＯ２（シリコン酸化物）、Ａｌ（アルミニウム）およびＡｌ２Ｏ３（アルミナ）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。板状基材１０は、ＳｉあるいはＡｌなどからなる不透明なものであってもよいし、ＳｉＯ２あるいはＡｌ２Ｏ３などからなる透明なものであってもよい。板状基材１０は、いずれか単体の材料で構成されていてもよいし、例えば、Ｓｉ基材を一部酸化することによりＳｉ部分とＳｉＯ２部分とを含むものであってもよいし、Ａｌ基材を一部酸化して、Ａｌ部分とＡｌ２Ｏ３部分とを含むものであってもよい。 The plate-like substrate 10 may be composed of one or more materials of Si (silicon), SiO 2 (silicon oxide), Al (aluminum), and Al 2 O 3 (alumina). preferable. The plate-like substrate 10 may be opaque made of Si or Al, or may be transparent made of SiO 2 or Al 2 O 3 . The plate-like substrate 10 may be composed of any single material, or may include, for example, a Si portion and a SiO 2 portion by partially oxidizing the Si substrate, The Al base material may be partially oxidized to include an Al portion and an Al 2 O 3 portion.
　Ｓｉ、ＳｉＯ２、ＡｌおよびＡｌ２Ｏ３などの無機物からなる検査デバイス１において、検査デバイス１の細孔１４の内壁面１４ａには疎水性高分子が結合されている。 In the inspection device 1 made of an inorganic material such as Si, SiO 2 , Al, and Al 2 O 3 , a hydrophobic polymer is bonded to the inner wall surface 14 a of the pore 14 of the inspection device 1.
　疎水性高分子とは、接触角８０°以上である高分子であり、例えば、ポリスチレン、ポリプロピレン、ポリエチレン、ポリテトラフロロエチレンおよびシリコンゴムなどが挙げられる。この中でもポリスチレンおよびポリプロピレンが好適である。 The hydrophobic polymer is a polymer having a contact angle of 80 ° or more, and examples thereof include polystyrene, polypropylene, polyethylene, polytetrafluoroethylene, and silicon rubber. Of these, polystyrene and polypropylene are preferred.
　内壁面１４ａへの疎水性高分子の結合の形態について図４Ａおよび図４Ｂを参照して説明する。図４Ａに疎水性高分子の細孔１４の内壁面１４ａへの結合工程を模式的に示す。図４Ａにおいては、検査デバイス１の１つの細孔１４の断面のみを示しているが、検査デバイス１中の全ての細孔１４に対する疎水性高分子の結合処理は同時に行うことができる。なお、後述する非検査領域に設けられる細孔１４内にも同時に疎水性高分子が付与されても差支えない。 The mode of binding of the hydrophobic polymer to the inner wall surface 14a will be described with reference to FIGS. 4A and 4B. FIG. 4A schematically shows a step of bonding the hydrophobic polymer pores 14 to the inner wall surface 14a. In FIG. 4A, only the cross section of one pore 14 of the inspection device 1 is shown, but the binding treatment of the hydrophobic polymer to all the pores 14 in the inspection device 1 can be performed simultaneously. It should be noted that a hydrophobic polymer may be simultaneously applied to the pores 14 provided in the non-inspection region described later.
　まず、無機物からなる板状基材１０の表面（細孔１４の内壁面１４ａ）に対して紫外線照射によるクリーニングを行う（Ａ１）。次に、水酸基（ＯＨ基）を有する疎水性高分子を含む溶液４５を細孔１４内に充填する（Ａ２）。その後、熱処理を行い脱水縮合させることにより、細孔１４の内壁面１４ａと疎水性高分子とを共有結合させる（Ａ３）。その後、結合していない疎水性高分子を溶剤により除去することにより、細孔１４の内壁面１４ａに疎水性高分子が結合されてなる化学修飾層（疎水性高分子層）４６が形成される。このようにして、疎水性高分子が内壁面１４ａに結合された細孔１４を備えた板状基材１０を得ることができる（Ａ４）。 First, the surface of the plate-like substrate 10 made of an inorganic material (inner wall surface 14a of the pore 14) is cleaned by ultraviolet irradiation (A1). Next, the pores 14 are filled with a solution 45 containing a hydrophobic polymer having a hydroxyl group (OH group) (A2). Thereafter, the inner wall surface 14a of the pores 14 and the hydrophobic polymer are covalently bonded by heat treatment and dehydration condensation (A3). Thereafter, the hydrophobic polymer that has not been bonded is removed with a solvent to form a chemically modified layer (hydrophobic polymer layer) 46 in which the hydrophobic polymer is bonded to the inner wall surface 14 a of the pore 14. . In this way, the plate-like substrate 10 having the pores 14 in which the hydrophobic polymer is bonded to the inner wall surface 14a can be obtained (A4).
　図４Ｂは、疎水性高分子と板状基材の結合状態および捕捉物質の吸着状態を模式的に示す概念図である。ここでは、疎水性高分子としてポリスチレンをＳｉからなる基材１０の細孔内壁面１４ａに結合する場合について説明する。ＯＨ基を備えたポリスチレンを含む溶液をＳｉ表面（内壁面１４ａ）に接触させた状態で熱処理を行う。これにより、ポリスチレンのＯＨ基と、Ｓｉ表面に自然形成されたＯＨ基とを脱水縮合させる。脱水縮合反応により水（Ｈ２Ｏ）が生成され、残った酸素原子（Ｏ）を介してポリスチレン（Ｒ）が無機物（Ｍ）と共有結合して、Ｒ－Ｏ－Ｍ結合が形成される。このようにして細孔１４の内壁面１４ａにポリスチレンが共有結合されてなる疎水性高分子層４１が形成される。 FIG. 4B is a conceptual diagram schematically showing the binding state between the hydrophobic polymer and the plate-like substrate and the adsorption state of the trapping substance. Here, a case where polystyrene is bonded as a hydrophobic polymer to the pore inner wall surface 14a of the substrate 10 made of Si will be described. Heat treatment is performed in a state where a solution containing polystyrene having an OH group is in contact with the Si surface (inner wall surface 14a). Thereby, the OH group of polystyrene and the OH group naturally formed on the Si surface are dehydrated and condensed. Water (H 2 O) is generated by the dehydration condensation reaction, and polystyrene (R) is covalently bonded to the inorganic substance (M) through the remaining oxygen atoms (O) to form R—O—M bonds. In this way, the hydrophobic polymer layer 41 formed by covalently bonding polystyrene to the inner wall surface 14a of the pore 14 is formed.
　捕捉物質３０である抗原または抗体は、タンパク質である。タンパク質分子は親水基を有するアミノ酸と疎水基を有するアミノ酸とが結合した高分子である。一般にタンパク質分子の表面は親水部と疎水部とによるモザイク状構造を有する。タンパク質の表面の疎水部は疎水性高分子層に容易に吸着する。抗原や抗体などのタンパク質分子からなる捕捉物質３０は、その種類に関わらず、親水部および疎水部を有しているので疎水性高分子に対して容易に吸着される。本検査デバイス１は複数の細孔１４の各内壁面１４ａに疎水性高分子が共有結合により結合されているので、異なる捕捉物質３０Ａ，３０Ｂ，３０Ｃ…を容易に物理吸着させることができ、作製が容易である。捕捉物質を内壁面に共有結合で固定化しようとすると、共有結合が可能な特定の分子しか固定化できず、所望の捕捉物質を固定化できない場合もあるが、疎水性高分子層４１への物理吸着はタンパク質分子であればほぼ全てに適用することができ、好ましい。 The antigen or antibody that is the capture substance 30 is a protein. A protein molecule is a polymer in which an amino acid having a hydrophilic group and an amino acid having a hydrophobic group are bonded. In general, the surface of a protein molecule has a mosaic structure composed of a hydrophilic part and a hydrophobic part. The hydrophobic part of the protein surface is easily adsorbed to the hydrophobic polymer layer. Regardless of the type, the capture substance 30 made of a protein molecule such as an antigen or an antibody has a hydrophilic part and a hydrophobic part, so that it is easily adsorbed to the hydrophobic polymer. In the present inspection device 1, since the hydrophobic polymer is covalently bonded to each inner wall surface 14a of the plurality of pores 14, different capture substances 30A, 30B, 30C,. Is easy. When trying to immobilize the capture substance on the inner wall surface with a covalent bond, only a specific molecule capable of covalent bond can be immobilized, and the desired capture substance may not be immobilized. Physical adsorption is preferable because it can be applied to almost all protein molecules.
　板状基材１０の厚さには、特に制限はないが、１００μｍ～２０００μｍ程度が好ましい。板状基材１０に設けられる細孔１４は、本実施形態に示すように、整列配置されていることが好ましいが、ランダムに配置されていてもよい。本実施形態においては、視認容易のために７行×７列の４９本の細孔を有するものとしたが、例えば、１０行×１０列の１００本の細孔を含むなど、検査デバイス１中に備えられる細孔の数に制限はない。また、例えば、ｎ行×ｍ列のｎ×ｍ本の細孔を１つの検査領域として、複数の検査領域を備えていてもよい（ｎ、ｍは、正の整数）。 The thickness of the plate-like substrate 10 is not particularly limited, but is preferably about 100 μm to 2000 μm. The pores 14 provided in the plate-like substrate 10 are preferably arranged in an aligned manner as shown in the present embodiment, but may be arranged randomly. In the present embodiment, 49 pores of 7 rows × 7 columns are provided for easy visual recognition, but the inspection device 1 includes, for example, 100 pores of 10 rows × 10 columns. There is no limit to the number of pores provided in the case. For example, n × m pores of n rows × m columns may be used as one inspection region, and a plurality of inspection regions may be provided (n and m are positive integers).
　細孔１４の開口および断面形状は本実形態においては正方形状であるが、正方形、長方形などの矩形に限らず、円形、楕円形、三角形あるいは五角形以上の多角形であってもよい。なお、作製上の都合により多角形状の角部には丸みがつく場合がある。細孔１４は、柱状で断面形状が変化しないものが一般的であるが、一部断面形状が変化したり、断面の大きさが変化したりしても構わない。 The opening and cross-sectional shape of the pore 14 are square in this embodiment, but are not limited to a rectangle such as a square or a rectangle, but may be a circle, an ellipse, a triangle, or a polygon that is a pentagon or more. Note that the corners of the polygon may be rounded due to manufacturing reasons. The pores 14 are generally columnar and the cross-sectional shape does not change, but the cross-sectional shape may partially change or the cross-sectional size may change.
　なお、細孔１４の少なくとも一方の面１１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一の面積を有する円の直径をいう。 It should be noted that the equivalent circle diameter of the opening in at least one surface 11 of the pore 14 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 the same area as the area of the opening region.
　本検査デバイス１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、抗体または抗原である。そして、細孔１４の内壁面１４ａに固定されている、抗原または抗体からなる捕捉物質３０としては、所望の被検物質と特異的に結合する物質を選択できる。特に、本検査デバイス１は、抗原の一種であるアレルゲンを捕捉物質として備えたアレルギー検査に好適である。 The test substance (target molecule) to be tested in the present testing device 1 is mainly a biological molecule, and is an antibody or an antigen. As the capture substance 30 made of an antigen or antibody fixed to the inner wall surface 14a of the pore 14, a substance that specifically binds to a desired test substance can be selected. In particular, the test device 1 is suitable for an allergy test provided with an allergen that is a kind of antigen as a capture substance.
　検査デバイス１においては、検査精度の向上を図るため、複数の細孔１４に共通して同一種の捕捉物質を備えた検査領域を備えることが好ましい。図５Ａ、図５Ｂに、それぞれ複数の検査領域を備えた設計変更例の検査デバイス１Ａ、１Ｂの平面模式図を示す。 In order to improve the inspection accuracy, the inspection device 1 preferably includes an inspection region provided with the same kind of trapping substance in common with the plurality of pores 14. 5A and 5B are schematic plan views of inspection devices 1A and 1B of a design change example each having a plurality of inspection regions.
　図５Ａにおいて、検査デバイス１Ａは検査領域２０Ａおよび検査領域２０Ｂを備えている。各検査領域２０Ａ，２０Ｂ中にはそれぞれの拡大図を示すように、３×３の９本の細孔１４が備えられている。検査領域２０Ａ中の細孔１４には捕捉物質３０Ａが物理吸着されており、検査領域２０Ｂ中の細孔１４には捕捉物質３０Ａとは異なる種類の捕捉物質３０Ｂが備えられている。 5A, the inspection device 1A includes an inspection area 20A and an inspection area 20B. In each of the inspection regions 20A and 20B, 9 × 3 × 9 pores 14 are provided as shown in the enlarged views. The capture substance 30A is physically adsorbed in the pores 14 in the inspection region 20A, and the capture material 30B of a type different from the capture material 30A is provided in the pores 14 in the inspection region 20B.
　図５Ｂにおいて、検査デバイス１Ｂは検査領域２０Ａ，２０Ｂ，２０Ｃおよび２０Ｄを備えている。各検査領域２０Ａ，２０Ｂ，２０Ｃおよび２０Ｄ中には図５Ａ中に拡大図で示したように、複数の細孔１４が備えられている。また、検査領域２０Ａ中には細孔１４には捕捉物質３０Ａが、検査領域２０Ｂ中の細孔１４には捕捉物質３０Ｂが、検査領域２０Ｃ中の細孔１４には捕捉物質３０Ｃが、検査領域２０Ｄ中の細孔１４には捕捉物質３０Ｄがそれぞれ物理吸着されている。そして、検査デバイス１Ｂには、この４つの領域を１セットとして、４セットが配置されている。 5B, the inspection device 1B includes inspection areas 20A, 20B, 20C, and 20D. Each inspection region 20A, 20B, 20C and 20D is provided with a plurality of pores 14 as shown in an enlarged view in FIG. 5A. Further, in the inspection region 20A, the trapping substance 30A is in the pore 14, the trapping material 30B is in the pore 14 in the inspection region 20B, and the trapping substance 30C is in the pore 14 in the inspection region 20C. The trapping substance 30D is physically adsorbed to the pores 14 in 20D. In the inspection device 1B, four sets are arranged with these four regions as one set.
　図５Ａおよび図５Ｂに示すように、同一種の捕捉物質を備えた複数の細孔を内包する検査領域を備えることにより、検査精度を向上できる。さらに、同一種の捕捉物質を備えた検査領域を複数、周期的に配置することにより、検査デバイス１上における精度バラツキを均等にして、さらなる検査精度の向上が可能となる。 As shown in FIGS. 5A and 5B, the inspection accuracy can be improved by providing an inspection region containing a plurality of pores including the same type of capture substance. Furthermore, by periodically arranging a plurality of inspection regions having the same type of capturing substance, it is possible to make the accuracy variation uniform on the inspection device 1 and further improve the inspection accuracy.
　近年、食物や花粉等によるアレルギーの検査に対する需要が高まっており、多項目のアレルギー検査を同時に実施可能とするバイオチップ（検査デバイス）において精度向上が求められている。例えば、図５Ｂの検査デバイス１Ｂの検査領域２０Ａ～２０Ｄに、食物アレルギーのアレルゲンである卵白、小麦、牛乳、ピーナッツを、それぞれ捕捉物質３０Ａ～３０Ｄとして、それぞれ備えることにより、これらのアレルゲンに対するＩｇＥ（Immunoglobulin E：免疫グロブリンＥ）抗体を同時に精度よく検出することが可能である。 In recent years, there is an increasing demand for testing for allergies caused by food, pollen, etc., and there is a need for improved accuracy in biochips (testing devices) that can simultaneously perform multi-item allergy testing. For example, by providing egg whites, wheat, milk, and peanuts, which are food allergy allergens, as capture substances 30A to 30D in the test regions 20A to 20D of the test device 1B of FIG. 5B, respectively, IgE for these allergens ( Immunoglobulin® E: Immunoglobulin E) It is possible to detect the antibody simultaneously and accurately.
　卵白、小麦、牛乳、ピーナッツなどは粗精製アレルゲンと言われ、これらの粗精製アレルゲン中には、個々にＩｇＥ抗体と結合する複数種のタンパク質が存在する。これらの粗精製アレルゲン中の個々のタンパク質をアレルゲンコンポーネントといい、アレルゲンコンポーネントには、臨床症状と関連性の強いアレルゲンコンポーネントとそうでないアレルゲンコンポーネントが存在する。そのため、粗精製アレルゲンに対する測定ではアレルギーの有無を判断できない場合には、臨床症状と関連性の高いアレルゲンコンポーネントに対するＩｇＥ測定を行うことが望まれる。そこで、１つのデバイス中には、粗精製アレルゲンが捕捉物質として備えられた検査領域と、その粗精製アレルゲン中の臨床症状と関連性の強いアレルゲンコンポーネントが捕捉物質として備えられた検査細孔とを備え、同時に検査可能とすることが好ましい。本発明においては、複数のアレルゲンコンポーネントを含有する粗精製アレルゲンも１種の捕捉物質と看做している。 Egg whites, wheat, milk, peanuts, etc. are said to be crudely purified allergens, and in these crudely purified allergens, there are multiple types of proteins that individually bind to IgE antibodies. Individual proteins in these crudely purified allergens are called allergen components, and allergen components include allergen components that are strongly associated with clinical symptoms and other allergen components. For this reason, if it is not possible to determine the presence or absence of allergy by measuring crudely purified allergen, it is desirable to perform IgE measurement on allergen components that are highly related to clinical symptoms. Therefore, in one device, a test region in which the crude purified allergen is provided as a capture substance, and a test pore in which an allergen component that is strongly associated with clinical symptoms in the crude purified allergen is provided as a capture substance. It is preferable to prepare and enable inspection at the same time. In the present invention, a crudely purified allergen containing a plurality of allergen components is also regarded as one kind of trapping substance.
　例えば、図５の検査デバイス１Ｂにおいて、卵白を捕捉物質３０Ａとして検査領域２０Ａに、卵白のアレルゲンコンポーネントであるオボムコイドを捕捉物質３０Ｂとして検査領域２０Ｂに、小麦を捕捉物質３０Ｃとして検査領域２０Ｃに、小麦のアレルゲンコンポーネントであるω－５グリアジンを捕捉物質３０Ｄとして検査領域２０Ｄにそれぞれ備える、などである。 For example, in the inspection device 1B of FIG. 5, egg white is used as the capturing substance 30A as the inspection area 20A, ovomucoid which is an egg white allergen component is used as the capturing substance 30B as the capturing area 20B, wheat is used as the capturing substance 30C as the inspection area 20C, wheat Ω-5 gliadin, which is an allergen component, is provided in the examination region 20D as a capture substance 30D.
　本発明の検査デバイスにおいては、一方の面から見た場合において、捕捉物質が物理吸着された複数の細孔を含む検査領域を複数備え、複数の検査領域のうち隣り合って配置された検査領域間は、隣り合って配置された検査領域中における細孔間隔よりも広い幅の非検査領域によって分離されていることが好ましい。このような実施形態を以下に説明する。 In the inspection device of the present invention, when viewed from one surface, the inspection device includes a plurality of inspection regions including a plurality of pores to which the trapping substance is physically adsorbed, and is disposed adjacent to each other among the plurality of inspection regions. The gap is preferably separated by a non-inspection region having a width wider than the pore interval in the adjacent inspection regions. Such an embodiment will be described below.
　図６は、第２の実施形態の検査デバイス２の平面図を示す。
　４つの細孔１４を含む検査領域２０が４つあり、検査領域２０間は非検査領域２１により隔てられて配置されている。図６において、右上がり斜線で示す細孔１４には捕捉物質３０Ａが物理吸着されており、左上がり斜線で示す細孔１４には捕捉物質３０Ｂが物理吸着されている。そして、斜線を付していない細孔１４は捕捉物質が付与されておらず、捕捉物質が存在していない（不存在である）非検査用細孔である。捕捉物質３０Ａが備えられた検査領域２０は図５Ａ，図５Ｂにおける検査領域２０Ａ、捕捉物質３０Ｂが備えられた検査領域２０は図５Ａ，図５Ｂにおける検査領域２０Ｂに相当するが、以下の実施形態においては単に検査領域２０と表記している。 FIG. 6 is a plan view of the inspection device 2 according to the second embodiment.
There are four inspection areas 20 including four pores 14, and the inspection areas 20 are separated by a non-inspection area 21. In FIG. 6, the trapping substance 30 </ b> A is physically adsorbed in the pores 14 indicated by the diagonal lines rising to the right, and the trapping substance 30 </ b> B is physically adsorbed in the pores 14 indicated by the diagonal lines rising to the left. The pores 14 not hatched are non-inspection pores to which no capture substance is applied and no capture substance is present (absent). The inspection region 20 provided with the capture substance 30A corresponds to the inspection region 20A in FIGS. 5A and 5B, and the inspection region 20 provided with the capture material 30B corresponds to the inspection region 20B in FIGS. 5A and 5B. In FIG. 4, the inspection area 20 is simply indicated.
　図６に示すように、１つの検査領域２０内の４つの細孔１４には同一種の捕捉物質が備えられ、捕捉物質３０Ａを備えた領域と、捕捉物質３０Ｂを備えた領域とが非検査領域２１を挟んで交互に配置されている。隣り合って配置された検査領域２０間の非検査領域２１の幅ｂは、その幅ｂを隔てて配置される検査領域２０中における細孔間隔ｃよりも広い。非検査領域２１の幅ｂは検査領域２０と非検査領域２１の並び方向における検査領域２０間の最短距離とする。また、細孔間隔ｃは、その並び方向に沿って配置された再隣接細孔間の最短距離とする。また、その並び方向に沿った検査領域２０の一辺の長さａが非検査領域２１の幅ｂよりも長いことが好ましい。すなわち、検査領域２０の一辺ａ、非検査領域２１の幅ｂおよび細孔間隔ｃはａ＞ｂ＞ｃの関係にあることが好ましい。 As shown in FIG. 6, the four pores 14 in one inspection region 20 are provided with the same kind of capture substance, and the region provided with the capture material 30A and the region provided with the capture material 30B are not inspected. The regions 21 are arranged alternately. The width b of the non-inspection region 21 between the inspection regions 20 arranged adjacent to each other is wider than the pore interval c in the inspection region 20 arranged with the width b therebetween. The width b of the non-inspection area 21 is the shortest distance between the inspection areas 20 in the alignment direction of the inspection area 20 and the non-inspection area 21. The pore interval c is the shortest distance between re-adjacent pores arranged along the direction of arrangement. Moreover, it is preferable that the length “a” of one side of the inspection region 20 along the arrangement direction is longer than the width “b” of the non-inspection region 21. That is, it is preferable that the side a of the inspection region 20, the width b of the non-inspection region 21, and the pore interval c have a relationship of a> b> c.
　このように、捕捉物質３０Ａに対する検査領域２０および捕捉物質３０Ｂに対する検査領域２０を備えているので、捕捉物質３０Ａおよび３０Ｂに対する検査を同時に行うことができる。また、検査領域２０間を非検査領域２１により隔てることにより、検査領域２０間におけるクロストークを抑制することができ、より精度の高い検査が可能となる。 Thus, since the inspection area 20 for the capture substance 30A and the inspection area 20 for the capture substance 30B are provided, the inspection for the capture substances 30A and 30B can be performed simultaneously. Further, by separating the inspection regions 20 by the non-inspection region 21, crosstalk between the inspection regions 20 can be suppressed, and a more accurate inspection can be performed.
　図７は、第３の実施形態の検査デバイス３の平面図を示す。
　第２の実施形態の検査デバイス２と同様の４つの検査領域２０を備える。本実施形態の検査デバイス３は、検査領域２０間を隔てる非検査領域２１において、細孔１４を備えていない点で第２の実施形態の検査デバイス２と異なる。このように、非検査領域２１には細孔１４が備えられていなくてもよく、第２の実施形態の検査デバイス２と同様の効果を得ることができる。本検査デバイス３においても、検査領域２０の一辺ａ、非検査領域２１の幅ｂおよび細孔間隔ｃはａ＞ｂ＞ｃの関係にあることが好ましい。 FIG. 7 shows a plan view of the inspection device 3 of the third embodiment.
The same four inspection regions 20 as the inspection device 2 of the second embodiment are provided. The inspection device 3 of the present embodiment is different from the inspection device 2 of the second embodiment in that the non-inspection region 21 that separates the inspection regions 20 is not provided with the pores 14. Thus, the non-inspection region 21 does not need to be provided with the pores 14, and the same effect as the inspection device 2 of the second embodiment can be obtained. Also in the present inspection device 3, it is preferable that the side a of the inspection region 20, the width b of the non-inspection region 21, and the pore interval c have a relationship of a>b> c.
　図８は、第４の実施形態の検査デバイス４の平面図を示す。
　第４の実施形態の検査デバイス４は４つの細孔１４を含む検査領域２０が縦横に６×６で配置されている。図８において、右上がり斜線で示す細孔１４には捕捉物質３０Ａが物理吸着されており、左上がり斜線で示す細孔１４には捕捉物質３０Ｂが物理吸着されている。また、横ストライプで示す細孔１４には捕捉物質３０Ｃが、縦ストライプで示す細孔１４には捕捉物質３０Ｄがそれぞれ物理吸着されている。そして、捕捉物質３０Ａ，３０Ｂ、３０Ｃおよび３０Ｄを各１領域ずつ含む領域を１セットとして、１つの検査デバイス４中に縦横に３×３セットが配置されている。本検査デバイス４では４つの捕捉物質３０Ａ，３０Ｂ，３０Ｃ，３０Ｄについての検査を同時に実施することができる。また、１デバイス中に複数セットを備えることにより、検査ばらつきを抑制し検出感度を高めることができる。 FIG. 8 shows a plan view of the inspection device 4 of the fourth embodiment.
In the inspection device 4 of the fourth embodiment, inspection regions 20 including four pores 14 are arranged 6 × 6 vertically and horizontally. In FIG. 8, the trapping substance 30 </ b> A is physically adsorbed in the pores 14 indicated by the diagonal lines rising to the right, and the trapping substance 30 </ b> B is physically adsorbed in the pores 14 indicated by the diagonal lines rising to the left. Further, the trapping substance 30C is physically adsorbed in the pores 14 indicated by the horizontal stripes, and the trapping substance 30D is physically adsorbed in the pores 14 indicated by the vertical stripes. Then, 3 × 3 sets are arranged vertically and horizontally in one inspection device 4 with one region including each of the capture substances 30A, 30B, 30C, and 30D as one set. In the present inspection device 4, it is possible to simultaneously perform the inspection on the four capture substances 30A, 30B, 30C, and 30D. In addition, by providing a plurality of sets in one device, it is possible to suppress inspection variations and increase detection sensitivity.
　本実施形態の検査デバイス４における非検査領域は第２の実施形態の検査デバイス２と同様に、捕捉物質３０を備えていない細孔１４を有する。また、検査領域２０の幅、非検査領域の幅および細孔間隔の関係についても検査デバイス２と同様である。 As in the inspection device 2 of the second embodiment, the non-inspection region in the inspection device 4 of the present embodiment has pores 14 that do not include the trapping substance 30. The relationship between the width of the inspection region 20, the width of the non-inspection region, and the pore interval is the same as that of the inspection device 2.
　図９は、第５の実施形態の検査デバイス５の断面図を示す。
　図示していないが、本実施形態の検査デバイス５の一方の面１１から見た平面図は、図６に示した第２の実施形態の検査デバイスの平面図と同様である。但し、本例においては、非検査領域２１には細孔に代えて、細孔１４の深さ方向に延び、少なくとも他方の面１２に非貫通である空洞部１６が備えられている。ここで、空洞部１６は、少なくとも他方の面１２側が閉塞した空間である。空洞部１６は、板状基材１０の一方の面１１から他方の面１２の手前まで延びて形成されており、板状基材１０の他方の面１２には空洞部１６に繋がる開口は設けられていない。ここで、「手前まで延びて形成される」とは他方の面１２に向かって延びているが、他方の面１２までは至っていないことを意味する。なお、空洞部１６の、一方の面１１から他方の面１２側に延びる長さ、すなわち、細孔１４の深さ方向に沿った深さは板状基材１０の厚みの半分以上であることが好ましく、さらには３／４以上であることがより好ましい。 FIG. 9 shows a cross-sectional view of the inspection device 5 of the fifth embodiment.
Although not shown, the plan view seen from one surface 11 of the inspection device 5 of the present embodiment is the same as the plan view of the inspection device of the second embodiment shown in FIG. However, in this example, the non-inspection region 21 is provided with a hollow portion 16 extending in the depth direction of the pore 14 and not penetrating at least on the other surface 12 instead of the pore. Here, the cavity 16 is a space where at least the other surface 12 is closed. The hollow portion 16 is formed to extend from one surface 11 of the plate-like base material 10 to the front of the other surface 12, and an opening connected to the hollow portion 16 is provided on the other surface 12 of the plate-like base material 10. It is not done. Here, “formed to extend to the front” means that it extends toward the other surface 12 but does not reach the other surface 12. The length of the cavity 16 extending from the one surface 11 to the other surface 12 side, that is, the depth along the depth direction of the pores 14 is at least half the thickness of the plate-like substrate 10. Is more preferable, and more preferably 3/4 or more.
　なお、非検査領域２１に空洞部１６を設ける場合には、上記実施形態のように、板状基材１０の一方の面１１から他方の面１２の手前まで伸びて形成されるものに限らない。図１０に第６の実施形態の検査デバイス６の断面図を示すように、空洞部１７は、板状基材１０の一方の面１１から他方の面１２に貫通する貫通孔４０を設けた後に、その他方の面１２側の開口を板状基材１０とは異なる材質からなる閉塞部材４２で塞ぐようにして形成されていてもよい。さらに、図１０に示すように、空洞部１７は一方の面１１側の開口も板状基材１０とは異なる材質の第２の閉塞部材４４で塞がれていても構わない。すなわち、空洞部１７は閉じた空間であってもよい。空洞部内部は真空であってもよいし、空気などの屈折率が略１である気体が封じ込められていてもよい。 In addition, when providing the cavity part 16 in the non-inspection area | region 21, it is not restricted to what is extended from the one surface 11 of the plate-shaped base material 10 to the front of the other surface 12 like the said embodiment. . As shown in the cross-sectional view of the inspection device 6 of the sixth embodiment in FIG. 10, the cavity portion 17 is provided with a through hole 40 penetrating from one surface 11 of the plate-like substrate 10 to the other surface 12. Alternatively, the opening on the other surface 12 side may be formed so as to be closed with a closing member 42 made of a material different from that of the plate-like substrate 10. Further, as shown in FIG. 10, the hollow portion 17 may have its opening on one surface 11 side closed by a second closing member 44 made of a material different from that of the plate-like substrate 10. That is, the hollow portion 17 may be a closed space. The inside of the cavity may be a vacuum, or a gas having a refractive index of about 1 such as air may be enclosed.
　なお、空洞部１７を構成する閉塞部材４２および第２の閉塞部材４４を構成する材料は、特に制限されないが、金属もしくは樹脂材料など、インクジェット装置により滴下後、固化もしくは硬化して閉塞部材として構成されうる材料が好適である。 The material constituting the closing member 42 and the second closing member 44 constituting the cavity portion 17 is not particularly limited, and is configured as a closing member by being solidified or cured after being dropped by an ink jet apparatus such as a metal or a resin material. Suitable materials are preferred.
　第２から第６の実施形態の検査デバイス２～６のように、複数の細孔１４を備えた検査領域２０を有する場合、少なくとも検査領域２０において板状基材１０が光透過性を有するものであることが好ましい。１本の細孔１４からの光開口より複数の細孔の開口をまとめて検査領域２０の全体を光開口とすることにより、光開口率が大きくなるため、デバイスの表面に出射される光取り出し光量を増加させることができる。 In the case where the inspection region 20 having the plurality of pores 14 is provided as in the inspection devices 2 to 6 of the second to sixth embodiments, at least the inspection region 20 has the plate-like substrate 10 having light transmittance It is preferable that Since the aperture of a plurality of pores is gathered from the aperture of light from one aperture 14 to make the entire inspection region 20 an optical aperture, the optical aperture ratio increases, so that the light emitted to the surface of the device is extracted. The amount of light can be increased.
　ここで、光透過性を有するとは、後述の検出光の透過率が５０％以上であることをいうが、具体的には、可視光波長域４００～７００ｎｍの平均透過率が５０％以上であれば光透過性を有すると看做すこととする。 Here, having light transmittance means that the transmittance of detection light described later is 50% or more. Specifically, the average transmittance in the visible light wavelength range of 400 to 700 nm is 50% or more. If there is, it will be regarded as having light transparency.
　また、上記第５および第６の実施形態の検査デバイス５、６の場合には、空洞部１６、１７に囲まれた領域全体が光透過性を有することが好ましい。 Further, in the case of the inspection devices 5 and 6 of the fifth and sixth embodiments, it is preferable that the entire region surrounded by the cavities 16 and 17 has light transmittance.
　上記第５および第６の実施形態で示した検査デバイス５、６はそれぞれ空洞部１６、１７を備えたことにより次のような効果を奏する。図１１は検査デバイス５の一部を拡大して示した模式図である。
　本検査デバイス５は、細孔１４の内壁面１４ａには疎水性高分子が結合しており、物理吸着により疎水性高分子に捕捉物質（図１１中では省略している。）が固定化されている。この捕捉物質に被検物質を結合させ、被検物質にさらに酵素により標識された標識物質を結合させ、標識物質を触媒とした化学発光を検出することにより被検物質の有無を検出する、あるいは被検物質の有無および量を検出する検査方法に用いられる。本検査デバイス１からの光信号検出は、細孔１４中にはバッファ液等の検査用溶液６１が充填された状態で実施される。なお、空洞部１６には溶液６１が入り込まないようにして用いられ、空洞部１６は空気層を構成する。 The inspection devices 5 and 6 shown in the fifth and sixth embodiments have the following effects due to the provision of the cavities 16 and 17, respectively. FIG. 11 is a schematic view showing a part of the inspection device 5 in an enlarged manner.
In the present inspection device 5, a hydrophobic polymer is bonded to the inner wall surface 14a of the pore 14, and a trapping substance (not shown in FIG. 11) is immobilized on the hydrophobic polymer by physical adsorption. ing. Detect the presence or absence of the test substance by binding the test substance to this capture substance, binding the label substance labeled with an enzyme to the test substance, and detecting chemiluminescence using the label substance as a catalyst, or It is used in a test method for detecting the presence and amount of a test substance. The detection of the optical signal from the present inspection device 1 is performed in a state where the pore 14 is filled with an inspection solution 61 such as a buffer solution. In addition, it uses so that the solution 61 may not enter into the cavity part 16, and the cavity part 16 comprises an air layer.
　検査領域２０の細孔１４を構成する基材は光透過性を有するので、シリコンの不透過な基材により構成された細孔１４と比較して、細孔１４内で発生した光の表面に出射する光開口率が増加する。また、光透過性を有する板状基材１０の屈折率と検査用溶液の屈折率は１．３～１．５程度の範囲で同等であるのに対して、空気層の屈折率は略１である。すなわち、板状基材１０と空気層との屈折率差は非常に大きいため、界面で大きな反射が生じる。また、板状基材１０は空気層に比べて屈折率が大きいため、板状基材１０側から空気層に向かって臨界角以上の入射角θ１で界面Ｃ１に入射する光は全反射して、板状基材１０の表面側に向かう。また、板状基材１０側から界面Ｃ１に臨界角より小さい入射角θ２で入射した場合であっても、入射光の一部は界面Ｃ１で正反射して板状基材１０の表面側に向かうこととなる。また、界面Ｃ１を透過した光は空気層から空洞部１６を構成する板状基材１０に入射し、この界面Ｃ２で少なくとも一部が正反射されて、やはり板状基材１０の表面側に向かう。このように、検査領域２０の周囲の非検査領域２１に空気層を設けることにより、細孔１４中で発生した光を板状基材１０の表面に向けて集光させる機能が付与される。結果として、検査デバイス１の表面から出射する光量を増加させ、光取り出し効率の向上効果を奏する。 Since the base material forming the pores 14 in the inspection region 20 has light transmittance, the surface of the light generated in the pores 14 is compared with the pores 14 formed of the base material impermeable to silicon. The exit aperture ratio increases. The refractive index of the light-transmitting plate-like substrate 10 and the refractive index of the test solution are equivalent in the range of about 1.3 to 1.5, whereas the refractive index of the air layer is about 1. It is. That is, since the refractive index difference between the plate-like substrate 10 and the air layer is very large, a large reflection occurs at the interface. Further, since the plate-like substrate 10 has a higher refractive index than the air layer, light incident on the interface C 1 at an incident angle θ 1 greater than the critical angle from the plate-like substrate 10 side toward the air layer is totally reflected. Then, it goes to the surface side of the plate-like substrate 10. Even when the incident light is incident on the interface C 1 from the plate-like substrate 10 side at an incident angle θ 2 smaller than the critical angle, a part of the incident light is specularly reflected at the interface C 1 and It will go to the surface side. Further, the light transmitted through the interface C 1 is incident on the plate-like base material 10 constituting the cavity 16 from the air layer, and at least a part of the light is regularly reflected at the interface C 2 , and the surface of the plate-like base material 10 is again formed. Head to the side. Thus, by providing an air layer in the non-inspection area 21 around the inspection area 20, the function of condensing light generated in the pores 14 toward the surface of the plate-like substrate 10 is given. As a result, the amount of light emitted from the surface of the inspection device 1 is increased, and the light extraction efficiency is improved.
　本実施形態の検査デバイス５では、板状基材と空気層との界面で光量をほとんど減衰させることなく反射させて、デバイスの表面側に光を集光させることができる。なお、第６の実施形態の検査デバイス６についても同様の効果を得ることができる。 In the inspection device 5 of the present embodiment, the amount of light can be reflected at the interface between the plate-like base material and the air layer with almost no attenuation, and the light can be condensed on the surface side of the device. The same effect can be obtained for the inspection device 6 of the sixth embodiment.
　本発明の検査デバイスの作製方法を説明する。 A method for producing the inspection device of the present invention will be described.
　複数の細孔を備えたＳｉＯ２からなる板状基材は下のようにして作製できる。
　まず、Ｓｉからなる板状基材を用意し、その一方の面側からウェットエッチングもしくはドライエッチングにより細孔を形成する。例えば、ＭＥＭＳ（Micro Electro Mechanical System）技術において公知のマイクロポーラスＳｉの製造プロセスを用いることができる。なお、図９に示したように非検査領域２１に空洞部１６を備えた構成とする場合には、細孔をエッチング形成する際には、細孔形成領域以外にはマスクをし、空洞部をエッチング形成する際には、空洞部形成領域以外にはマスクをして順次形成すればよい。空洞部は、一方の面側からエッチングを行い、他面の手前でエッチングを止めることにより形成することができる。 A plate-like substrate made of SiO 2 having a plurality of pores can be produced as follows.
First, a plate-like substrate made of Si is prepared, and pores are formed from one surface side by wet etching or dry etching. For example, a microporous Si manufacturing process known in MEMS (Micro Electro Mechanical System) technology can be used. In addition, when it is set as the structure provided with the cavity part 16 in the non-inspection area | region 21, as shown in FIG. 9, when etching a pore, it masks other than a pore formation area, and a cavity part When etching is performed, the mask may be formed sequentially with a mask other than the cavity forming region. The cavity can be formed by etching from one side and stopping the etching before the other side.
　上記のようにして形成した細孔を有するＳｉからなる基材を酸化処理することによりＳｉＯ２に変化させることにより、基材全体をもしくは部分的に透明化することができる。酸化処理としては、例えば温度１１００℃で所定時間熱酸化処理を行う等である。熱酸化処理時間は板状基材の大きさ、形状および所望の酸化処理領域に応じて適宜調整すればよい。 By changing the base material made of Si having pores formed as described above to SiO 2 by oxidation treatment, the whole base material can be made transparent or partially transparent. As the oxidation treatment, for example, thermal oxidation treatment is performed at a temperature of 1100 ° C. for a predetermined time. The thermal oxidation treatment time may be appropriately adjusted according to the size and shape of the plate-like substrate and the desired oxidation treatment region.
　なお、図１０に示した空洞部１７のように貫通孔４０と閉塞部材４２、４４により構成する場合には、まず、空洞部の一部を構成する貫通孔４０を細孔１４と同時にエッチングにより形成する。その後、上記の熱酸化処理によりＳｉ基材をＳｉＯ２化させた後、空洞部の一部を構成する貫通孔４０の他方の面側の開口を基材とは異なる材質の閉塞部材４２で塞ぐ。同様に貫通孔４０の一方の面側の開口を第２の閉塞部材４４で塞ぐ。これにより閉じた空間となる空洞部１７を形成することができる。閉塞部材４２は貫通孔４０の他方の面側の開口を塞ぐことができればよく、貫通孔４０内部に一部侵入して形成されていても構わない。閉塞部材４２は、例えば、インクジェット装置を用いて貫通孔の他方の面側の開口を塞ぐように金属もしくは樹脂材料を滴下させ、固化あるいは硬化して形成することができる。 When the through hole 40 and the blocking members 42 and 44 are formed as in the hollow portion 17 shown in FIG. 10, first, the through hole 40 constituting a part of the hollow portion is etched simultaneously with the pore 14. Form. Thereafter, the Si base material is changed to SiO 2 by the above thermal oxidation treatment, and then the opening on the other surface side of the through hole 40 constituting a part of the cavity is closed with a closing member 42 made of a material different from the base material. . Similarly, the opening on one surface side of the through hole 40 is closed with the second closing member 44. Thereby, the hollow part 17 used as the closed space can be formed. The blocking member 42 only needs to be able to block the opening on the other surface side of the through hole 40, and may be formed so as to partially enter the through hole 40. The closing member 42 can be formed, for example, by dripping a metal or a resin material so as to close the opening on the other surface side of the through hole using an ink jet device, and solidifying or curing.
　一方、複数の細孔を有するＡｌからなる板状基材は、Ａｌの板状基材にドリルなどの機械的な手法で細孔を形成して作製することもできるし、エッチング等により細孔を形成して作製することもできる。また、Ａｌからなる板状基材に対して陽極酸化処理を施すことにより、複数の細孔を有するＡｌ２Ｏ３からなる板状基材を得ることができる。なお、細孔を有する板状基材１０の作製方法は、これらに限らず、多孔性基板を形成する種々の公知の手法を用いることができる。 On the other hand, a plate-like substrate made of Al having a plurality of pores can be produced by forming pores in an Al plate-like substrate by a mechanical method such as a drill, or by etching or the like. Can also be produced. Further, by performing the anodic oxidation process to the plate-shaped substrates made of Al, it is possible to obtain a plate-shaped substrate made of Al 2 O 3 having a plurality of pores. In addition, the production methods of the plate-like substrate 10 having pores are not limited to these, and various known methods for forming a porous substrate can be used.
　以上のようにして、作製された、複数の細孔を備えた板状基材の細孔の内壁面に被検物質と特異的に結合する特異的結合物質である捕捉物質を固定化することにより、検査デバイスを作製することができる。 Immobilizing the capture substance, which is a specific binding substance that specifically binds to the test substance, on the inner wall surface of the pores of the plate-like substrate having a plurality of pores produced as described above Thus, an inspection device can be manufactured.
　細孔の内壁面への捕捉物質の固定は、既述の通り細孔の内壁面に疎水性高分子による化学修飾層を形成し、その後、その疎水性高分子への捕捉物質の物理吸着によって実施することができる。 As described above, the trapping substance is fixed to the inner wall surface of the pore by forming a chemically modified layer with a hydrophobic polymer on the inner wall surface of the pore, and then by physical adsorption of the trapping substance on the hydrophobic polymer. Can be implemented.
　疎水性高分子の細孔内壁面への付与および捕捉物質の物理吸着の具体例を説明する。まず、疎水性高分子の細孔内壁面への付与としては、Ｓｉからなる板状基材にポリスチレンを共有結合させる場合の具体例を説明する（図４Ａ参照）。
　細孔１４が設けられたＳｉからなる板状基材１０に対して、紫外線を５分間照射してクレーニング（ＵＶ（Ultra Violet洗浄）を行う。板状基材１０の表面にＯＨ末端基を持つ分子量１０００～１００００程度のポリスチレンをトルエン溶媒に１質量％で溶解させたポリスチレン溶液をディップコート等により塗布する。これにより細孔１４内にポリスチレン溶液が充填される。これを、１６０～１８０℃で数時間から３日間程度熱処理をすることで脱水縮合させ、細孔１４の内壁面１４ａとポリスチレンとを結合させる。その後、内壁面１４ａに結合していないポリスチレンをトルエンで除去することにより、１０ｎｍ以下の化学修飾層４６を形成することができる。 Specific examples of the application of the hydrophobic polymer to the pore inner wall surface and the physical adsorption of the trapping substance will be described. First, a specific example in which polystyrene is covalently bonded to a plate-like substrate made of Si will be described as application of the hydrophobic polymer to the inner wall surface of the pores (see FIG. 4A).
The plate-like substrate 10 made of Si provided with the pores 14 is subjected to ultraviolet ray irradiation for 5 minutes and subjected to craying (UV (Ultra Violet cleaning). OH end groups are formed on the surface of the plate-like substrate 10. A polystyrene solution in which polystyrene having a molecular weight of about 1000 to 10,000 is dissolved in a toluene solvent at 1% by mass is applied by dip coating, etc. This fills the pores 14 with the polystyrene solution, which is 160 to 180 ° C. Then, heat treatment is performed for several hours to 3 days to dehydrate and condense, and the inner wall surface 14a of the pores 14 and polystyrene are bonded to each other, and then polystyrene not bonded to the inner wall surface 14a is removed with toluene to 10 nm. The following chemically modified layer 46 can be formed.
　捕捉物質としてアレルゲンをポリスチレンに物理吸着させる方法は、次の通りである。アレルゲン抽出物を１０μg/ｍＬになるようＰＢＳ（Phosphate buffered saline；リン酸緩衝生理食塩水）で希釈し、スポッターでアレルゲンを物理吸着させる所定の細孔１４に分注する。その後、３７℃で２時間放置後、液を捨て１質量％のＢＳＡ（Bovine serum albumin；ウシ血清アルブミン）ブロッキング溶液を注入した後、４℃で１２時間放置する。以上の工程により、細孔１４内の疎水性高分子にアレルゲンを物理吸着させることができる。細孔１４内のアレルゲンが物理吸着されていない部分に検査時に物理吸着が生じるのを防ぐために、ブロッキングが施されてなる検査デバイスを得ることができる。
　なお、検査デバイスの細孔毎、あるいは複数の細孔を含む検査領域毎に、異なるアレルゲンを同様の手順にて物理吸着させることが可能である。 A method for physically adsorbing allergen on polystyrene as a capture substance is as follows. The allergen extract is diluted with PBS (Phosphate buffered saline) so as to be 10 μg / mL, and dispensed into predetermined pores 14 on which the allergen is physically adsorbed by a spotter. Then, after leaving at 37 ° C. for 2 hours, the solution is discarded and 1% by mass of BSA (Bovine serum albumin) blocking solution is injected, and then left at 4 ° C. for 12 hours. Through the above steps, the allergen can be physically adsorbed to the hydrophobic polymer in the pores 14. In order to prevent physical adsorption from occurring at the time of inspection in a portion where the allergen in the pores 14 is not physically adsorbed, an inspection device in which blocking is applied can be obtained.
Note that different allergens can be physically adsorbed in the same procedure for each pore of the inspection device or for each inspection region including a plurality of pores.
　次に、本発明の検査デバイスを備えた検査装置および本発明の検査デバイスを用いた検査方法を説明する。 Next, an inspection apparatus provided with the inspection device of the present invention and an inspection method using the inspection device of the present invention will be described.
　図１２は、本発明の一実施形態の検査装置５０の構成を模式的に示す図である。本実施形態の検査装置５０は、上記検査デバイス５と、この検査デバイス５の細孔１４中に検査用溶液を供給する溶液供給部６０と、検査デバイス５の一方の面１１側に配置された光検出器７０とを備えている。光検出器７０は、検査デバイス５から出射される光を検出するものであり、ここでは、検査デバイス５の一方の面１１側に配置されているが、他方の面１２側に配置されるものであってもよい。検査装置５０においては検査デバイス５に限らず、上記のいずれの検査デバイスを備えてもよい。 FIG. 12 is a diagram schematically showing the configuration of the inspection apparatus 50 according to an embodiment of the present invention. The inspection apparatus 50 according to the present embodiment is disposed on the inspection device 5, the solution supply unit 60 that supplies the inspection solution into the pores 14 of the inspection device 5, and the one surface 11 side of the inspection device 5. And a photodetector 70. The light detector 70 detects light emitted from the inspection device 5. Here, the light detector 70 is disposed on the one surface 11 side of the inspection device 5, but is disposed on the other surface 12 side. It may be. The inspection apparatus 50 is not limited to the inspection device 5 and may include any of the inspection devices described above.
　図１３は溶液供給部６０の概略構成を示す図である。
　溶液供給部６０は、検査デバイス５の他方の面１２側に設置される検査用溶液６１を貯留する貯留部６２と、貯留部６２の上部に設置されて、貯留部６２に貯留されている検査用溶液６１を吸引するピペット部６４と、このピペット部６４上に設置される検査デバイス５の一方の面１１側に配置される減加圧空間部６６および減加圧空間部６６の圧力を減圧もしくは加圧するためのポンプ６８を備えている。 FIG. 13 is a diagram illustrating a schematic configuration of the solution supply unit 60.
The solution supply unit 60 stores the inspection solution 61 installed on the other surface 12 side of the inspection device 5, and the inspection installed in the upper part of the storage unit 62 and stored in the storage unit 62. The pipette part 64 that sucks the solution 61 for use, and the pressure of the depressurizing space part 66 and the depressurizing space part 66 arranged on the one surface 11 side of the inspection device 5 installed on the pipette part 64 are reduced. Alternatively, a pump 68 for pressurization is provided.
　ピペット部６４の先端が検査用溶液６１中に浸漬された状態で、ポンプ６８により減加圧空間部６６を減圧することにより検査用溶液６１がピペット部６４を介して検査デバイス５の細孔１４内に供給される。なお、ポンプ６８による減加圧空間部６６の減圧および加圧により、検査デバイス５の細孔１４への検査用溶液６１の供給および、細孔１４からの検査用溶液６１の排出を行うことができる。 In a state where the tip of the pipette part 64 is immersed in the inspection solution 61, the reduced pressure space 66 is decompressed by the pump 68 so that the inspection solution 61 passes through the pipette part 64 and the pores 14 of the inspection device 5. Supplied in. Note that the inspection solution 61 is supplied to the pores 14 of the inspection device 5 and the inspection solution 61 is discharged from the pores 14 by the pressure reduction and pressurization of the reduced pressure space 66 by the pump 68. it can.
　ここで、検査用溶液６１は、光検出時に細孔１４中に供給される溶液であるが、溶液供給部６０は、検査工程において細孔１４中に供給すべき検体液、標識溶液、洗浄液等の供給にも用いられる。すなわち、溶液供給部６０は検査の工程毎に必要な溶液を細孔１４に供給するものである。なお、空洞部１６は他方の面１２側が非開口であるため、供給される溶液は他方の面１２側から空洞部１６に浸入することはない。そして、既述の空洞部１６を備えた効果を得るために、空洞部１６が開口されている一方の面１１からも溶液が空洞部に入りこまないように溶液供給部６０は溶液の供給を制御する。図１３に示すように、溶液供給部６０は、減加圧空間部６６の一部に空洞部１６の開口を覆う蓋部６７を備えた、一方の面１１の細孔１４の開口から溢れた溶液が空洞部１６に入り込まないような構成であってもよい。 Here, the test solution 61 is a solution supplied into the pores 14 at the time of light detection, but the solution supply unit 60 includes a sample solution, a label solution, a cleaning solution, and the like to be supplied into the pores 14 in the test process. It is also used for supply. That is, the solution supply unit 60 supplies a necessary solution to the pores 14 for each inspection process. The cavity 16 is not open on the other surface 12 side, so that the supplied solution does not enter the cavity 16 from the other surface 12 side. In order to obtain the effect provided with the cavity 16 described above, the solution supply unit 60 supplies the solution so that the solution does not enter the cavity from one surface 11 where the cavity 16 is opened. Control. As shown in FIG. 13, the solution supply unit 60 was provided with a lid portion 67 that covers the opening of the cavity portion 16 in a part of the depressurizing space portion 66, and overflowed from the opening of the pores 14 on one surface 11. The configuration may be such that the solution does not enter the cavity 16.
　以下、上記検査装置５０を用いた本発明の検査方法について説明する。
　本発明において検査デバイスから出射され、光検出器により検出される光は、例えば、被検物質に付与された標識が励起されて生じる蛍光、もしくは標識が反応液の基質として作用することで生じる発光反応による発光（以下において、「化学発光」という。）など、標識に起因する光信号である。なお、被検物質が自家蛍光を生じるものであれば、標識は不要であり、自家蛍光を検出すればよい。また、ここで光信号とは、蛍光、化学発光光のほか、吸光度（比色）を含むものとする。 Hereinafter, the inspection method of the present invention using the inspection apparatus 50 will be described.
In the present invention, the light emitted from the inspection device and detected by the photodetector is, for example, fluorescence generated when the label attached to the test substance is excited, or luminescence generated by the label acting as a substrate of the reaction solution It is an optical signal resulting from a label, such as luminescence by reaction (hereinafter referred to as “chemiluminescence”). In addition, if the test substance generates autofluorescence, no label is necessary, and autofluorescence may be detected. Here, the optical signal includes absorbance (colorimetric) in addition to fluorescence and chemiluminescence light.
　図１４は検査工程を模式的に示す図である。
　検査デバイス５の細孔１４の内壁面１４ａには疎水性高分子が結合しており、アレルゲンなどの捕捉物質３０（３０Ａあるいは３０Ｂ）が疎水性高分子に物理吸着により固定されている（Ｓ１）。この検査デバイス５の細孔１４に被検物質（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）を含む検体液を供給して、被検物質３２を捕捉物質３０に結合させる（Ｓ２）。検体液の供給には、上述の溶液供給部６０を用いる。貯留部６２に検体液を貯留させた状態で、ポンプを動作させてピペット部６４による検体液の吸引および排出を繰り返すことにより、検体液を細孔１４内の捕捉物質３０に効率よく接触させることができる。 FIG. 14 is a diagram schematically showing the inspection process.
A hydrophobic polymer is bonded to the inner wall surface 14a of the pore 14 of the inspection device 5, and a capture substance 30 (30A or 30B) such as an allergen is fixed to the hydrophobic polymer by physical adsorption (S1). . A specimen liquid containing a test substance (for example, a specific IgE antibody that specifically binds to the allergen) is supplied to the pores 14 of the test device 5, and the test substance 32 is bound to the capture substance 30 (S2). ). The above-described solution supply unit 60 is used for supplying the sample liquid. The sample liquid is efficiently brought into contact with the capture substance 30 in the pores 14 by repeatedly pumping and discharging the sample liquid by the pipette unit 64 while the sample liquid is stored in the storage unit 62. Can do.
　次に、検体液を排出した後、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１４に供給して、被検物質３２に標識物質３５を結合させる（Ｓ３）。標識溶液の細孔１４へ供給は上記検体液の供給と同様に溶液供給部６０により行う。 Next, after discharging the sample liquid, a labeled solution containing a labeled substance 35 in which a label F is added to a substance 33 (for example, a secondary antibody) that specifically binds to the test substance 32 is supplied to the pores 14. Then, the labeling substance 35 is bound to the test substance 32 (S3). The label solution is supplied to the pores 14 by the solution supply unit 60 in the same manner as the sample solution.
　なお、上記では、被検物質３２の捕捉物質３０への結合の後に、標識物質３５を被検物質３２に結合させることとしたが、検査装置５０外において、あらかじめ検体液と標識溶液を混合して被検物質３２と標識物質３５を結合させ、その混合液を細孔１４内に供給するようにしてもよい。この場合、混合液の細孔１４への供給により、細孔１４内に固定された捕捉物質３０に、標識物質３５が結合された被検物質３２を結合させることができる。 In the above description, the labeling substance 35 is bound to the test substance 32 after the test substance 32 is bound to the capture substance 30. However, the sample liquid and the labeling solution are mixed in advance outside the test apparatus 50. Thus, the test substance 32 and the labeling substance 35 may be combined to supply the mixed liquid into the pores 14. In this case, by supplying the mixed liquid to the pores 14, the test substance 32 to which the labeling substance 35 is bound can be bound to the capture substance 30 fixed in the pores 14.
　そして、標識溶液もしくは混合液を排出した後、検体液の供給と同様の方法で、溶液供給部６０を用いて細孔１４に洗浄液を供給して、細孔１４中に非特異吸着している被検物質３２や標識物質３５を除去する。 Then, after discharging the labeling solution or the mixed solution, the cleaning solution is supplied to the pores 14 using the solution supply unit 60 in the same manner as the supply of the sample solution, and nonspecific adsorption is performed in the pores 14. The test substance 32 and the labeling substance 35 are removed.
　最後に、洗浄液を排出して、細孔１４内にバッファ溶液等の検査用溶液を充填させた状態で、標識Ｆを基質として作用する発光反応から生じる光を光検出器７０により検出する（Ｓ４）。なお、光検出器７０による検査デバイス５からの出射光の検出時、空洞部１６は空気層を構成している。 Finally, the cleaning solution is discharged, and light generated from a luminescence reaction that acts using the label F as a substrate is detected by the photodetector 70 in a state where the inspection solution such as a buffer solution is filled in the pores 14 (S4). ). When the light emitted from the inspection device 5 is detected by the photodetector 70, the cavity 16 forms an air layer.
　なお、標識Ｆが蛍光色素、量子ドットなどの蛍光標識である場合には、バッファ溶液を検査用溶液として細孔１４内に供給し、細孔１４内をバッファ溶液で満たした状態で、蛍光測定を行う。具体的には、蛍光標識を励起する波長の光を励起光として検査デバイスに照射し、その励起光により励起された標識からの蛍光を検出する。なお、蛍光標識の検出の際に用いられる光検出器７０には、励起光照射部が備えられている。 When the label F is a fluorescent label such as a fluorescent dye or a quantum dot, the fluorescence measurement is performed in a state where the buffer solution is supplied into the pore 14 as a test solution and the pore 14 is filled with the buffer solution. I do. Specifically, the inspection device is irradiated with light having a wavelength that excites the fluorescent label as excitation light, and fluorescence from the label excited by the excitation light is detected. The photodetector 70 used for detecting the fluorescent label is provided with an excitation light irradiation unit.
　一方、上述のように、標識Ｆがルミノールなどの化学発光基質を触媒する酵素標識である場合には、上記洗浄処理の後、酵素標識を触媒として化学反応が促進される化学発光基質を含む反応液を検査用溶液として細孔１４中に供給する。そして、光検出器７０では、細孔１４にこの反応液を供給にすることにより、細孔１４の捕捉物質３０に捕捉された被検物質３２に付与された酵素標識が反応液中の物質の化学反応を触媒して生じる発光を検出する。この場合、反応液により細孔１４が満たされた状態で発光検出を行う。 On the other hand, as described above, when the label F is an enzyme label that catalyzes a chemiluminescent substrate such as luminol, a reaction including a chemiluminescent substrate that promotes a chemical reaction using the enzyme label as a catalyst after the washing treatment. The liquid is supplied into the pores 14 as a test solution. Then, in the photodetector 70, by supplying this reaction solution to the pores 14, the enzyme label imparted to the test substance 32 captured by the capture material 30 in the pores 14 becomes the substance in the reaction solution. Luminescence generated by catalyzing a chemical reaction is detected. In this case, luminescence detection is performed in a state where the pores 14 are filled with the reaction solution.
　化学発光を生じる酵素標識としては、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と反応する酵素などが挙げられる。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。 Examples of enzyme labels that generate chemiluminescence include enzymes that react with chemiluminescent substrates such as luminol, lophine, lucigenin and oxalate. When HRP (horseradish peroxidase) enzyme is used as a label, a reaction liquid (luminol reaction liquid) containing a luminol-based chemiluminescent substrate in which HRP functions as a catalyst is used, and an ALP (alkaline phosphatase) enzyme is used. It is preferable to use a reaction solution containing a dioxetane 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。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 catalyzes the oxidation of luminol in the presence of hydrogen peroxide. The reaction solution preferably contains a sensitizer that sensitizes 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、蛍光を検出するものとしてもよい。 In light detection using an enzyme label, not only the chemiluminescent substrate described above but also a reaction solution containing a luminescent substrate or a fluorescent substrate may be used to detect a color reaction (absorption) or fluorescence.
　いずれの検出方法であっても、本発明の検査デバイスを用いることにより、細孔中で生じた光は検査デバイスの表面から効率よく出射され、光の取り出し効率が高いので、高精度な光検出が可能である。 Regardless of the detection method, by using the inspection device of the present invention, the light generated in the pores is efficiently emitted from the surface of the inspection device, and the light extraction efficiency is high. Is possible.
　１，１Ａ，１Ｂ，２，３，４，５，６　　検査デバイス
　１０　板状基材
　１１　板状基材（検査デバイス）の一方の面
　１２　板状基材（検査デバイス）の他方の面
　１４　細孔
　１４ａ　細孔の内壁面
　１６，１７　空洞部
　２０（２０Ａ，２０Ｂ，２０Ｃ，２０Ｄ）　検査領域
　３０（３０Ａ、３０Ｂ、３０Ｃ，３０Ｄ）　捕捉物質
　３２　被検物質
　３３　被検物質に特異的に結合する物質
　３５　標識物質
　４０　貫通孔
　４２　閉塞部材
　４４　第２の閉塞部材
　４５　疎水性高分子を含む溶液
　４６　化学修飾層（疎水性高分子層）
　５０　検査装置
　６０　溶液供給部
　６１　検査用溶液
　６２　貯留部
　６４　ピペット部
　６６　減加圧空間部
　６７　蓋部
　６８　ポンプ
　７０　光検出器
　Ｆ　標識 1, 1A, 1B, 2, 3, 4, 5, 6 Inspection device 10 Plate-like substrate 11 One surface of plate-like substrate (inspection device) 12 Other surface of plate-like substrate (inspection device) 14 Fine Hole 14a Inner wall surface 16, 17 Cavity 20 (20A, 20B, 20C, 20D) Inspection region 30 (30A, 30B, 30C, 30D) Captured substance 32 Test substance 33 Specific binding to test substance Substance 35 Labeling substance 40 Through-hole 42 Blocking member 44 Second blocking member 45 Solution containing hydrophobic polymer 46 Chemical modification layer (hydrophobic polymer layer)
DESCRIPTION OF SYMBOLS 50 Inspection apparatus 60 Solution supply part 61 Test solution 62 Storage part 64 Pipette part 66 Depressurization space part 67 Cover part 68 Pump 70 Photo detector F Marker

Claims (15)

  1. 　無機物からなる板状基材の一方の面から他方の面に貫通する複数の細孔を備えた検査デバイスであって、
    　少なくとも２つ以上の細孔の内壁面に疎水性高分子が結合しており、
    　互いに異なる細孔の前記疎水性高分子に、被検物質と特異的に結合する、抗原または抗体からなる互いに異なる捕捉物質が物理吸着されている検査デバイス。     An inspection device comprising a plurality of pores penetrating from one surface of a plate-like substrate made of an inorganic material to the other surface,
    A hydrophobic polymer is bonded to the inner wall surface of at least two or more pores,
    A test device in which different capture substances composed of an antigen or an antibody that specifically bind to a test substance are physically adsorbed to the hydrophobic polymer having different pores.
  2. 　前記疎水性高分子と前記板状基材は、前記疎水性高分子をＲ、前記板状基材を構成する前記無機物をＭとしたとき、酸素原子Ｏを介して、Ｒ－Ｏ－Ｍ結合で共有結合している請求項１記載の検査デバイス。 The hydrophobic polymer and the plate-like substrate are R—O—M bonds through an oxygen atom O, where R is the hydrophobic polymer and M is the inorganic substance constituting the plate-like substrate. The inspection device according to claim 1, wherein the inspection device is covalently bonded.
  3. 　前記疎水性高分子はポリスチレンまたはポリプロピレンである請求項１または２に記載の検査デバイス。 3. The inspection device according to claim 1, wherein the hydrophobic polymer is polystyrene or polypropylene.
  4. 　前記無機物がＳｉ、ＳｉＯ２、ＡｌおよびＡｌ２Ｏ３のうちの１種もしくは２種以上である請求項１から３のいずれか１項に記載の検査デバイス。 The inspection device according to any one of claims 1 to 3, wherein the inorganic substance is one or more of Si, SiO 2 , Al, and Al 2 O 3 .
  5. 　前記一方の面から見た場合において、前記捕捉物質が吸着された複数の細孔を含む検査領域を複数備え、該複数の検査領域のうち隣り合って配置された検査領域間は、該隣り合って配置された検査領域中における細孔間隔よりも広い幅の非検査領域によって分離されており、
    　１つの前記検査領域中の細孔には同一種の捕捉物質が物理吸着されてなり、
    　前記隣り合って配置された検査領域のうち一方の検査領域の細孔と他方の検査領域の細孔には互いに異なる捕捉物質が物理吸着されている請求項１から４のいずれか１項に記載の検査デバイス。     When viewed from the one surface, a plurality of inspection regions including a plurality of pores to which the trapping substance has been adsorbed are provided, and inspection regions arranged adjacent to each other among the plurality of inspection regions are adjacent to each other. Separated by a non-inspection area with a width wider than the pore spacing in the inspection area arranged
    The same kind of trapping substance is physically adsorbed in the pores in one of the inspection regions,
    5. The trapping material different from each other is physically adsorbed in the pores of one inspection region and the pores of the other inspection region among the inspection regions arranged adjacent to each other. Inspection device.
  6. 　前記非検査領域は、前記複数の細孔のうちの前記捕捉物質が不存在である非検査用細孔が配置された領域である請求項５に記載の検査デバイス。 6. The inspection device according to claim 5, wherein the non-inspection region is a region where non-inspection pores in which the trapping substance is absent from the plurality of pores are arranged.
  7. 　前記非検査領域に、前記細孔の深さ方向に延び、少なくとも前記他方の面に非貫通である空洞部を備えている請求項５に記載の検査デバイス。 6. The inspection device according to claim 5, wherein the non-inspection region includes a cavity that extends in the depth direction of the pore and is not penetrating at least on the other surface.
  8. 　前記非検査領域は、前記細孔を備えていない前記板状基材の部分である請求項５に記載の検査デバイス。 The inspection device according to claim 5, wherein the non-inspection region is a portion of the plate-like base material that does not include the pores.
  9. 　前記一方の面において、前記非検査領域と前記検査領域の並び方向における前記検査領域の一辺が、前記非検査領域の前記幅よりも長い請求項５から８のいずれか１項に記載の検査デバイス。 9. The inspection device according to claim 5, wherein one side of the inspection area in an arrangement direction of the non-inspection area and the inspection area is longer than the width of the non-inspection area on the one surface. .
  10. 　前記一方の検査領域と前記他方の検査領域とが周期的に配置されている請求項５から９のいずれか１項に記載の検査デバイス。 The inspection device according to any one of claims 5 to 9, wherein the one inspection region and the other inspection region are periodically arranged.
  11. 　前記互いに異なる捕捉物質として、粗精製アレルゲンと、該粗精製アレルゲン中に含まれる少なくとも１つのアレルゲンコンポーネントとを含む請求項１から１０いずれか１項に記載の検査デバイス。 The inspection device according to any one of claims 1 to 10, wherein the different capture substances include a crude purified allergen and at least one allergen component contained in the crude purified allergen.
  12. 　請求項１から１１のいずれか１項に記載の検査デバイスと、
    　前記検査デバイスの前記細孔中に検査用溶液を供給する溶液供給部と、
    　前記検査デバイスの前記一方の面側または前記他方の面側に配置され、前記検査デバイスから出射される光を検出する光検出器とを備えた検査装置。     The inspection device according to any one of claims 1 to 11,
    A solution supply unit for supplying a test solution into the pores of the test device;
    An inspection apparatus comprising: a photodetector that is disposed on the one surface side or the other surface side of the inspection device and detects light emitted from the inspection device.
  13. 　請求項１から１１のいずれか１項に記載の検査デバイスの前記少なくとも一部の細孔に、前記被検物質を含有する検体液を供給して、前記被検物質を前記捕捉物質に結合させ、
    　前記被検物質と特異的に結合する標識物質を該被検物質に結合させ、
    　前記細孔に検査用溶液を供給して該細孔に前記検査用溶液を留めた状態で、前記検査デバイスから出射される光を検出する検査方法。     A sample liquid containing the test substance is supplied to the at least some of the pores of the test device according to any one of claims 1 to 11, and the test substance is bound to the capture substance. ,
    Binding a labeling substance that specifically binds to the test substance to the test substance,
    An inspection method for detecting light emitted from the inspection device in a state where an inspection solution is supplied to the pores and the inspection solution is retained in the pores.
  14. 　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反応液を用い、
    　前記出射される光として、前記反応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１３記載の検査方法。     Using a substance containing an enzyme label as the labeling substance,
    As the test solution, a reaction solution containing a substrate that is catalyzed by the enzyme label and reacts,
    The test | inspection method of Claim 13 which detects the light which arises when the said substrate in the said reaction liquid is catalyzed by the said enzyme label | marker as the said emitted light.
  15. 　前記標識物質として蛍光標識を含む物質を用い、
    　該蛍光標識を励起させる励起光を前記検査デバイスに照射し、
    　前記出射される光として、前記励起光の照射により前記標識物質から生じる蛍光を検出する請求項１３記載の検査方法。     Using a substance containing a fluorescent label as the labeling substance,
    Irradiating the inspection device with excitation light for exciting the fluorescent label;
    The inspection method according to claim 13, wherein fluorescence emitted from the labeling substance by irradiation with the excitation light is detected as the emitted light.
PCT/JP2017/010099 2016-03-30 2017-03-14 Inspection device, inspection apparatus, and inspection method WO2017169716A1 (en)

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Citations (5)

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JP2007279027A (en) * 2006-03-14 2007-10-25 Fujifilm Corp Method of inspecting biosensor surface
JP4125244B2 (en) * 2002-04-19 2008-07-30 インフィネオン テクノロジーズ アクチエンゲゼルシャフト Apparatus comprising locally oxidized porous silicon and method for producing the same
JP2014206460A (en) * 2013-04-12 2014-10-30 三菱レイヨン株式会社 Protein immobilization gel microarray
JP2015034797A (en) * 2013-08-09 2015-02-19 日本電信電話株式会社 Antibody immobilization carrier and production method thereof

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Publication number Priority date Publication date Assignee Title
JP2004510996A (en) * 2000-10-10 2004-04-08 バイオトローブ・インコーポレイテツド Instruments for assay, synthesis, and storage, and methods of making, using, and manipulating them
JP4125244B2 (en) * 2002-04-19 2008-07-30 インフィネオン テクノロジーズ アクチエンゲゼルシャフト Apparatus comprising locally oxidized porous silicon and method for producing the same
JP2007279027A (en) * 2006-03-14 2007-10-25 Fujifilm Corp Method of inspecting biosensor surface
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