WO2017169716A1 - Dispositif d'inspection, appareil d'inspection et procédé d'inspection - Google Patents

Dispositif d'inspection, appareil d'inspection et procédé d'inspection 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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English (en)
Japanese (ja)
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弘隆 渡野
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富士フイルム株式会社
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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.

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Abstract

Le problème décrit par la présente invention est de fournir : un dispositif d'inspection ayant une configuration qui permet d'immobiliser facilement des substances capturées de multiples types différents dans un seul dispositif ; un appareil d'inspection équipé du dispositif d'inspection ; et un procédé d'inspection. La solution selon l'invention concerne un dispositif d'inspection (1) ayant une pluralité de pores (14) qui pénètrent d'une face (11) à l'autre face (12) d'un matériau de base de type plaque (10) constitué d'une substance inorganique. Un polymère hydrophobe est lié aux faces de paroi interne (14a) d'au moins deux pores (14), et des substances capturées différentes (30A, 30B, 30C, etc.) comprenant des antigènes ou des anticorps sont physisorbées sur les polymères hydrophobes de différents pores (14).
PCT/JP2017/010099 2016-03-30 2017-03-14 Dispositif d'inspection, appareil d'inspection et procédé d'inspection WO2017169716A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2004510996A (ja) * 2000-10-10 2004-04-08 バイオトローブ・インコーポレイテツド アッセイ、合成、および保存用の器具、ならびに、その作製、使用、および操作の方法
JP2007279027A (ja) * 2006-03-14 2007-10-25 Fujifilm Corp バイオセンサー表面の検査方法
JP4125244B2 (ja) * 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 局部的に酸化された多孔質シリコンからなる装置、および、その製造方法
JP2014206460A (ja) * 2013-04-12 2014-10-30 三菱レイヨン株式会社 タンパク質固定ゲルマイクロアレイ
JP2015034797A (ja) * 2013-08-09 2015-02-19 日本電信電話株式会社 抗体固定化担体およびその製造方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2004510996A (ja) * 2000-10-10 2004-04-08 バイオトローブ・インコーポレイテツド アッセイ、合成、および保存用の器具、ならびに、その作製、使用、および操作の方法
JP4125244B2 (ja) * 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 局部的に酸化された多孔質シリコンからなる装置、および、その製造方法
JP2007279027A (ja) * 2006-03-14 2007-10-25 Fujifilm Corp バイオセンサー表面の検査方法
JP2014206460A (ja) * 2013-04-12 2014-10-30 三菱レイヨン株式会社 タンパク質固定ゲルマイクロアレイ
JP2015034797A (ja) * 2013-08-09 2015-02-19 日本電信電話株式会社 抗体固定化担体およびその製造方法

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