WO2020171158A1 - Puce de réaction en phase solide et procédé de mesure l'utilisant - Google Patents

Puce de réaction en phase solide et procédé de mesure l'utilisant Download PDF

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Publication number
WO2020171158A1
WO2020171158A1 PCT/JP2020/006695 JP2020006695W WO2020171158A1 WO 2020171158 A1 WO2020171158 A1 WO 2020171158A1 JP 2020006695 W JP2020006695 W JP 2020006695W WO 2020171158 A1 WO2020171158 A1 WO 2020171158A1
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Prior art keywords
substance
phase reaction
reaction chip
solid
binding
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PCT/JP2020/006695
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English (en)
Japanese (ja)
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昭雄 中原
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株式会社パートナーファーム
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Priority to CN202080015179.1A priority Critical patent/CN113454462A/zh
Priority to KR1020217026444A priority patent/KR20210127945A/ko
Priority to JP2021502126A priority patent/JP7205940B2/ja
Publication of WO2020171158A1 publication Critical patent/WO2020171158A1/fr

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    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • 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
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • 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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/04—Closures and closing means
    • B01L2300/041—Connecting closures to device or container
    • B01L2300/042—Caps; Plugs
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0803—Disc shape

Definitions

  • the present invention relates to a solid-phase reaction chip and a measuring method using the same.
  • type I allergies that cause diseases such as bronchial asthma, allergic rhinitis, urticaria, and anaphylactic shock are caused by an overreaction of a specific antigen (allergen) with an IgE antibody against it.
  • a test for detecting an IgE antibody contained in a sample liquid is performed in order to identify a causative allergen that causes an allergic disease.
  • CAP having an immunofluorescence method as a measurement principle
  • MAST having an immunochemiluminescence method as a measurement principle
  • a solid-phase reaction chip is used that performs the detection of a measurement target substance bound to a binding substance on the solid phase.
  • the solid-phase reaction chip is also called a biochip in the field of biochemistry, and for example, an mRNA chip, a cDNA chip, a micro PCR chip, a protein chip and the like are known.
  • an immunoassay microchip using an antigen-antibody reaction has also been proposed for the diagnosis of allergic diseases.
  • antigens of various allergens are treated as independent spots, that is, spaced spots.
  • a biochip analysis method that uses a mounted biochip to automate the process of detecting a reaction between a sample liquid and an antigen after the sample liquid is collected and to quickly obtain a measurement result.
  • CAP which is a conventional technique, is a single-item method in which measurement is basically performed for each type of allergen, and a sample liquid is required for each measurement, so that the physical burden on the subject is large and the throughput related to the measurement is also high. Not good.
  • MAST is a multi-item method in which, for example, about 30 types of allergens are measured at once. Although the number of allergens that can be measured simultaneously is larger than in the single-item method, the operation is complicated and it takes about 5 hours to obtain the measurement results.
  • Patent Document 1 requires a suction nozzle for sucking each liquid supplied from each nozzle such as a cleaning nozzle, an antibody nozzle, and a reagent nozzle, and by the time the suction of each liquid is completed. As time is required, there is a risk that the suction force may be insufficient due to a malfunction of the suction pump, or that the deposits attached to the suction nozzle may attach to the biochip and cause contamination.
  • the applicant of the present application includes a liquid absorbing member on the outer circumference of a rotating body formed by stacking a first base portion and a second base portion, and accommodates the liquid absorbing member.
  • a solid-phase reaction chip consisting of a cover member and applied for a patent (Patent Document 2).
  • Patent Document 2 a plurality of binding substances having a specific binding ability with respect to a measurement target substance contained in a sample liquid are fixed to a surface of the second base portion facing the first base portion. It is possible to realize simultaneous multi-item measurement with a small amount of sample liquid by changing the method, and according to the invention of Patent Document 2, a measurement target substance contained in the sample liquid can be quickly and simply operated. Can be detected.
  • the solid-phase reaction chip according to Patent Document 2 requires a cover member that houses a rotating body formed by stacking the first base portion and the second base portion.
  • the cover member plays a role of forming the solid-phase reaction chip outer shape and holding the rotating body through the liquid absorbing member.
  • the rotating body is displaced inside the cover member as it rotates at a high speed, affecting the measurement accuracy.
  • the circular rotating body is in surface contact with the liquid absorbing member formed in an annular shape, there is a problem that a liquid pool is generated at the contact surface and the liquid absorbing ability is reduced.
  • the present invention has been made to solve such a problem, and an object of the present invention is to overcome the shortcomings of a solid-phase reaction chip having a liquid-absorbing member inside, and to perform a sample with a quick and simple operation. It is an object of the present invention to provide a solid-phase reaction chip capable of accurately detecting a substance to be measured contained in a liquid and a measuring method using the same.
  • a solid-phase reaction chip is a solid-phase reaction chip formed as a rotating body by fitting and integrating an upper lid member and a lower lid member, Alternatively, an immobilization unit that is provided on the inner bottom surface side of any one of the lower lid members and that immobilizes a plurality of binding substances having a specific binding ability with respect to the measurement target substance contained in the sample liquid, and the upper lid member.
  • An opening is provided that supplies the sample liquid to the surface of the immobilization unit, and a liquid absorption unit that is provided outside the immobilization unit and that absorbs the sample liquid supplied through the opening.
  • the immobilization part has a plurality of immobilization regions divided by the ridges that bulge toward the inner bottom surface direction side facing each other, and at least one apex is included in the outer edge part of each immobilization region. It has a feature.
  • the measurement method according to the present invention the solid-phase reaction chip, introducing the sample liquid through the opening, a binding step of binding the measurement target substance and the binding substance, the binding An addition step of adding a reaction solution containing a physiologically active substance having a specific binding ability to the measurement target substance bound to a substance and having an enzymatic activity, and introducing a washing liquid through the opening, and then a solid phase It is characterized by comprising a removing step of removing the washing liquid by rotating the reaction chip at a predetermined rotation speed, and a measuring step of measuring the enzyme activity of the physiologically active substance.
  • the measurement method according to the present invention with respect to the solid-phase reaction chip, the sample liquid is introduced through the opening, and the substance to be measured and the binding substance immobilized on the immobilization unit are
  • a labeled substance having a specific binding ability with respect to the substance to be measured and labeled with a signal generating substance is bound to the binding substance in the first binding step. It is characterized by comprising a second binding step of binding to the substance to be measured and a measuring step of measuring a signal generated by the signal generating substance.
  • phase reaction chip having a liquid-absorbing member inside, and to detect a substance to be measured contained in a sample liquid with high accuracy by a quick and simple operation.
  • a phase reaction chip and a measuring method using the same can be provided.
  • FIG. 3 is a component diagram of each member constituting the solid-phase reaction chip 100.
  • FIG. (A) is a plan view of the upper lid member 10 when viewed from the inner bottom surface 11 side, and (b) is a side sectional view of the upper lid member 10.
  • (A) is a plan view of the lower lid member 20 as viewed from the inner bottom surface 21 side, and (b) is a side sectional view of the lower lid member 20.
  • FIG. 2 is a side sectional view of the solid-phase reaction chip 100 shown in FIG. 1. It is a flow chart explaining the measuring method concerning this embodiment.
  • 3 is a block diagram illustrating a configuration example of an automatic measurement device 200.
  • FIG. 9A is a plan view illustrating an upper lid member 102 according to a modified example
  • FIG. 9B is a plan view illustrating a lower lid member 302 according to a modified example.
  • (A) is a perspective view explaining the solid-phase reaction chip 50 which concerns on a modification
  • (b) is a perspective view explaining the solid-phase reaction chip 60 which concerns on a modification. It is a figure which shows the coloring state of the colorimetric spot measured by the measuring method which concerns on this embodiment. It is a figure which shows the coloring state of the colorimetric spot measured by the measuring method which concerns on this embodiment.
  • a solid-phase reaction chip according to the present invention is a solid-phase reaction chip formed as a rotating body by fitting and integrating an upper lid member and a lower lid member, and the solid state reaction chip is provided inside either the upper lid member or the lower lid member.
  • An immobilization unit that is provided on the bottom surface and that immobilizes a plurality of binding substances that have specific binding ability to the measurement target substance contained in the sample liquid, and that is provided on the upper lid member, and the sample liquid is attached to the surface of the immobilization unit. It has an opening for supplying and an absorbing part which is provided outside the immobilizing part and absorbs the sample liquid supplied through the opening, and the immobilizing part is bulged toward the inner bottom face direction facing the immobilizing part. It has a plurality of immobilization regions sectioned by ridges, and the outer edge of each immobilization region includes at least one apex. The details will be described below.
  • FIG. 1 is a perspective view for explaining an overview of the solid-phase reaction chip 100 according to the present embodiment
  • FIG. 2 is a component diagram of each member constituting the solid-phase reaction chip 100.
  • the solid-phase reaction chip 100 according to the present embodiment includes an upper lid member 10 and a lower lid member 20 that is fitted and integrated with the upper lid member 10 to form a rotating body together with the upper lid member 10, and the upper lid member 10 and the lower lid member.
  • An internal space 30 formed by fitting with 20 absorbs a sample liquid, a reaction liquid, a cleaning liquid, etc. (hereinafter, may be simply referred to as a liquid) supplied through the opening 14.
  • the member 40 is provided along the outer periphery 27 of the immobilizing portion 23 described later.
  • FIG. 3A is a plan view of the upper lid member 10 when viewed from the inner bottom surface 11 side
  • FIG. 3B is a side sectional view of the upper lid member 10.
  • the upper lid member 10 according to the present embodiment has a substantially circular shape, and the upper lid fitting wall is configured to be fittable with the lower lid member 20 along the peripheral edge (circumference) of the inner bottom surface 11. 12 are erected.
  • the inner bottom surface 11 of the upper lid member 10 has the same quadrangular shape as the planar view shape of the immobilizing portion 23 of the lower lid member 20, and the immobilizing portion facing surface 13 facing the immobilizing portion surface 24 is formed. ..
  • the immobilization portion facing surface 13 forms a space between the immobilization portion surface 24 and the immobilization portion surface 24 by contact with a raised portion 26 provided on the immobilization portion surface 24, which will be described later, and is supplied through the opening 14.
  • the liquid thus formed can be spread on the surface 24 of the immobilizing portion.
  • the fixing portion facing surface 13 can be formed with a predetermined protrusion width a from the inner bottom surface 11 or can be formed as a smooth surface on the inner bottom surface 11. It can also be formed directly.
  • the planar view shape of the fixing portion facing surface 13 may be the same as the planar view shape of the entire fixing portion 23, or may be different.
  • the opening 14 is provided substantially in the center of the surface of the upper lid member 10 and coaxially with the rotation axis of the lower lid member 20.
  • the opening 14 has an opening formed in a tapered shape, and is electrically connected to the surface of the fixing portion facing surface 13.
  • FIG. 4A is a plan view of the lower lid member 20 as viewed from the inner bottom surface 21 side
  • FIG. 4B is a side sectional view of the lower lid member 20.
  • the lower lid member 20 according to the present embodiment has a substantially perfect circular shape that is the same as the upper lid member 10, and can be fitted to the upper lid member 10 inside the peripheral edge (circumference) of the inner bottom surface 21.
  • the lower lid fitting wall 22 is erected so that On the inner bottom surface 21 of the lower lid member 20, a fixing portion 23 having a quadrangular shape in plan view is formed.
  • An immobilization region 25 for immobilizing a plurality of binding substances having a specific binding ability with respect to a substance to be measured is formed on the immobilization portion surface 24 of the immobilization unit 23.
  • the immobilization portion surface 24 is divided into four immobilization regions 25 by the ridge portions 26 that bulge toward the inner bottom surface 11 side of the opposing upper lid member 10.
  • the raised portion 26 provided on the immobilizing portion surface 24 abuts on the immobilizing portion facing surface 13 of the upper lid member 10, so that between the immobilizing portion surface 24 and the immobilizing portion facing surface 13.
  • a void is formed by the above, and the liquid supplied through the opening 14 is introduced into the void (immobilized region 25).
  • At least one apex 230 is included in the outer edge portion 28 of each immobilization region 25 delimited by the raised portion 26.
  • one apex 230 having an internal angle of 90° is provided on the outer edge portion 28 of each immobilization region 25, and the immobilization portion 23 having a quadrangular shape as the overall planar shape of the immobilization portion 23.
  • the example of is shown.
  • the vertices included in the outer edge portion 28 of the immobilization region 25 indicate inner angles of 30° to 162°, and the number of vertices is not particularly limited as long as it is one or more.
  • the four immobilization regions 25 included in the immobilization unit 23 By forming the four immobilization regions 25 included in the immobilization unit 23 in such a shape, a stable rotation state can be obtained when the solid-phase reaction chip 100 is rotated, and the solid state reaction chip 100 is opened through the opening 14.
  • the supplied liquid can be uniformly spread in the space between the immobilizing portion surface 24 and the immobilizing portion facing surface 13.
  • spots 250 each having a diameter of about 100 ⁇ m to 1 mm are formed in 4 ⁇ 4 rows (16 spots) per region.
  • the number of binding substances immobilized in the immobilization region 25 is not limited to the 64 types described above, and is, for example, 2 ⁇ 2 rows (16 types in total) to 6 ⁇ 6 rows (144 types in total). As described above, it can be appropriately changed according to the measurement item.
  • the method of immobilizing the binding substance is not based on the above-mentioned spots, but is provided at a position corresponding to each immobilization position, for example, U bottom (spherical shape), V bottom, flat bottom (round shape, square shape), etc.
  • a method of directly or indirectly immobilizing the binding substance in the recess (well) having the bottom of the above may be adopted.
  • the protrusion width b from the inner bottom surface 21 of the immobilization portion 23 is set so that the gap formed between the immobilization portion 23 and the confronting surface 13 has a predetermined gap width.
  • the height of the raised portion 26 may be taken into consideration and may be appropriately set according to the protrusion width a of the fixing portion facing surface 13 formed on the upper lid member 10.
  • FIG. 5 is a side sectional view of the solid-phase reaction chip 100 shown in FIG.
  • the void width c of the void formed between the immobilizing portion surface 24 and the immobilizing portion facing surface 13 is preferably between 0.05 mm and 0.30 mm, and among them, More preferably, the gap width c is between 0.08 mm and 0.15 mm.
  • the gap width c can be appropriately changed by adjusting the protrusion width b of the fixing portion 23, the protrusion width a of the fixing portion facing surface 13, and the height of the raised portion 26. Accordingly, the liquid amount of the liquid supplied to the void can be set to a desired liquid amount.
  • the upper lid member 10 and the lower lid member 20 having the above configurations are not particularly limited as long as they are impermeable to liquids and non-adsorbing to proteins and the like, and examples thereof include polyethylene, polycarbonate, and polyethylene terephthalate.
  • Plastic materials such as vinyl chloride, polystyrene, ABS resin, polyamide, tetrafluoroethylene, polypropylene, unsaturated polyester, epoxy, etc., for example, injection molding, insert molding, blow molding, extrusion molding, compression molding, or transfer It can be manufactured by a molding method such as molding.
  • the color and transparency of the material are not particularly limited, but the upper lid member 10 is preferably transparent and the lower lid member 20 is preferably non-transparent in order to suppress reflection during measurement.
  • the fitting and integration of the upper lid member 10 and the lower lid member 20 is not particularly limited, but the upper lid fitting wall 12 of the upper lid member 10 and the lower lid fitting wall 22 of the lower lid member 20 are ultrasonically coupled. Welding, vibration welding, hot plate welding, high frequency welding, hot air welding, laser welding, welding/welding such as welding, bonding of adhesives, adhesive tapes, etc., joining by machine fastening using tapping screws, bolts and nuts, etc.
  • the fitting and integration can be achieved with.
  • the liquid absorbing member 40 housed in the internal space 30 formed by fitting the upper lid member 10 and the lower lid member 20 has a thin plate-like member joined at both ends thereof. It can be formed as an annular member and is provided along the outer side (outer periphery 27) of the immobilizing portion 23.
  • the liquid absorbing member 40 is provided to absorb the liquid that is removed by the centrifugal force generated by the rotation. Examples of materials used for manufacturing the liquid absorbing member 40 include polyester fibers such as polyethylene terephthalate and polyethylene terebutyrate, polyolefin fibers such as polyethylene and polypropylene, polyurethane, and composite fibers obtained by combining these.
  • Fibers such as pulp fibers, cotton fibers, hemp fibers and other plant fibers, silk fibers, rayon fibers and other recycled fibers, woven fabrics, non-woven fabrics, papers, and other materials that are excellent in water absorption and water retention can be used. Further, it is possible to use a porous matrix composed of a cellulose-based material such as pulp fiber or rayon fiber, or a polysaccharide such as acetate (acetyl cellulose) produced by reacting wood pulp with acetic acid.
  • a cellulose-based material such as pulp fiber or rayon fiber
  • a polysaccharide such as acetate (acetyl cellulose) produced by reacting wood pulp with acetic acid.
  • a binding step of introducing a sample liquid into the solid-phase reaction chip 100 through the opening 14 to bond the measurement target substance and the binding substance, and the measurement target substance bound to the binding substance An addition step of adding a reaction solution containing a physiologically active substance having a specific binding ability and an enzymatic activity, and a removal step of removing the reaction solution by rotating the solid-phase reaction chip at a predetermined rotation speed, And a measuring step for measuring the enzyme activity of the physiologically active substance.
  • the substance to be measured is an IgE antibody that specifically binds to an allergen
  • the allergen as a binding substance that is immobilized as a binding substance in the immobilization region 25 of the immobilization unit surface 24 of the immobilization unit 23 is not particularly limited, and examples thereof include house dust 1(2) and mosquito leopard mite.
  • Inhalation system such as, Japanese cedar, Japanese cypress, alder (genus), birch (genus), duckweed, ragweed, mugwort, alternaria, aspergillus, malassezia (genus), cat (dandruff), dog (dandruff), cockroach, moth, latex ⁇ Other allergens, such as milk, egg white, opomcoid, rice, wheat (fruit), buckwheat, soybean, peanut, apple, kiwi, sesame, beef, chicken, shrimp, crab, mackerel, salmon, tuna, etc., Any allergen can be selected as long as it can be examined as an allergen test item at a medical institution or the like.
  • the immobilization of the allergen to the immobilization region 25 can be performed by generally used physical adsorption or chemical bonding.
  • an allergen solution containing the allergen of the measurement item is placed at a desired position in the immobilization region 25, or, for example, a U-bottom (spherical shape), V-bottom, flat bottom (round shape, square shape)-shaped depression (well).
  • the allergen is immobilized on the immobilization region 25 by spotting or stamping on ().
  • the allergen may be directly immobilized on the immobilization region 25, or the allergen may be immobilized via a spacer substance. Since a total of 64 spot positions are provided in the immobilization region 25 according to the present embodiment, it is possible to measure 64 measurement items at once by immobilizing different allergens on each spot. You can
  • the physiologically active substance having a specific binding ability to the IgE antibody as a measurement target substance and having an enzymatic activity is not particularly limited, and examples thereof include alkaline phosphatase, ⁇ -galactosidase, glucose oxidase, urease, It is preferable to use an anti-IgE antibody labeled with creatine kinase, uricase, glucose-6-phosphate dehydrogenase, peroxidase or the like.
  • HRP-labeled antibody obtained by labeling an anti-IgE antibody with horseradish peroxidase (HRP) will be described.
  • the generated signal can be obtained as a colorimetric signal or a chemiluminescent signal depending on the substrate.
  • the substrate used for generating the colorimetric signal for example, tetramethylbenzidine and its derivative, o-phenylenediamine, triarylmethanes, imidazole leuco dyes and the like can be used.
  • the chemiluminescent signal generation include acridinium salts, dioxetanes, luciferin, lucigenin, and oxalyl chloride.
  • the solid-phase reaction chip 100 is configured.
  • the sample liquid (30 ⁇ l to 60 ⁇ l) is supplied through the opening 14 of the upper lid member 10 (step S100).
  • the sample liquid can be appropriately selected from biological samples such as whole blood, serum, plasma, tear fluid, urine, saliva, and spinal fluid.
  • the sample liquid is spread over the entire immobilization region 25, and the IgE antibody having a specific binding ability binds to the allergen, which is the binding substance immobilized on each spot.
  • the solid-phase reaction chip 100 is preferably placed under a temperature environment in the range of 35°C to 40°C.
  • the IgE antibody can be efficiently bound to the allergen by performing a series of operations in a constant temperature bath capable of maintaining the temperature around 37°C.
  • step S101 after supplying a cleaning liquid (30 ⁇ l to 60 ⁇ l) containing a surfactant such as tween 20 through the opening 14, the cleaning liquid is removed by rotating at a predetermined rotation speed (2000 to 2500 rpm/min). Yes (step S101).
  • step S102 the reaction liquid (30 ⁇ l to 60 ⁇ l) containing the HRP-labeled antibody is supplied through the opening 14 (step S102).
  • a washing solution (30 ⁇ l to 60 ⁇ l) containing a surfactant such as tween 20 is supplied through the opening 14, and then a predetermined rotation speed (2000 to The cleaning liquid is removed by rotating at 2500 rpm/min) (step S103).
  • step S104 After the washing in step S103 is completed, a substrate such as tetramethylbenzidine (TMB) is added, and the spot that has emitted a colorimetric signal is measured using an imaging means such as CCD (step S104).
  • TMB tetramethylbenzidine
  • the automatic measuring device 200 includes a dispensing unit 201 capable of sucking/discharging a solution from at least a set reagent cartridge, and a centrifuge rotating at a predetermined number of revolutions per minute while holding the solid-phase reaction chip 100.
  • An operation unit 204 including a separation unit 202, an image pickup unit 203 including an image pickup unit such as a CCD, an input unit such as a touch panel, and a display unit such as an LCD (Liquid Crystal Display) that displays device information or measurement results.
  • LCD Liquid Crystal Display
  • An image analysis unit 205 that analyzes an image (colorimetric signal emission spot, etc.) captured by the image capturing unit 203; a measurement result generation unit 206 that generates a measurement result from the analysis result of the image analysis unit 205; And a control unit 207 for controlling the physical properties of the solid phase reaction chip 100.
  • the final measurement result is obtained in about 20 minutes, so that the throughput is high, and the operation can be intuitively performed according to the screen displayed via the operation unit 204. Therefore, there is an advantage that no skill is required. Further, since the device itself can be downsized, it can be applied as a device for a clinical site immediate examination.
  • the measurement target substance was an IgE antibody that specifically binds to the allergen, and the allergen was immobilized as a binding substance in the immobilization region 25.
  • the invention is not limited to this, and the substance to be measured is, for example, a genotype derived from human papillovirus (HPV), and is immobilized on the immobilization region 32c of the second base 32 as a binding substance. It is also possible to use a genomic DNA probe derived from HPV. In this case, there are 13 types of high risk types such as 16 type, 18 type, 31 type, 33 type, 35 type, 39 type, 45 type, 51 type, 52 type, 56 type, 58 type, 59 type and 68 type. It is also possible to construct it as a measurement system for detecting HPV to be classified.
  • the present invention for example, a preoperative infection test marker, a liver function test marker, a renal function test marker, an autoimmune hepatitis marker, a heart disease marker, a sepsis marker, a thyroid marker, or a specific drug contained in blood It is also possible to use, etc. as the substance to be measured.
  • Fig. 11 (1) preoperative infection test marker (1. Syphilis TP, 2. HIV Ag/Ab combo, 3. Anti-HCV, 4. HBsAg), (2) liver function test marker (1) Anti-HBc, 2. Anti-HBs, 3. Anti-HBe, 4. HBeAg, 5. Anti-HBc IgM, (3) Renal function test marker (1. BUN, 2. CRE, 3. UA), (4) autoimmune hepatitis markers (1. IgG, 2. antinuclear antibody), and (5) heart disease markers (1. troponin, 2. myoglobin, 3. biochemical myocardial markers such as CK-MB, 4) The color-developed state of the colorimetric spot obtained when each of the (heart-type fatty acid-binding proteins) was measured is shown.
  • preoperative infection test marker (1. Syphilis TP, 2. HIV Ag/Ab combo, 3. Anti-HCV, 4. HBsAg
  • liver function test marker (1) Anti-HBc, 2. Anti-HBs, 3. Anti-HBe, 4. HBeAg, 5. Anti
  • FIG. 11 and FIG. 12 (1) 2. HIV Ag/Ab combo and 4. 3. HBsAg and (7) typical examples of allergy (allergen) Egg white, 6. Milk, 7. Wheat, 8. Soybean, and 9. The result was false positive for some of the cypresses, but for all other items measured this time, a superior colorimetric signal was observed compared to the control. From these results, it was shown that the measurement system using the solid-phase reaction chip according to the present invention is a measurement system suitable for so-called screening tests, regardless of the application item to be measured.
  • test markers such as AST (GOT), ALT (GPT) and ⁇ -GTP, which are used as liver function test markers.
  • AST GAT
  • ALT GTP
  • ⁇ -GTP liver function test markers.
  • infectious diseases designated infectious diseases, new infectious diseases, and other infectious disease markers, or inflammatory markers such as leukocytes, C-reactive protein, and serum amyloid A protein, as well as other disease test markers.
  • the present invention includes, for example, insecticides, acaricides, nematicides, fungicides, herbicides, insecticides, insecticides, plant growth regulators, repellents.
  • Pesticides such as drugs, attractants, spreaders, stimulants, narcotic analgesics, anabolic agents, diuretics, peptides and glycoprotein hormones and their analogs, blood doping, alcohol, marijuana, local anesthetics, cortico It is also possible to develop testing and measurement of doping drugs such as steroids (corticosteroids) and beta blockers.
  • the measurement system using an enzyme label is an excellent measurement system from the viewpoint of reaction specificity and signal amplification.
  • the enzyme has a large molecular weight, there are problems that it affects the antigen-antibody reaction, for example, and if the enzyme deactivates, it will not function as a measurement system. The reproducibility of was sometimes decreased.
  • the solid-phase reaction chip according to the present invention can also construct a measurement system using a labeling substance labeled with a signal generating substance other than an enzyme, it will be described below.
  • the measurement method using a labeled substance labeled with a signal-generating substance other than an enzyme is that the sample liquid is introduced into the solid-phase reaction chip through the opening and immobilized on the measurement target substance and the immobilization unit. And a binding substance that has a specific binding ability for the substance to be measured and is labeled with a signal generating substance in the first binding step. It is characterized by comprising a second binding step of binding to the substance to be measured and a measuring step of measuring a signal generated by the signal generating substance.
  • a cleaning step may be provided between the steps or between any of the steps to remove the cleaning solution by adding the cleaning solution and rotating the solid-phase reaction chip at a predetermined rotation speed.
  • the labeling substance having a specific binding ability to the measurement target substance and labeled with the signal generating substance any of a radioisotope, a lanthanoid element, a free radical derivative, a chemiluminescent substance or a fluorescent emitting substance is used.
  • An antibody, a protein such as protein A/G, lectin, avidin, streptavidin, a nucleic acid, or a fragment thereof can be used.
  • the binding of the signal generating substance to the labeling substance is not particularly limited, and examples thereof include covalent bond, ionic bond, hydrogen bond, coordinate bond, physical adsorption, chemical adsorption and the like.
  • a linker composed of an organic molecule such as a polyethylene glycol chain may be provided.
  • the labeled antibody is an anti-antibody labeled with either a radioisotope, a lanthanoid element, a free radical derivative, a chemiluminescent substance or a fluorescent luminescent substance. It is preferred to use IgE antibodies.
  • a radioisotope is used as the signal generating substance
  • 125 I, 131 I, 3 H and the like can be mentioned, and 125 I is preferably used from the viewpoint of sensitivity and half-life.
  • iodine When iodine is oxidized, it easily binds to a tyrosine residue, a histidine residue, etc. in an antibody (protein) as a new electron reagent, and is therefore suitable for labeling.
  • elements with atomic numbers 57 to 71 (La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu) are used.
  • the chelate of Eu (europium) with the atomic number 63 has a large wavelength difference between the excitation light (340 nm) and the emitted light (615 nm) and has a long emitted light (fluorescence) lifetime, and thus time-resolved fluorescence measurement is performed. be able to.
  • a free radical derivative When a free radical derivative is used as the signal-generating substance, examples thereof include piperidine-N-oxide derivative, pyrrolidine-N-oxide derivative, oxazolidine-N-oxide derivative, and the like. Labeling with these free radical derivatives The change in the ESR spectrum of the antibody is used as an index.
  • a chemiluminescent substance for example, a luminol derivative, acridinium salt, dioxetane, luciferin, lucigenin, oxalyl chloride, indoxyl derivative and the like can be mentioned, and a labeled antibody labeled with these chemiluminescent substance Is subjected to a chemiluminescence reaction in the presence of an enzyme such as microperoxidase or peroxidase, H 2 O 2 or in the presence of an alkaline condition or H 2 O 2 , and this is measured.
  • an enzyme such as microperoxidase or peroxidase, H 2 O 2 or in the presence of an alkaline condition or H 2 O 2 , and this is measured.
  • a fluorescent light emitting substance When a fluorescent light emitting substance is used as the signal generating substance, it can be any of a fluorescent dye, a quantum dot, or a fluorescent particle (bead) composed of a fluorescent dye or a quantum dot and a particle.
  • the fluorescent luminescent material referred to here is one of visible light to near infrared light having a wavelength in the range of 400 nm to 1100 nm when excited by ultraviolet light to near infrared light having a wavelength in the range of 200 nm to 700 nm. It shows radiated light (light emission).
  • fluorescent dyes include fluorescein dye molecules, rhodamine dye molecules, ALexa Fluor (Invitrogen) dye molecules, BODIPY (Invitrogen) dye molecules, cascade dye molecules, coumarin dye molecules, and eosin dyes.
  • fluorescent dyes include dye molecules, NBD-based dye molecules, pyrene-based dye molecules, cyanine-based dye molecules, aromatic hydrocarbon-based molecules and the like.
  • a quantum dot containing a II-VI group compound, a III-V group compound, a IV group element or the like can be used, and these compounds may be used alone or in combination of plural kinds. You may combine.
  • CdSe, CdS, CdTe, ZnSe, ZnS, ZnTe, InP, InN, InAs, InGaP, GaAs, Si, Ge and the like can be mentioned.
  • core/shell type quantum dots shelled with other nanomaterials are prepared, and the surface of the quantum dots is made of an organic substance with high biocompatibility such as polyethylene glycol or amphipathic polymer.
  • a coated material can also be used in the present invention.
  • fluorescent particles composed of fluorescent dyes or quantum dots and particles include inorganic particles such as silica and alumina, or polystyrene and resin particles (resin polymer) made of an organic polymer such as poly(meth)acrylic acid ester.
  • the fluorescent dye or the quantum dot may be bonded, partially bonded, or encapsulated inside the particles.
  • the method of introducing the fluorescent dye or the like into the resin particles is not particularly limited, a method of polymerizing the monomer after binding the fluorescent dye to the monomer that is a raw material of the resin, a fluorescent dye in the polymer after forming the polymer.
  • the average particle diameter of the fluorescent particles is not particularly limited as long as it has a size that does not affect the immune reaction and provides sufficient brightness, but it is preferably in the range of 1 nm to 100 ⁇ m.
  • An example of the fluorescent particles is a combination of a fluorescent substance and particles that emit visible light to near infrared light (emission) when excited by ultraviolet light to near infrared light. There is no particular limitation so long as it is, for example, a combination of the above-mentioned lanthanoid element and resin particles.
  • the fluorescent particles that can be used in the present invention can be prepared by a known method, or commercially available fluorescent particles may be used.
  • Patent Document 3 describes a method for synthesizing fluorescent dye labeling resin particles in which a fluorescent dye is bound to a resin particle made of a resin by a bond other than a covalent bond, and various fluorescent dyes can be used by using a desired fluorescent dye. Fluorescent particles can be created.
  • Patent Document 4 discloses 1,1-dimethyl-2,3,4,5-tetraphenylsilole-encapsulated polystyrene microparticles, 1,1,2,3,4,5-hexaphenylsilole-encapsulated polystyrene microparticles, 1,1- A particle composed of a network polymer such as dimethyl-2,5-dianisyl-3,4-diphenylsilole-encapsulated polystyrene microparticles and 1,1,2,3,4,5-hexaphenylsilole-encapsulated polystyrene microparticles, which has an aggregation-induced luminescence activity Fluorescent resin fine particles containing a compound (silole) are described.
  • the fluorescent resin fine particles according to the present invention are fluorescent resin fine particles formed by encapsulating these compounds in a reticulated polymer. Therefore, it is expected that the measurement can be performed with higher efficiency than in the case of using the conventional ⁇ -conjugated fluorescent substance.
  • a labeling substance labeled with a signal generating substance such as a radioisotope, a lanthanoid element, a free radical derivative, a chemiluminescent substance or a fluorescent emitting substance
  • a highly efficient measurement system having a higher efficiency can be constructed.
  • a radioisotope, a lanthanoid element, a free radical derivative, or a fluorescent substance is used as a signal-generating substance, unlike the system for measuring enzyme activity, the time and effort of adding a substrate (solution) at the measurement step can be omitted. Therefore, the measurement can be performed more quickly and with high reproducibility.
  • the outer edge portion 28 of the four fixing regions 25 included in the fixing portion 23 has one apex having an inner angle of 90°, and the shape of the entire fixing portion 23 in plan view is The example of the rectangular shape has been described. However, the present invention is not limited to this shape, and other modifications such as another polygonal shape such as a triangular shape or an octagonal shape as a planar view shape of the entire fixing portion are possible.
  • FIG. 8A is an example of the upper lid member 101 in which the opening 141 and the fixing portion facing surface 131 are formed, and FIG. 8B is engageable with the upper lid member 101 and has three fixing regions.
  • FIG. 9A is an example of the upper lid member 102 in which the opening 142 and the fixing portion facing surface 132 are formed, and FIG. 9B is engageable with the upper lid member 102 and four fixing members are provided.
  • FIGS. 4, 8 and 9 are merely examples, and in the present invention, the angles within the vertices, the number of vertices, the number of sides forming the vertices, the ratio of the side lengths, straight lines, or curves.
  • the line type and the like of the sides can be appropriately selected, and the number of immobilization regions formed on the surface of the immobilization portion based on these selections is not limited.
  • the shape in which the upper lid member and the lower lid member are fitted and integrated together can be appropriately changed.
  • a solid-phase reaction chip 50 in which an upper lid member 103 and a lower lid member 303 having a quadrangular shape in plan view are fitted and integrated or as shown in FIG. 10( b ),
  • a combination such as a solid-phase reaction chip 60 in which an upper lid member 104 and a lower lid member 304, which are hexagonal in plan view, are integrally fitted is also possible.
  • the combination of the immobilizing portion and the lid member can be appropriately selected in view of various conditions such as high measurement accuracy, reproducibility, ease of manufacturing, and manufacturing cost.
  • the cover member that has been conventionally required is eliminated, and at least one apex is formed at the outer edge portion of the immobilization region for immobilizing the binding substance, so that the inner portion is sucked.
  • a solid-phase reaction chip capable of overcoming the drawbacks of a solid-phase reaction chip equipped with a liquid member and accurately detecting a substance to be measured contained in a sample liquid by a quick and simple operation, and a measurement using the same A method can be provided.

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Abstract

Le problème décrit par la présente invention est de fournir une puce de réaction en phase solide qui surmonte les inconvénients des puces de réaction en phase solide qui sont équipées à l'intérieur d'un élément d'absorption de liquide et qui est capable de détecter, avec une excellente précision, une substance cible contenue dans un échantillon liquide au moyen d'une opération rapide et simple ; et un procédé de mesure qui utilise la puce de réaction en phase solide. La solution selon l'invention porte sur une puce de réaction en phase solide formée en tant que corps rotatif par un élément de couvercle supérieur et un élément de couvercle inférieur qui s'adaptent l'un à l'autre de façon à être d'un seul tenant, la puce de réaction en phase solide comprenant une partie d'immobilisation qui est disposée sur le côté de surface inférieure intérieure soit de l'élément de couvercle supérieur soit de l'élément de couvercle inférieur et immobilise une pluralité de substances de liaison comprenant des capacités de liaison spécifiques à une substance cible de mesure comprise dans un liquide d'échantillon, une ouverture qui est disposée sur l'élément de couvercle supérieur et qui fournit le liquide d'échantillon à la surface de partie d'immobilisation et une partie d'absorption de liquide qui est disposée à l'extérieur de la partie d'immobilisation et absorbe le liquide d'échantillon fourni par l'ouverture, la partie d'immobilisation ayant une pluralité de régions d'immobilisation séparées par des saillies qui font saillie de façon à se faire face les une aux autres sur le côté de surface inférieure intérieure et la partie de bord externe des régions d'immobilisation comprenant au moins un sommet. L'invention concerne en outre un procédé de mesure utilisant la puce de réaction en phase solide.
PCT/JP2020/006695 2019-02-20 2020-02-20 Puce de réaction en phase solide et procédé de mesure l'utilisant WO2020171158A1 (fr)

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