WO2018199168A1 - Membrane carrier, method for producing same, and liquid sample test kit - Google Patents

Membrane carrier, method for producing same, and liquid sample test kit Download PDF

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Publication number
WO2018199168A1
WO2018199168A1 PCT/JP2018/016815 JP2018016815W WO2018199168A1 WO 2018199168 A1 WO2018199168 A1 WO 2018199168A1 JP 2018016815 W JP2018016815 W JP 2018016815W WO 2018199168 A1 WO2018199168 A1 WO 2018199168A1
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Prior art keywords
substance
charge
detection
flow path
membrane carrier
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PCT/JP2018/016815
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French (fr)
Japanese (ja)
Inventor
佳那恵 原
門田　健次
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デンカ株式会社
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Application filed by デンカ株式会社 filed Critical デンカ株式会社
Priority to JP2019514578A priority Critical patent/JP7025413B2/en
Publication of WO2018199168A1 publication Critical patent/WO2018199168A1/en

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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

Definitions

  • the present invention relates to a membrane carrier, a manufacturing method thereof, and a liquid sample inspection kit.
  • POCT Point of Care Test
  • reagents have been attracting attention, which measure antigen morbidity, pregnancy, blood glucose levels, etc. by using antigen-antibody reactions and the like.
  • POCT is a test performed beside the subject in the POCT guidelines emitted by the Japan Society for Clinical Laboratory Automation, or a test performed by the subject himself, shortening the test time and testing on the spot (test visible to the subject) Is defined as an inspection that has the advantage of being possible.
  • POCT reagents are characterized by rapid testing, simple usage, and inexpensive reagents. Because of these characteristics, it is often used for medical examinations and regular medical examinations at a mild stage, and it is an important diagnostic tool in home medical care that is expected to increase in the future.
  • determination is performed by introducing a liquid sample such as blood into a test kit and detecting a specific substance to be detected contained therein.
  • An immunochromatography method is often used as a method for detecting a specific substance to be detected from a liquid sample.
  • the immunochromatography method is that the liquid dropped on the membrane carrier of the test kit moves on the membrane carrier, and the substance to be detected and the subject to be detected in the liquid sample suspended or dissolved in the liquid sample.
  • Labeled particles bound with antibodies or antigen-binding fragments thereof that specifically react with the substance bind to each other, and these bind specifically with the substance immobilized in the test kit (hereinafter referred to as detection substance). This is a method of detecting a change in color or mass that occurs as a result.
  • the visual sensitivity in the immunochromatography method may decrease.
  • the detection substance for example, solid phase protein
  • An antibody is composed of a base Fc site and a Fab site that reacts with an antigen.
  • the outermost surface of the flow path of the POCT reagent is a random surface composed of a Fab site and an Fc site.
  • the outermost surface of the POCT reagent channel is the Fab site, and the adhesive surface between the channel and the antibody is the Fc site. Therefore, in the POCT reagent in which the orientation of the antibody is controlled, the probability of reaction between the Fab site and the antigen on the outermost surface of the flow path is higher than that in the current non-controlled POCT reagent, so that the visual sensitivity in the immunochromatography method is increased. I think that. That is, it is expected that the visual sensitivity of the POCT reagent is increased by controlling the orientation of the detection substance.
  • Patent Document 1 when immobilizing a micro object such as a protein by light irradiation on a solid phase containing a photoresponsive component, the micro object can be fixed with a fixed orientation with respect to the solid phase. By providing an operable component in the solid phase, the orientation of the micro object is controlled and immobilized on the solid phase.
  • a POCT reagent is produced using the light immobilization method of Patent Document 1, the light stability of the POCT reagent is lowered, and thus practical application is difficult.
  • Patent Document 2 and Patent Document 3 show that protein crystal growth is promoted by placing a concentrated protein aqueous solution in an electric field. However, Patent Document 2 and Patent Document 3 do not describe a liquid sample inspection kit.
  • Patent Document 4 succeeds in adhering microparticles to the surface of ceramic particles by utilizing the charge of a cationic polymer and an anionic polymer as an electrical effect.
  • a specific method is to apply an aqueous solution of a cationic polymer and an anionic polymer to the surface of the ceramic particles, thereby imparting a charge to the surface of the ceramic particles and generating electrostatic charges between the charged fine particles. It is strongly bonded by mechanical interaction.
  • Patent Document 4 does not describe a liquid sample inspection kit.
  • Patent Document 5 is a membrane carrier for a test kit that detects a substance to be detected in a liquid sample, and is provided with at least one flow path capable of transporting the liquid sample, and the liquid sample is provided on the bottom surface of the flow path.
  • a membrane carrier for a liquid sample test kit is described, which is provided with a microstructure that causes a capillary action for transporting the sample.
  • Patent Document 5 does not describe the orientation control of the detection substance.
  • an object of the present invention is to provide a membrane carrier capable of highly sensitive determination.
  • the present invention is as follows.
  • a flow path, a charge imparting substance, and a detection substance are provided, the detection substance is held in the flow path via the charge imparting substance, and the mass ratio of the charge imparting substance to the detection substance (charge imparting substance / detection substance) is A membrane carrier that is 0.01-100.
  • the charge-imparting substance is at least one selected from the group consisting of a cationic polymer and an anionic polymer.
  • the liquid sample inspection kit (hereinafter also simply referred to as “inspection kit”) detects a substance to be detected in the liquid sample.
  • FIG. 1 is a schematic top view of an inspection kit.
  • the test kit 10 includes a membrane carrier 4 and a housing 10 a that houses the membrane carrier 4.
  • the membrane carrier 4 has, on its surface, a dropping zone 4x where a liquid sample is dropped and a detection zone 4y for detecting a substance to be detected in the liquid sample.
  • the dripping zone 4x is exposed at the first opening 10b of the housing 10a.
  • the detection zone 4y is exposed at the second opening 10c of the housing 10a.
  • the dripping zone 4x and the detection zone 4y are located on the upstream side and the downstream side in the transport direction of the liquid sample, respectively.
  • the membrane carrier is a membrane carrier for a liquid sample inspection kit that detects a substance to be detected in the liquid sample.
  • the substance to be detected is not limited at all, and may be any substance capable of antigen-antibody reaction with an antibody such as various pathogens and various clinical markers.
  • viral antigens such as influenza virus, norovirus, adenovirus, RS virus, HAV, HBs, HIV, bacterial antigens such as MRSA, group A streptococci, group B streptococci, legionella, toxins produced by bacteria, etc.
  • hormones such as Mycoplasma, Chlamydia trachomatis, human chorionic gonadotropin, C-reactive protein, myoglobin, cardiac troponin, various tumor markers, agricultural chemicals, and environmental hormones.
  • the substance to be detected may be an antigen capable of inducing an immune reaction alone, or a hapten capable of binding an antibody by an antigen-antibody reaction although it cannot induce an immune reaction alone.
  • the substance to be detected may be in a suspended or dissolved state in the liquid sample.
  • the liquid sample may be, for example, a sample in which the substance to be detected is suspended or dissolved in a buffer solution.
  • FIG. 2 is a schematic top view of the membrane carrier.
  • the membrane carrier 4 includes a flow path 2, a charge imparting substance 1, and a detection substance 3.
  • the detection substance 3 is held in the flow path 2 via the charge imparting substance 1.
  • a region where the detection substance 3 exists constitutes a detection zone.
  • the liquid sample is transported from the dropping zone to the detection zone via the flow path 2.
  • FIG. 3 is a schematic cross-sectional view (cross-sectional view taken along the line II-II in FIG. 2) of the membrane carrier of one embodiment.
  • a membrane carrier 4A shown in FIG. 3 is composed of a flow path 2, a charge-providing substance 1 present on at least a part of the main surface of the flow path 2, and a detection substance 3 present on the charge-provided substance 1. ing.
  • the membrane carrier 4A is obtained, for example, by applying the charge-providing substance 1 to at least a part of the surface of the flow path 2 and further applying the detection substance 3 on the charge-providing substance 1.
  • FIG. 4 is a schematic cross-sectional view of a membrane carrier according to another embodiment.
  • a membrane carrier 4B shown in FIG. 4 includes a flow path 2, a charge-providing substance 1 present in at least a part of the flow path 2, and a detection substance 3 present on the charge-providing substance 1 and on the surface of the flow path 2. It consists of and.
  • the membrane carrier 4B is formed on the surface of the flow path 2 after applying the charge imparting substance 1 to at least a part of the surface of the flow path 2 and infiltrating the charge imparting substance 1 into the flow path 2. It is obtained by further applying the detection substance 3 to the area where the charge imparting substance 1 is applied.
  • FIG. 5 is a schematic cross-sectional view of a membrane carrier of another embodiment.
  • a membrane carrier 4C shown in FIG. 5 includes a flow path 2, a charge-providing substance 1 present in at least a part of the flow path 2, and a detection substance present on the charge-providing substance 1 and in and on the surface of the flow path 2. 3. That is, the detection substance 3 exists inside the flow path 2 and is also exposed on the surface of the flow path 2.
  • the membrane carrier 4C is formed on the surface of the flow channel 2 after applying the charge imparting material 1 to at least a part of the surface of the flow channel 2 and infiltrating the charge imparting material 1 into the flow channel 2. It is obtained by further applying the detection substance 3 to the area where the charge imparting substance 1 is applied and infiltrating the detection substance 3 into the flow path 2.
  • the channel 2 functions as a channel for transporting the liquid sample in the membrane carrier 4.
  • the surface of the flow path 2 may be flat or curved.
  • a fine structure (uneven structure) may be formed on the surface of the flow path 2.
  • the fine structure is provided on the surface of the flow path 2
  • the liquid sample is transported through the fine structure by the capillary action of the fine structure.
  • the flow path 2 may be comprised with the porous base material.
  • the liquid sample is composed of a porous substrate, the liquid sample is transported by moving through the pores of the porous substrate by capillary action.
  • the overall shape of the flow path is not particularly limited, but may be, for example, a polygon such as a quadrangle, a circle, or an ellipse.
  • the vertical width (length in the short direction) of the flow path may be, for example, 2 mm or more and 100 mm or less
  • the horizontal width (length in the longitudinal direction) of the flow path is, for example, 2 mm. It may be 100 mm or less.
  • the thickness of the flow path 2 is preferably 100 ⁇ m to 5 mm, more preferably 500 ⁇ m to 2 mm. When the uneven structure is provided on the flow path 2, the thickness of the flow path 2 does not include the height of the convex portion.
  • the material forming the flow path 2 is preferably a resin, and more preferably a thermoplastic resin.
  • the thermoplastic resin include nitrocellulose, polyester resin, polyolefin resin, polystyrene resin, polycarbonate resin, fluorine resin, and acrylic resin.
  • PET polyethylene terephthalate
  • COP cycloolefin polymer
  • PP polypropylene
  • PS polystyrene
  • MS methyl methacrylate-styrene copolymer
  • MBS methyl methacrylate-butadiene-styrene copolymer
  • MABS Methyl methacrylate-acrylonitrile-butadiene-styrene
  • SBC polycarbonate
  • PC polyvinylidene fluoride
  • PVDF polymethyl methacrylate
  • the material of the channel 2 is preferably nitrocellulose.
  • a nitrocellulose membrane can be used as the flow path 2.
  • the material of the flow path 2 is preferably a thermoplastic resin.
  • the fine structure has a concavo-convex structure and has a plurality of convex portions.
  • the flow path 2 has a concavo-convex structure (fine structure) on at least a part of the surface from the viewpoint of facilitating highly sensitive inspection. That is, it is preferable that the membrane carrier 4 includes the flow path 2 provided with an uneven structure on at least a part of the surface.
  • the concavo-convex structure may be located at least between the dropping zone and the detection zone. An uneven structure may be provided over the entire surface of the flow path 2.
  • the fine structure can be formed by imprinting.
  • thermal imprinting is a method for producing a substrate having a fine structure by pressing a mold (mold) having a fine structure against the substrate and transferring the fine structure to a substrate softened by heating. This thermal imprint enables nano-order microfabrication.
  • the mold (mold) having a fine structure has an uneven structure.
  • a structure in which the recesses 5 are regularly arranged in a triangular arrangement on the surface of the mold 7 is preferable.
  • a structure in which the convex portions 6 are regularly arranged in each direction as protrusions on the surface of the flow path 2 is preferable.
  • the structure in which the convex portions 6 are regularly arranged as protrusions is preferably a structure in which the protrusions 6 are arranged in a planar lattice pattern.
  • the planar lattice is preferably one or more of an orthorhombic lattice, a hexagonal lattice, a tetragonal lattice, a rectangular lattice, and a parallel lattice, and more preferably a hexagonal lattice. Of the hexagonal lattice, an equilateral triangular lattice is preferable.
  • FIG. 8 shows a structure in which the convex portions 6 are arranged in an equilateral triangular lattice shape.
  • the convex part 6 is preferably made of the same material as the flow path 2.
  • the cross section of the convex portion 6 is preferably a triangle, and more preferably a regular triangle.
  • a cone shape or a pyramid shape is preferable, and a cone shape is more preferable.
  • the pitch 11 of the convex portions 6 is preferably 1 to 300 ⁇ m, more preferably 5 to 100 ⁇ m, and most preferably 10 to 50 ⁇ m.
  • the pitch 11 of the convex portions 6 is 1 ⁇ m or more, the light reflectivity is more excellent.
  • the pitch 11 of the convex portions 6 is 300 ⁇ m or less, the transparency is more excellent.
  • the pitch 11 of the convex portions 6 refers to the dimension between the convex portions 6 and 6 adjacent to each other (the distance between the centers of the adjacent convex portions 6).
  • the pitch 11 means the dimension between the vertex of a cone and the vertex of a cone as shown in FIG.
  • the height of the convex portion 6 is preferably 1 to 1000 ⁇ m, and more preferably 10 to 50 ⁇ m. If it is 1 ⁇ m or more, the flow path volume becomes large, and it does not take a long time to develop the liquid sample. When the thickness is 1000 ⁇ m or less, it does not take much time and cost to produce the fine structure, and the production of the fine structure becomes easy.
  • the height of the convex portion 6 is defined as the maximum length of the convex portion 6 in the direction orthogonal to the bottom surface of the convex portion 6. That is, the height of the convex portion 6 is a dimension from the bottom surface of the convex portion 6 to the apex of the convex portion 6.
  • the diameter of the bottom surface of the convex portion 6 is preferably 1 to 1000 ⁇ m, and more preferably 10 to 50 ⁇ m.
  • the diameter of the bottom surface of the convex portion 6 is 1 ⁇ m or more, the microfabrication cost of the mold is reduced, and it is easy to uniformly produce an infinite number of fine structures on the surface of the flow path 2 of the membrane carrier 4 having a large area. Capillary force necessary to move is increased.
  • the diameter of the bottom surface of the convex portion 6 is 1000 ⁇ m or less, the volume of the metal scraped from the metal member during the production of the mold is reduced, the production cost of the mold and the membrane carrier 4 is reduced, and the area of the flow path in the membrane carrier 4 , The membrane carrier 4 is downsized, and the membrane carrier 4 itself can be easily transported.
  • the diameter of the bottom surface of the convex portion 6 is defined as the representative length at the bottom surface of the convex portion 6.
  • the representative length at the bottom is the diameter when the shape of the bottom is a circle, the length of the shortest side when it is a triangle or a quadrangle, the length of the longest diagonal line when it is a pentagon or more polygon, and other shapes In this case, the maximum length at the bottom is used.
  • the convex part 6 is a cone (refer FIG. 8) or a cylinder
  • the diameter of the bottom face of the convex part 6 is a diameter of the bottom face (circle) of the convex part 6.
  • a polymer is preferable.
  • the polymer is preferably at least one selected from the group consisting of cationic polymers and anionic polymers.
  • the cationic polymer is a polymer compound having a positive charge in an aqueous solution
  • the anionic polymer is a polymer compound having a negative charge in an aqueous solution.
  • the weight average molecular weight of the polymer is preferably 10,000 to 500,000, more preferably 50,000 to 200,000.
  • the weight average molecular weight is a value in terms of standard polyethylene oxide measured by a gel permeation chromatography (GPC) method.
  • GPC gel permeation chromatography
  • the weight average molecular weight can be obtained by preparing a calibration curve with commercially available standard polyethylene oxide using GPC system (SC-8010 manufactured by Tosoh Corporation) using water as a solvent.
  • cationic polymer for example, poly (diallyldimethylammonium chloride) (PDDA) can be preferably used.
  • PDDA poly (diallyldimethylammonium chloride)
  • Other cationic polymers that can be used in the present embodiment include polyethyleneimine (PEI), polyvinylamine (PVAm), poly (vinylpyrrolidone / N, N-dimethylaminoethylacrylic acid) copolymer, and the like. It is done. However, it is an example as a cationic polymer, and is not limited thereto.
  • anionic polymer for example, polystyrene sulfonic acid (PSS) can be preferably used.
  • Other anionic polymers that can be used in the present embodiment include polyvinyl sulfate (PVS), polyacrylic acid (PAA), polymethacrylic acid (PMA), and the like.
  • PVS polyvinyl sulfate
  • PAA polyacrylic acid
  • PMA polymethacrylic acid
  • this is an example of an anionic polymer and is not limited thereto.
  • the charge imparting substance may exist in at least a part of the detection zone, may exist throughout the detection zone, or may exist outside the detection zone.
  • the detection substance may be, for example, a solid phase protein.
  • the solid phase protein include antibodies.
  • the antibody is an antibody that reacts with a substance to be detected by antigen-antibody reaction, and may be a polyclonal antibody or a monoclonal antibody.
  • the detection substance only needs to be present on the charge imparting substance, may be present in a part of the region where the charge imparting substance is present, or may be present throughout.
  • the mass ratio of the charge imparting substance to the detection substance is preferably 0.01 to 100, more preferably 0.1 to 10.
  • the charge-providing substance / detecting substance may be 0.01 or more, 0.1 or more, 0.3 or more, 0.5 or 0.8 or more, and is 100 or less, 10 or less, 8 or less, 5 or less, or 3 or less. It may be.
  • the detection substance 3b can be strongly fixed.
  • the charge imparting substance / detection substance is 100 or less, non-specific interaction with the substance to be detected does not occur.
  • the mass ratio of the charge imparting substance to the detection substance is defined as the mass ratio in the region where both the charge imparting substance and the detection substance exist.
  • the mechanism of the orientation control of the detection substance will be described with reference to FIG. 6, taking a solid phase protein as an example.
  • the solid phase protein constituting the detection substance 3 has a solid phase protein base portion 3a and a solid phase protein end portion 3b.
  • the charge-imparting substance 1 exhibits an electrostatic interaction with the surface charge localized in the solid phase protein base 3a.
  • the solid phase protein base 3a faces the direction of the charge imparting substance 1 (channel 2) and the solid phase protein end 3b is located on the outermost surface, the orientation of the detection substance (solid phase protein) 3 can be controlled. It becomes.
  • the detection substance may be oriented by an electric field generated by electrostatic interaction between the detection substance and the charge imparting substance. In one embodiment, it can also be said that the detection substance is oriented by the potential of the surface of the substrate (flow path) holding the detection substance.
  • the detection zone on the flow path shows a color change when a substance to be detected is detected.
  • the color change in the detection zone occurs when the detection target substance is held in the detection zone by the detection substance (reacts with the detection substance).
  • the color change may be a color change that can be confirmed by an optical method.
  • the optical method there are mainly two methods: a visual determination and a method of measuring fluorescence intensity.
  • a visual determination the color difference between two color stimuli (described in JIS Z8781-4: 2013) when the color before detection and after detection is measured in the color system of CIE1976L * a * b * color space.
  • ⁇ E a color change occurs such that ⁇ E) is 0.5 or more.
  • this color difference is 0.5 or more, it becomes easy to visually confirm the color difference.
  • the ratio (Fl1 / Fl2) 10 of the fluorescence intensity (Fl1) in the detection zone and the fluorescence intensity (Fl2) in the upstream region and downstream region adjacent to the detection zone. It is preferable that a color change that is 1 or more occurs. When this ratio is 10/1 or more, separation of signal and noise becomes easy.
  • the film carrier has a step of applying a charge-imparting substance on at least a part of the surface of the flow path (first application step), and a step of applying a detection substance on the applied charge-imparting material (second application step). It can manufacture by the method provided with these.
  • the charge imparting substance is applied to at least a part of the surface of the flow path.
  • the charge imparting substance may be applied to at least a part of the detection zone, the charge imparting substance may be applied over the entire detection zone, or may be applied outside the detection zone.
  • the charge imparting substance may be implemented by applying an aqueous solution containing the charge imparting substance on the charge imparting substance.
  • a step (first drying step) of volatilizing the solvent in the aqueous solution containing the charge imparting substance is provided between the first coating step and the second coating step.
  • the first drying step can be performed using a vacuum dryer or the like.
  • the concentration of the charge-providing substance in the aqueous solution containing the charge-providing substance is preferably 0.01 to 20% by mass, and more preferably 0.1 to 5% by mass.
  • the detection substance is applied on the applied charge imparting substance.
  • the detection substance may be applied to a part of the charge-providing substance, or may be applied over the entire region where the charge-providing substance exists, or may be applied so as to protrude from the area where the charge-providing substance exists.
  • the detection substance can be held on the charge imparting substance by applying a solution containing the detection substance (detection substance solution) on the charge imparting substance.
  • a step of volatilizing the solvent in the detection substance solution is provided after the second application step.
  • the detection substance solution is preferably an aqueous solution in which the detection substance is dissolved in purified water.
  • other compounds such as sodium chloride and trishydroxymethylaminomethane may be added to the detection substance solution.
  • the concentration of the detection substance in the detection substance solution is preferably 0.00001 to 1 w / v%, more preferably 0.0001 to 0.1 w / v%.
  • the appropriate charge imparting substance 1 can vary depending on the pH of the detection substance solution.
  • the charge imparting substance 1 to be used is preferably a cationic polymer in terms of enhancing electrostatic interaction.
  • the charge imparting substance 1 to be used is preferably an anionic polymer in terms of enhancing electrostatic interaction.
  • the pH of the detection substance solution is 7.0, either a cationic polymer or an anionic polymer can be used as the charge imparting substance 1.
  • a step of forming an uneven structure in the flow path by imprinting may be further provided.
  • the ratio of the application amount of the charge-imparting substance to the application amount of the detection substance is preferably 0.01 to 100, more preferably 0.1 to 10.
  • the charge-providing substance / detecting substance may be 0.01 or more, 0.1 or more, 0.3 or more, 0.5 or 0.8 or more, and is 100 or less, 10 or less, 8 or less, 5 or less, or 3 or less. It may be.
  • the charge application amount / detection material application amount is a value calculated based on the mass (solid content) of each substance.
  • the membrane carrier 4 of the present embodiment has a limit magnification that can be visually determined compared to a membrane carrier in which no charge-providing substance is present and a membrane carrier in which the mass ratio of the charge-providing substance to the detection substance is outside the range of 0.01 to 100. Since it is large, it can be evaluated as a membrane carrier capable of highly sensitive determination.
  • a charge imparting substance such as a cationic polymer or an anionic polymer is applied to the surface of the base material (flow path 2), so that the base material surface is charged and charged. Due to the electrostatic interaction that occurs between the detection substance (for example, solid phase protein), the charge-providing substance strongly adheres the detection substance.
  • the orientation of the detection substance can be controlled by using the localized charge existing in the detection substance. In the present embodiment, the orientation of the detection substance can be controlled using the electric field generated by the charge imparting substance.
  • PDDA diallyldimethylammonium chloride
  • Anti-influenza A virus NP antibody suspension in which anti-influenza A virus NP antibody is suspended in water as a solid phase protein in the area where PDDA is applied on the nitrocellulose membrane (the concentration of anti-influenza A virus NP antibody is 0 .0033 w / v%) was applied so as to have the same area as that of PDDA, and dried well under warm air.
  • the solid phase protein coating amount is a mass (solid content) obtained by subtracting the mass of water from the mass of the anti-influenza A virus NP antibody suspension.
  • Example 1 the mass ratio of the charge-imparting substance to the solid phase protein (charge-giving substance coating amount / solid-phase protein coating amount) (solid content conversion ratio) was 1.
  • a purified anti-influenza A virus NP antibody (an antibody different from the above) was used.
  • the purified anti-influenza A virus NP antibody is labeled with 0.2 ⁇ m red latex particles (SC-042-R manufactured by JSR Life Sciences) with a covalent bond, and added to a Tris buffer containing sugar, surfactant and protein.
  • An anti-A-type labeling body was prepared by suspending it so that the concentration of latex particles was 0.025 w / v%, and performing sonication to sufficiently disperse and float.
  • a specimen suspension attached to Quick Navi-Flu manufactured by Denka Seken Co., Ltd. was used as a diluted solution.
  • the dilution factor of influenza A virus A / Beijing / 32/92 (H3N2) is increased from 2 ⁇ 10 4
  • the dilution factor (visual determination) that makes it impossible to visually check the presence of a colored line 10 minutes after the start of the test Possible limit magnification).
  • Table 1 shows the results when the flow path is a membrane.
  • Example 2 The experiment was performed under the same conditions as in Example 1 except that the mass ratio of the charge-imparting substance to the solid-phase protein in Example 1 (charge-imparting substance application amount / solid-phase protein application amount) was 100.
  • Example 3 The experiment was performed under the same conditions as in Example 1 except that the mass ratio of the charge-imparting substance to the solid-phase protein in Example 1 (charged substance application amount / solid-phase protein application amount) was 0.01.
  • PSS polystyrene sulfonic acid
  • Example 5 The same conditions as in Example 1 except that the charge-providing substance in Example 1 is PSS and the mass ratio of the charge-providing substance to the solid-phase protein is 100 (charge-providing substance coating amount / solid-phase protein coating amount). The experiment was conducted.
  • Example 6 The charge imparting substance in Example 1 is PSS, and the mass ratio of the charge imparting substance to the solid phase protein (charge imparting substance coating amount / solid phase protein coating amount) is 0.01. The experiment was conducted under conditions.
  • Example 1 The same as Example 1 except that the mass ratio of the charge-imparting substance to the solid phase protein (charge-giving substance application amount / solid-phase protein application amount) was set to 0 without applying the charge-giving substance in Example 1. The experiment was conducted under the following conditions.
  • Example 2 The experiment was performed under the same conditions as in Example 1 except that the charge-providing substance in Example 1 was set to a mass ratio of the charge-providing substance to the solid phase protein (charged substance coating amount / solid phase protein coating amount) of 1000. It was.
  • Example 3 The charge imparting substance in Example 1 is PSS, and the mass ratio of the charge imparting substance with respect to the solid phase protein (charge imparting substance coating amount / solid phase protein coating amount) is 0.001. The experiment was conducted under conditions.
  • Example 7 ⁇ Mold preparation> The mold was produced by laser processing and mechanical cutting. This mold is made of aluminum alloy A5052. A conical recess having a diameter of 25 ⁇ m, a pitch of 30 ⁇ m, and a depth of 30 ⁇ m is machined in the center of the mold in a triangular array form as shown in FIG. 7 in a range of 3 cm ⁇ 3 cm. A release treatment was performed on the uneven surface of the mold in order to easily and reliably peel off the mold and the thermoplastic resin when transferred. The mold release treatment was performed by immersing in OPTOOL HD-2100TH manufactured by Daikin Industries, Ltd. for about 1 minute, drying, and allowing to stand overnight.
  • OPTOOL HD-2100TH manufactured by Daikin Industries, Ltd.
  • thermoplastic resin polystyrene (PS, Denka styrene sheet manufactured by Denka Co., Ltd., film thickness: 300 ⁇ m) was used.
  • Thermal imprinting was used as a processing method, and an X-300 manufactured by SCIVAX was used as the apparatus.
  • the molding temperature was 120 ° C.
  • the applied pressure was 5.5 MPa
  • transfer was performed for 10 minutes.
  • the thermoplastic plastic and the mold were cooled to 80 ° C. while applying pressure, and then the pressure was removed to produce a flow path having a conical convex portion on the surface.
  • conical convex portions 6 having a pitch of 30 ⁇ m, a diameter of 25 ⁇ m, and a height of 30 ⁇ m are processed into a 3 cm ⁇ 3 cm range in a triangular arrangement form as shown in FIG.
  • Example 2 The subsequent operation was conducted under the same conditions as in Example 1 except that the flow path in Example 1 was made of polystyrene.
  • Table 2 shows the results when the flow path is made of polystyrene.
  • Example 8 The experiment was performed under the same conditions as in Example 7, except that the mass ratio of the charge-imparting substance to the solid-phase protein in Example 7 (charge-imparting substance coating amount / solid-phase protein coating amount) was 100.
  • Example 9 The experiment was performed under the same conditions as in Example 7 except that the mass ratio of the charge-imparting substance to the solid-phase protein in Example 7 (charged substance application amount / solid-phase protein application amount) was 0.01.
  • Example 10 The experiment was performed under the same conditions as in Example 7 except that PSS was used as the charge-providing substance in Example 7.
  • Example 11 The same conditions as in Example 7 except that the charge-providing substance in Example 7 was PSS and the mass ratio of the charge-providing substance to the solid phase protein (charge-providing substance coating amount / solid-phase protein coating amount) was 100. The experiment was conducted.
  • Example 7 is the same as Example 7 except that the charge-providing substance in Example 7 is PSS, and the mass ratio of the charge-providing substance to the solid-phase protein (charge-providing substance coating amount / solid-phase protein coating amount) is 0.01. The experiment was conducted under conditions.
  • Example 7 is the same as Example 7 except that the mass ratio of the charge-providing substance to the solid phase protein (charge-applying substance application amount / solid-phase protein application quantity) was set to 0 without applying the charge-providing substance in Example 7. The experiment was conducted under the following conditions.
  • Example 5 The experiment was performed under the same conditions as in Example 7, except that the mass ratio of the charge-imparting substance to the solid-phase protein in Example 7 (charge-imparting substance coating amount / solid-phase protein coating amount) was 0.0001.
  • Example 6 Under the same conditions as in Example 7, except that the charge-providing substance in Example 7 was PSS and the mass ratio of the charge-providing substance to the solid phase protein (charged substance coating amount / solid phase protein coating amount) was 10,000. The experiment was conducted.
  • a highly sensitive liquid sample inspection kit in which the orientation of the detection substance is controlled can be obtained.
  • the liquid sample test kit of this embodiment has a test score 1.5 times higher than that of a liquid sample test kit that does not use this embodiment, and can achieve high sensitivity.
  • SYMBOLS 1 Charge provision substance 2 Flow path 3 Detection substance 3a Solid phase protein base 3b Solid phase protein edge part 4,4A, 4B, 4C Membrane carrier 4x Dropping zone 4y Detection zone 5 Recess 6 Protrusion 7 Mold (mold) 10 Liquid Sample Inspection Kit 10a Case 10b First Opening 10c Second Opening 11 Pitch

Abstract

The present invention provides a membrane carrier having a flow path, a charge-imparting substance, and a detection substance. The detection substance is retained in the flow path via the charge-imparting substance. The mass ratio of the charge-imparting substance to the detection substance (charge-imparting substance/detection substance) is 0.01 to 100.

Description

膜担体及びその製造方法並びに液体試料検査キットMembrane carrier, manufacturing method thereof, and liquid sample inspection kit
　本発明は、膜担体及びその製造方法並びに液体試料検査キットに関する。 The present invention relates to a membrane carrier, a manufacturing method thereof, and a liquid sample inspection kit.
　近年、抗原抗体反応等を用いることで、感染症への罹患や妊娠、血糖値等を測定する、Ｐｏｉｎｔ　ｏｆ　Ｃａｒｅ　Ｔｅｓｔ（ＰＯＣＴ、臨床現場即時検査）試薬が注目を集めている。ＰＯＣＴとは、日本臨床検査自動化学会が発光したＰＯＣＴガイドラインにおいて、被験者の傍らで行われる検査、或いは、被験者自らが行う検査であり、検査時間の短縮及びその場での検査（被験者に見える検査）が可能という利点を有する検査と定義されている。 In recent years, Point of Care Test (POCT, immediate on-site clinical examination) reagents have been attracting attention, which measure antigen morbidity, pregnancy, blood glucose levels, etc. by using antigen-antibody reactions and the like. POCT is a test performed beside the subject in the POCT guidelines emitted by the Japan Society for Clinical Laboratory Automation, or a test performed by the subject himself, shortening the test time and testing on the spot (test visible to the subject) Is defined as an inspection that has the advantage of being possible.
　ＰＯＣＴ試薬には、迅速検査が可能、使用方法が簡便、試薬が安価という特徴がある。これらの特徴から、症状が軽度の段階での診察や定期診察等に多く使用されており、今後増加することが予想される在宅医療においても重要な診察ツールとなっている。 POCT reagents are characterized by rapid testing, simple usage, and inexpensive reagents. Because of these characteristics, it is often used for medical examinations and regular medical examinations at a mild stage, and it is an important diagnostic tool in home medical care that is expected to increase in the future.
　多くのＰＯＣＴ試薬では、血液等の液体試料を検査キットに導入し、その中に含まれる特定の被検出物質を検出することで判定を行っている。液体試料から特定の被検出物質を検出する方法としてイムノクロマトグラフィ法がよく用いられている。イムノクロマトグラフィ法とは、検査キットの膜担体上に滴下された液体が膜担体上を移動する中で、被検出物質と、液体試料中に浮遊又は溶解した状態にある、液体試料中の被検出物質と特異的に反応する抗体又はその抗原結合性断片を結合した標識粒子とが、結合し、更にこれらが検査キット中に固定化された物質（以下、検出物質という）と特異的に結合し、その結果生じた色又は質量の変化等を検出するという手法である。 In many POCT reagents, determination is performed by introducing a liquid sample such as blood into a test kit and detecting a specific substance to be detected contained therein. An immunochromatography method is often used as a method for detecting a specific substance to be detected from a liquid sample. The immunochromatography method is that the liquid dropped on the membrane carrier of the test kit moves on the membrane carrier, and the substance to be detected and the subject to be detected in the liquid sample suspended or dissolved in the liquid sample. Labeled particles bound with antibodies or antigen-binding fragments thereof that specifically react with the substance bind to each other, and these bind specifically with the substance immobilized in the test kit (hereinafter referred to as detection substance). This is a method of detecting a change in color or mass that occurs as a result.
　現行のＰＯＣＴ試薬は、検出物質（例えば、固相タンパク質）の配向を制御できていないために、イムノクロマトグラフィ法での目視感度が低下する場合があった。一例として検出物質が抗体、被検出物質が抗原であるＰＯＣＴを考える。抗体は基部であるＦｃ部位と、抗原と反応するＦａｂ部位から構成される。この時、抗体の配向を制御できていない現行のＰＯＣＴ試薬では、ＰＯＣＴ試薬の流路の最表面がＦａｂ部位とＦｃ部位から成るランダムな表面となる。一方、抗体の配向が制御されたＰＯＣＴ試薬においては、ＰＯＣＴ試薬の流路の最表面がＦａｂ部位となり、かつ流路と抗体の接着面がＦｃ部位となる。従って、抗体の配向が制御されたＰＯＣＴ試薬では現行の制御されていないＰＯＣＴ試薬に比べ、流路の最表面においてＦａｂ部位と抗原の反応確率が上昇するため、イムノクロマトグラフィ法での目視感度が上昇すると考えられる。即ち、検出物質の配向制御によりＰＯＣＴ試薬の目視感度が上昇すると期待される。 Since the current POCT reagent does not control the orientation of the detection substance (for example, solid phase protein), the visual sensitivity in the immunochromatography method may decrease. As an example, consider POCT in which the detection substance is an antibody and the detection substance is an antigen. An antibody is composed of a base Fc site and a Fab site that reacts with an antigen. At this time, in the current POCT reagent in which the orientation of the antibody cannot be controlled, the outermost surface of the flow path of the POCT reagent is a random surface composed of a Fab site and an Fc site. On the other hand, in the POCT reagent in which the orientation of the antibody is controlled, the outermost surface of the POCT reagent channel is the Fab site, and the adhesive surface between the channel and the antibody is the Fc site. Therefore, in the POCT reagent in which the orientation of the antibody is controlled, the probability of reaction between the Fab site and the antigen on the outermost surface of the flow path is higher than that in the current non-controlled POCT reagent, so that the visual sensitivity in the immunochromatography method is increased. I think that. That is, it is expected that the visual sensitivity of the POCT reagent is increased by controlling the orientation of the detection substance.
　例えば、特許文献１では、光応答性成分を含有する固相に光照射により、タンパク質等の微小物体を固定化するのに際して、微小物体が固相に対して一定の配向性で固定可能な相互作用可能な成分を固相に備えさせることで、微小物体の配向性を制御して固相に固定化している。しかしながら、特許文献１の光固定化方法を用いてＰＯＣＴ試薬を製造した場合、ＰＯＣＴ試薬の光安定性が低下するため、実用化は難しい。 For example, in Patent Document 1, when immobilizing a micro object such as a protein by light irradiation on a solid phase containing a photoresponsive component, the micro object can be fixed with a fixed orientation with respect to the solid phase. By providing an operable component in the solid phase, the orientation of the micro object is controlled and immobilized on the solid phase. However, when a POCT reagent is produced using the light immobilization method of Patent Document 1, the light stability of the POCT reagent is lowered, and thus practical application is difficult.
　特許文献２及び特許文献３では、濃厚タンパク質水溶液を電場中に設置することにより、タンパク質の結晶成長が促進されると示されている。しかし、特許文献２及び特許文献３は、液体試料検査キットについて、記載がない。 Patent Document 2 and Patent Document 3 show that protein crystal growth is promoted by placing a concentrated protein aqueous solution in an electric field. However, Patent Document 2 and Patent Document 3 do not describe a liquid sample inspection kit.
　特許文献４では、電気的効果としてカチオン性高分子及びアニオン性高分子が有する電荷を利用することで、セラミックス粒子表面への微小粒子の接着に成功している。具体的な手法としては、セラミックス粒子表面にカチオン性高分子及びアニオン性高分子の水溶液を塗布することで、セラミックス粒子表面に電荷を付与しており、帯電した微小粒子との間に生じる静電的相互作用により強く接着させている。しかし、特許文献４は、液体試料検査キットについて、記載がない。 Patent Document 4 succeeds in adhering microparticles to the surface of ceramic particles by utilizing the charge of a cationic polymer and an anionic polymer as an electrical effect. A specific method is to apply an aqueous solution of a cationic polymer and an anionic polymer to the surface of the ceramic particles, thereby imparting a charge to the surface of the ceramic particles and generating electrostatic charges between the charged fine particles. It is strongly bonded by mechanical interaction. However, Patent Document 4 does not describe a liquid sample inspection kit.
　特許文献５は、液体試料中の被検出物質を検出する検査キット用の膜担体であって、前記液体試料を輸送できる少なくとも一つの流路が設けられ、前記流路の底面に、前記液体試料を輸送するための毛細管作用を生じせしめる微細構造が設けられている、液体試料検査キット用膜担体を記載している。しかし、特許文献５は、検出物質の配向制御について、記載がない。 Patent Document 5 is a membrane carrier for a test kit that detects a substance to be detected in a liquid sample, and is provided with at least one flow path capable of transporting the liquid sample, and the liquid sample is provided on the bottom surface of the flow path. A membrane carrier for a liquid sample test kit is described, which is provided with a microstructure that causes a capillary action for transporting the sample. However, Patent Document 5 does not describe the orientation control of the detection substance.
特許第４８８７８６３号公報Japanese Patent No. 4888863 特許第５８５８２７４号公報Japanese Patent No. 5858274 特許第５６２６９１４号公報Japanese Patent No. 5626914 特開２０１０－６４９４５号公報JP 2010-64945 A 国際公開第２０１６／０９８７４０号International Publication No. 2016/098740
　本発明は、上記事情を鑑みて、高感度な判定が可能な膜担体の提供を目的とする。 In view of the above circumstances, an object of the present invention is to provide a membrane carrier capable of highly sensitive determination.
　即ち、本発明は、以下の通りである。
（１）流路と電荷付与物質と検出物質とを備え、検出物質は電荷付与物質を介して流路に保持され、検出物質に対する電荷付与物質の質量比（電荷付与物質／検出物質）が、０．０１～１００である、膜担体。
（２）検出物質が、検出物質と電荷付与物質との静電的相互作用により生じる電界によって配向している、（１）に記載の膜担体。
（３）電荷付与物質が、カチオン性高分子及びアニオン性高分子からなる群より選択される１種以上である、（１）又は（２）に記載の膜担体。
（４）電荷付与物質が、ポリ（ジアリルジメチルアンモニウムクロライド）である、（１）～（３）のいずれかに記載の膜担体。
（５）電荷付与物質が、ポリスチレンスルホン酸である、（１）～（４）のいずれかに記載の膜担体。
（６）流路が、樹脂で形成されている、（１）～（５）のいずれかに記載の膜担体。
（７）流路が、凹凸構造を有する、（１）～（６）のいずれかに記載の膜担体。
（８）（１）～（７）のいずれかに記載の膜担体を有する、液体試料検査キット。
（９）流路上に電荷付与物質を塗布する工程と、塗布された電荷付与物質上に検出物質を塗布する工程と、を備え、検出物質の塗布量に対する電荷付与物質の塗布量（電荷付与物質塗布量／検出物質塗布量）が０．０１～１００である、膜担体の製造方法。
（１０）電荷付与物質を塗布する工程の前に、インプリントにより流路に凹凸構造を形成する工程を更に備える、（９）に記載の膜担体の製造方法。 That is, the present invention is as follows.
(1) A flow path, a charge imparting substance, and a detection substance are provided, the detection substance is held in the flow path via the charge imparting substance, and the mass ratio of the charge imparting substance to the detection substance (charge imparting substance / detection substance) is A membrane carrier that is 0.01-100.
(2) The film carrier according to (1), wherein the detection substance is oriented by an electric field generated by an electrostatic interaction between the detection substance and the charge imparting substance.
(3) The membrane carrier according to (1) or (2), wherein the charge-imparting substance is at least one selected from the group consisting of a cationic polymer and an anionic polymer.
(4) The membrane carrier according to any one of (1) to (3), wherein the charge-imparting substance is poly (diallyldimethylammonium chloride).
(5) The membrane carrier according to any one of (1) to (4), wherein the charge-imparting substance is polystyrene sulfonic acid.
(6) The membrane carrier according to any one of (1) to (5), wherein the flow path is formed of a resin.
(7) The membrane carrier according to any one of (1) to (6), wherein the flow path has an uneven structure.
(8) A liquid sample inspection kit comprising the membrane carrier according to any one of (1) to (7).
(9) A step of applying a charge-providing substance on the flow path and a step of applying a detection substance on the applied charge-providing substance, wherein the application amount of the charge-providing substance relative to the application amount of the detection substance (charge-providing substance) A method for producing a membrane carrier, wherein the coating amount / the detection substance coating amount) is from 0.01 to 100.
(10) The method for producing a film carrier according to (9), further comprising a step of forming an uneven structure in the flow path by imprinting before the step of applying the charge-imparting substance.
　本発明によれば、高感度な判定が可能な膜担体の提供が可能となる。 According to the present invention, it is possible to provide a membrane carrier capable of highly sensitive determination.
本発明による実施形態の一例であり、検査キットの模式的な上面図である。It is an example of embodiment by this invention, and is a typical top view of a test | inspection kit. 本発明による実施形態の一例であり、膜担体の模式的な俯瞰図（上面図）である。It is an example of embodiment by this invention, and is a typical bird's-eye view (top view) of a membrane carrier. 本発明による実施形態の一例であり、膜担体の模式的な断面図である。It is an example of embodiment by this invention and is typical sectional drawing of a membrane carrier. 本発明による実施形態の一例であり、膜担体の模式的な断面図である。It is an example of embodiment by this invention and is typical sectional drawing of a membrane carrier. 本発明による実施形態の一例であり、膜担体の模式的な断面図である。It is an example of embodiment by this invention and is typical sectional drawing of a membrane carrier. 検出物質の配向制御を説明するための、膜担体の要部を示す断面図である。It is sectional drawing which shows the principal part of a film | membrane carrier for demonstrating orientation control of a detection substance. 本発明による実施形態の一例であり、微細構造を形成するための金型（モールド）の概略図である。It is an example of embodiment by this invention, and is the schematic of the metal mold | die (mold) for forming a fine structure. 本発明による実施形態の一例であり、膜担体の拡大斜視図である。It is an example of embodiment by this invention, and is an expansion perspective view of a film | membrane carrier.
　以下、本発明の実施形態について説明する。 Hereinafter, embodiments of the present invention will be described.
　本実施形態に係る液体試料検査キット（以下、単に「検査キット」ともいう）は、液体試料中の被検出物質を検出する。図１は、検査キットの模式的な上面図である。例えば、図１に示すように、検査キット１０は、膜担体４と、膜担体４を収容する筐体１０ａと、を備える。膜担体４は、その表面に、液体試料が滴下される滴下ゾーン４ｘと、液体試料中の被検出物質を検出するための検知ゾーン４ｙと、を有している。滴下ゾーン４ｘは、筐体１０ａの第一開口部１０ｂにおいて露出している。検知ゾーン４ｙは、筐体１０ａの第二開口部１０ｃにおいて露出している。膜担体４において、滴下ゾーン４ｘ及び検知ゾーン４ｙは、それぞれ液体試料の輸送方向の上流側及び下流側に位置する。 The liquid sample inspection kit (hereinafter also simply referred to as “inspection kit”) according to the present embodiment detects a substance to be detected in the liquid sample. FIG. 1 is a schematic top view of an inspection kit. For example, as shown in FIG. 1, the test kit 10 includes a membrane carrier 4 and a housing 10 a that houses the membrane carrier 4. The membrane carrier 4 has, on its surface, a dropping zone 4x where a liquid sample is dropped and a detection zone 4y for detecting a substance to be detected in the liquid sample. The dripping zone 4x is exposed at the first opening 10b of the housing 10a. The detection zone 4y is exposed at the second opening 10c of the housing 10a. In the membrane carrier 4, the dripping zone 4x and the detection zone 4y are located on the upstream side and the downstream side in the transport direction of the liquid sample, respectively.
　膜担体は、一実施形態において、液体試料中の被検出物質を検出する液体試料検査キット用の膜担体である。 In one embodiment, the membrane carrier is a membrane carrier for a liquid sample inspection kit that detects a substance to be detected in the liquid sample.
　ここで、被検出物質としては、何ら限定されるものではなく、各種病原体、各種臨床マーカー等、抗体と抗原抗体反応することが可能ないかなる物質であってもよい。具体例としては、インフルエンザウイルス、ノロウイルス、アデノウイルス、ＲＳウイルス、ＨＡＶ、ＨＢｓ、ＨＩＶ等のウイルス抗原、ＭＲＳＡ、Ａ群溶連菌、Ｂ群溶連菌、レジオネラ属菌等の細菌抗原、細菌等が産生する毒素、マイコプラズマ、クラミジア・トラコマティス、ヒト絨毛性ゴナドトロピン等のホルモン、Ｃ反応性タンパク質、ミオグロビン、心筋トロポニン、各種腫瘍マーカー、農薬、及び環境ホルモン等を例示できるが、これらに限定されるものではない。特に、インフルエンザウイルス、ノロウイルス、Ｃ反応性タンパク質、ミオグロビン、及び心筋トロポニンのような検出と治療措置に急を要する項目の場合にはその有用性が特に大きい。被検出物質は、単独で免疫反応を誘起できる抗原であってもよいし、単独では免疫反応を誘起できないが抗体と抗原抗体反応により結合することが可能なハプテンであってもよい。被検出物質は、液体試料中で浮遊又は溶解した状態にあってよい。液体試料は、例えば、上記被検出物質を緩衝液に浮遊又は溶解させた試料であってよい。 Here, the substance to be detected is not limited at all, and may be any substance capable of antigen-antibody reaction with an antibody such as various pathogens and various clinical markers. Specific examples include viral antigens such as influenza virus, norovirus, adenovirus, RS virus, HAV, HBs, HIV, bacterial antigens such as MRSA, group A streptococci, group B streptococci, legionella, toxins produced by bacteria, etc. Examples include, but are not limited to, hormones such as Mycoplasma, Chlamydia trachomatis, human chorionic gonadotropin, C-reactive protein, myoglobin, cardiac troponin, various tumor markers, agricultural chemicals, and environmental hormones. In particular, it is particularly useful for items that require urgent detection and treatment measures such as influenza virus, norovirus, C-reactive protein, myoglobin, and cardiac troponin. The substance to be detected may be an antigen capable of inducing an immune reaction alone, or a hapten capable of binding an antibody by an antigen-antibody reaction although it cannot induce an immune reaction alone. The substance to be detected may be in a suspended or dissolved state in the liquid sample. The liquid sample may be, for example, a sample in which the substance to be detected is suspended or dissolved in a buffer solution.
　図２は、膜担体の模式的な上面図である。例えば、図２に示すように、膜担体４は、流路２と電荷付与物質１と検出物質３とを備えている。検出物質３は電荷付与物質１を介して流路２に保持されている。検出物質３が存在する領域が、検知ゾーンを構成している。液体試料は、流路２を介して、滴下ゾーンから検知ゾーンへ輸送される。 FIG. 2 is a schematic top view of the membrane carrier. For example, as shown in FIG. 2, the membrane carrier 4 includes a flow path 2, a charge imparting substance 1, and a detection substance 3. The detection substance 3 is held in the flow path 2 via the charge imparting substance 1. A region where the detection substance 3 exists constitutes a detection zone. The liquid sample is transported from the dropping zone to the detection zone via the flow path 2.
　図３は、一実施形態の膜担体の模式的な断面図（図２のＩＩ－ＩＩ線に沿った断面図）である。図３に示す膜担体４Ａは、流路２と、流路２の主面上の少なくとも一部に存在する電荷付与物質１と、該電荷付与物質１上に存在する検出物質３とから構成されている。この膜担体４Ａは、例えば、流路２の表面の少なくとも一部に電荷付与物質１を塗布し、該電荷付与物質１上に検出物質３を更に塗布することにより得られる。 FIG. 3 is a schematic cross-sectional view (cross-sectional view taken along the line II-II in FIG. 2) of the membrane carrier of one embodiment. A membrane carrier 4A shown in FIG. 3 is composed of a flow path 2, a charge-providing substance 1 present on at least a part of the main surface of the flow path 2, and a detection substance 3 present on the charge-provided substance 1. ing. The membrane carrier 4A is obtained, for example, by applying the charge-providing substance 1 to at least a part of the surface of the flow path 2 and further applying the detection substance 3 on the charge-providing substance 1.
　図４は、他の実施形態の膜担体の模式的な断面図である。図４に示す膜担体４Ｂは、流路２と、流路２の内部の少なくとも一部に存在する電荷付与物質１と、電荷付与物質１上かつ流路２の表面上に存在する検出物質３とから構成されている。この膜担体４Ｂは、例えば、流路２の表面の少なくとも一部に電荷付与物質１を塗布し、該電荷付与物質１を流路２の内部に浸潤させた後、流路２の表面上の電荷付与物質１を塗布した領域に、検出物質３を更に塗布することにより得られる。 FIG. 4 is a schematic cross-sectional view of a membrane carrier according to another embodiment. A membrane carrier 4B shown in FIG. 4 includes a flow path 2, a charge-providing substance 1 present in at least a part of the flow path 2, and a detection substance 3 present on the charge-providing substance 1 and on the surface of the flow path 2. It consists of and. For example, the membrane carrier 4B is formed on the surface of the flow path 2 after applying the charge imparting substance 1 to at least a part of the surface of the flow path 2 and infiltrating the charge imparting substance 1 into the flow path 2. It is obtained by further applying the detection substance 3 to the area where the charge imparting substance 1 is applied.
　図５は、他の実施形態の膜担体の模式的な断面図である。図５に示す膜担体４Ｃは、流路２と、流路２の内部の少なくとも一部に存在する電荷付与物質１と、電荷付与物質１上かつ流路２の内部及び表面に存在する検出物質３とから構成されている。つまり、検出物質３は、流路２の内部に存在すると共に、流路２の表面にも露出している。この膜担体４Ｃは、例えば、流路２の表面の少なくとも一部に電荷付与物質１を塗布し、該電荷付与物質１を流路２の内部に浸潤させた後、流路２の表面上の電荷付与物質１を塗布した領域に、検出物質３を更に塗布し、該検出物質３を流路２の内部に浸潤させることにより得られる。 FIG. 5 is a schematic cross-sectional view of a membrane carrier of another embodiment. A membrane carrier 4C shown in FIG. 5 includes a flow path 2, a charge-providing substance 1 present in at least a part of the flow path 2, and a detection substance present on the charge-providing substance 1 and in and on the surface of the flow path 2. 3. That is, the detection substance 3 exists inside the flow path 2 and is also exposed on the surface of the flow path 2. For example, the membrane carrier 4C is formed on the surface of the flow channel 2 after applying the charge imparting material 1 to at least a part of the surface of the flow channel 2 and infiltrating the charge imparting material 1 into the flow channel 2. It is obtained by further applying the detection substance 3 to the area where the charge imparting substance 1 is applied and infiltrating the detection substance 3 into the flow path 2.
　流路２は、膜担体４において、液体試料を輸送するための流路として機能する。流路２の表面は、平坦又は曲面であってもよい。流路２の表面上には、微細構造（凹凸構造）が形成されていてもよい。流路２の表面上に微細構造が設けられている場合、微細構造の毛細管作用により、液体試料は、微細構造を介して輸送される。また、流路２は、多孔質基材で構成されていてもよい。液体試料が多孔質基材で構成されている場合、液体試料は、多孔質基材の孔の中を毛細管作用によって移動することにより、輸送される。 The channel 2 functions as a channel for transporting the liquid sample in the membrane carrier 4. The surface of the flow path 2 may be flat or curved. A fine structure (uneven structure) may be formed on the surface of the flow path 2. When the fine structure is provided on the surface of the flow path 2, the liquid sample is transported through the fine structure by the capillary action of the fine structure. Moreover, the flow path 2 may be comprised with the porous base material. When the liquid sample is composed of a porous substrate, the liquid sample is transported by moving through the pores of the porous substrate by capillary action.
　流路の全体の形状は、特に限定されないが、例えば、四角形等の多角形、円形、又は楕円形であってよい。流路が四角形である場合、流路の縦幅（短手方向の長さ）は、例えば、２ｍｍ以上１００ｍｍ以下であってよく、流路の横幅（長手方向の長さ）は、例えば、２ｍｍ以上１００ｍｍ以下であってよい。 The overall shape of the flow path is not particularly limited, but may be, for example, a polygon such as a quadrangle, a circle, or an ellipse. When the flow path is a quadrangle, the vertical width (length in the short direction) of the flow path may be, for example, 2 mm or more and 100 mm or less, and the horizontal width (length in the longitudinal direction) of the flow path is, for example, 2 mm. It may be 100 mm or less.
　流路２の厚さは１００μｍ～５ｍｍが好ましく、５００μｍ～２ｍｍがより好ましい。流路２上に凹凸構造が設けられている場合、流路２の厚さは、凸部の高さを含まない。 The thickness of the flow path 2 is preferably 100 μm to 5 mm, more preferably 500 μm to 2 mm. When the uneven structure is provided on the flow path 2, the thickness of the flow path 2 does not include the height of the convex portion.
　流路２を形成する材料は、樹脂であることが好ましく、熱可塑性樹脂であることがより好ましい。熱可塑性樹脂としては、例えば、ニトロセルロース、ポリエステル系樹脂、ポリオレフィン系樹脂、ポリスチレン系樹脂、ポリカーボネート系樹脂、フッ素系樹脂、アクリル系樹脂等が挙げられる。具体的には、ポリエチレンテレフタレート（ＰＥＴ）、シクロオレフィンポリマー（ＣＯＰ）、ポリプロピレン（ＰＰ）、ポリスチレン（ＰＳ）、メチルメタクリレート―スチレン共重合体（ＭＳ）、メチルメタクリレート―ブタジエン―スチレン共重合体（ＭＢＳ）、メチルメタクリレート―アクリロニトリル―ブタジエン―スチレン（ＭＡＢＳ）、スチレンブロック共重合体（ＳＢＣ）、ポリカーボネート（ＰＣ）、ポリフッ化ビニリデン（ＰＶＤＦ）、ポリメタクリル酸メチル（ＰＭＭＡ）等が挙げられる。 The material forming the flow path 2 is preferably a resin, and more preferably a thermoplastic resin. Examples of the thermoplastic resin include nitrocellulose, polyester resin, polyolefin resin, polystyrene resin, polycarbonate resin, fluorine resin, and acrylic resin. Specifically, polyethylene terephthalate (PET), cycloolefin polymer (COP), polypropylene (PP), polystyrene (PS), methyl methacrylate-styrene copolymer (MS), methyl methacrylate-butadiene-styrene copolymer (MBS) ), Methyl methacrylate-acrylonitrile-butadiene-styrene (MABS), styrene block copolymer (SBC), polycarbonate (PC), polyvinylidene fluoride (PVDF), polymethyl methacrylate (PMMA), and the like.
　流路２が多孔質基材で構成されている場合、流路２の材料はニトロセルロースであることが好ましい。この場合、流路２として、ニトロセルロースメンブレンを用いることができる。 When the channel 2 is composed of a porous substrate, the material of the channel 2 is preferably nitrocellulose. In this case, a nitrocellulose membrane can be used as the flow path 2.
　流路２が表面の少なくとも一部に凹凸構造（微細構造）が設けられた基材で構成されている場合、流路２の材料は、熱可塑性樹脂であることが好ましい。微細構造は凹凸構造を有する構造であり、複数の凸部を有している。 When the flow path 2 is composed of a base material provided with an uneven structure (fine structure) on at least a part of the surface, the material of the flow path 2 is preferably a thermoplastic resin. The fine structure has a concavo-convex structure and has a plurality of convex portions.
　流路２は、高感度な検査がより一層容易になる観点から、少なくとも表面の一部に凹凸構造（微細構造）を有することが好ましい。つまり、膜担体４は、少なくとも表面の一部に凹凸構造が設けられた流路２を備えていることが好ましい。凹凸構造は、少なくとも滴下ゾーンと検知ゾーンとの間に位置していてよい。流路２の表面全体にわたり、凹凸構造が設けられていてもよい。 It is preferable that the flow path 2 has a concavo-convex structure (fine structure) on at least a part of the surface from the viewpoint of facilitating highly sensitive inspection. That is, it is preferable that the membrane carrier 4 includes the flow path 2 provided with an uneven structure on at least a part of the surface. The concavo-convex structure may be located at least between the dropping zone and the detection zone. An uneven structure may be provided over the entire surface of the flow path 2.
　微細構造は、インプリントにより形成することができる。インプリントにより微細構造を形成する方法としては、熱インプリントが好ましい。熱インプリントとは、微細構造を有する金型（モールド）を基材に押し当て、加熱により柔らかくした基材に微細構造を転写することにより、微細構造を有する基材を作製する方法である。この熱インプリントではナノオーダーの微細加工が可能である。 The fine structure can be formed by imprinting. As a method for forming a fine structure by imprinting, thermal imprinting is preferable. Thermal imprinting is a method for producing a substrate having a fine structure by pressing a mold (mold) having a fine structure against the substrate and transferring the fine structure to a substrate softened by heating. This thermal imprint enables nano-order microfabrication.
　微細構造を有する金型（モールド）は、凹凸構造を有する。金型における凹凸構造としては、図７に示すように、金型７の表面に、凹部５が三角配列形式にて、規則的に整列する構造が好ましい。 The mold (mold) having a fine structure has an uneven structure. As the concavo-convex structure in the mold, as shown in FIG. 7, a structure in which the recesses 5 are regularly arranged in a triangular arrangement on the surface of the mold 7 is preferable.
　流路２における凹凸構造としては、図８に示すように、流路２の表面に、凸部６が突起として各方向に規則的に配列する構造が好ましい。凸部６が突起として規則的に配列する構造としては、平面格子状に配列する構造が好ましい。平面格子としては、斜方格子、六角格子、正方格子、矩形格子、平行体格子のうちの１種以上が好ましく、六角格子がより好ましい。六角格子の中では、正三角形格子が好ましい。図８は、凸部６が正三角形格子状に配列する構造を示す。 As the concavo-convex structure in the flow path 2, as shown in FIG. 8, a structure in which the convex portions 6 are regularly arranged in each direction as protrusions on the surface of the flow path 2 is preferable. The structure in which the convex portions 6 are regularly arranged as protrusions is preferably a structure in which the protrusions 6 are arranged in a planar lattice pattern. The planar lattice is preferably one or more of an orthorhombic lattice, a hexagonal lattice, a tetragonal lattice, a rectangular lattice, and a parallel lattice, and more preferably a hexagonal lattice. Of the hexagonal lattice, an equilateral triangular lattice is preferable. FIG. 8 shows a structure in which the convex portions 6 are arranged in an equilateral triangular lattice shape.
　凸部６は流路２と同じ材料からなることが好ましい。凸部６の形状としては、錐体又は柱体が好ましく、錐体がより好ましい。錐体としては、図８のように、凸部６の断面は三角形であることが好ましく、正三角形がより好ましい。断面が三角形である錐体としては、円錐形状又は角錐形状が好ましく、円錐形状がより好ましい。 The convex part 6 is preferably made of the same material as the flow path 2. As a shape of the convex part 6, a cone or a column is preferable, and a cone is more preferable. As a cone, as shown in FIG. 8, the cross section of the convex portion 6 is preferably a triangle, and more preferably a regular triangle. As the cone having a triangular cross section, a cone shape or a pyramid shape is preferable, and a cone shape is more preferable.
　凸部６のピッチ１１は、１～３００μｍが好ましく、５～１００μｍがより好ましく、１０～５０μｍが最も好ましい。凸部６のピッチ１１が１μｍ以上である場合、光反射性がより優れたものとなる。凸部６のピッチ１１が３００μｍ以下である場合、透明性がより優れたものとなる。 The pitch 11 of the convex portions 6 is preferably 1 to 300 μm, more preferably 5 to 100 μm, and most preferably 10 to 50 μm. When the pitch 11 of the convex portions 6 is 1 μm or more, the light reflectivity is more excellent. When the pitch 11 of the convex portions 6 is 300 μm or less, the transparency is more excellent.
　凸部６のピッチ１１は、互いに隣接する、凸部６と凸部６の間の寸法（隣接する凸部６の中心間距離）をいう。凸部６が錐体である場合、ピッチ１１は、図８のように、錐体の頂点と錐体の頂点の間の寸法をいう。 The pitch 11 of the convex portions 6 refers to the dimension between the convex portions 6 and 6 adjacent to each other (the distance between the centers of the adjacent convex portions 6). When the convex part 6 is a cone, the pitch 11 means the dimension between the vertex of a cone and the vertex of a cone as shown in FIG.
　凸部６の高さは、１～１０００μｍが好ましく、１０～５０μｍがより好ましい。１μｍ以上だと、流路体積が大きくなり、液体試料を展開するのに長い時間を要しない。１０００μｍ以下だと、微細構造を作製するのに多大な時間とコストがかからず、微細構造の製造が容易になる。凸部６の高さは凸部６の底面に直交する方向における凸部６の最大長さとして定義される。つまり、凸部６の高さは、凸部６の底面から凸部６の頂点までの寸法をいう。 The height of the convex portion 6 is preferably 1 to 1000 μm, and more preferably 10 to 50 μm. If it is 1 μm or more, the flow path volume becomes large, and it does not take a long time to develop the liquid sample. When the thickness is 1000 μm or less, it does not take much time and cost to produce the fine structure, and the production of the fine structure becomes easy. The height of the convex portion 6 is defined as the maximum length of the convex portion 6 in the direction orthogonal to the bottom surface of the convex portion 6. That is, the height of the convex portion 6 is a dimension from the bottom surface of the convex portion 6 to the apex of the convex portion 6.
　凸部６の底面の径は、１～１０００μｍが好ましく、１０～５０μｍがより好ましい。凸部６の底面の径が１μｍ以上である場合、モールドの微細加工費が安くなり、面積の大きい膜担体４の流路２の表面に無数の微細構造を均一に作製し易く、液体試料を移動させるのに必要な毛細管力が強まる。凸部６の底面の径が１０００μｍ以下である場合、モールドの作製時に金属部材から削りだす金属の体積が小さくなり、モールド及び膜担体４の作製費用が安くなり、膜担体４における流路の面積が小さくなり、膜担体４が小型化して、膜担体４自体の運搬が容易になる。 The diameter of the bottom surface of the convex portion 6 is preferably 1 to 1000 μm, and more preferably 10 to 50 μm. When the diameter of the bottom surface of the convex portion 6 is 1 μm or more, the microfabrication cost of the mold is reduced, and it is easy to uniformly produce an infinite number of fine structures on the surface of the flow path 2 of the membrane carrier 4 having a large area. Capillary force necessary to move is increased. When the diameter of the bottom surface of the convex portion 6 is 1000 μm or less, the volume of the metal scraped from the metal member during the production of the mold is reduced, the production cost of the mold and the membrane carrier 4 is reduced, and the area of the flow path in the membrane carrier 4 , The membrane carrier 4 is downsized, and the membrane carrier 4 itself can be easily transported.
　凸部６の底面の径は、凸部６の底面における代表長さとして定義される。底面における代表長さは、底面の形状が円の場合は直径、三角形又は四角形の場合は最も短い一辺の長さ、五角形以上の多角形の場合は最も長い対角線の長さ、それ以外の形状の場合は底面における最大の長さとする。凸部６が円錐（図８参照）又は円柱である場合、凸部６の底面の径は、凸部６の底面（円）の直径である。 The diameter of the bottom surface of the convex portion 6 is defined as the representative length at the bottom surface of the convex portion 6. The representative length at the bottom is the diameter when the shape of the bottom is a circle, the length of the shortest side when it is a triangle or a quadrangle, the length of the longest diagonal line when it is a pentagon or more polygon, and other shapes In this case, the maximum length at the bottom is used. When the convex part 6 is a cone (refer FIG. 8) or a cylinder, the diameter of the bottom face of the convex part 6 is a diameter of the bottom face (circle) of the convex part 6. FIG.
　電荷付与物質１としては、高分子が好ましい。高分子としては、カチオン性高分子及びアニオン性高分子からなる群より選択される１種以上が好ましい。カチオン性高分子とは水溶液中で正電荷を有する高分子化合物であり、アニオン性高分子とは水溶液中で負電荷を有する高分子化合物である。電荷付与物質として、カチオン性高分子又はアニオン性高分子を流路２に塗布することで、流路２の表面に正又は負の電荷を付与できる。 As the charge imparting substance 1, a polymer is preferable. The polymer is preferably at least one selected from the group consisting of cationic polymers and anionic polymers. The cationic polymer is a polymer compound having a positive charge in an aqueous solution, and the anionic polymer is a polymer compound having a negative charge in an aqueous solution. By applying a cationic polymer or an anionic polymer to the flow channel 2 as a charge imparting substance, a positive or negative charge can be imparted to the surface of the flow channel 2.
　高分子の重量平均分子量は、１００００～５０００００が好ましく、５００００～２０００００がより好ましい。重量平均分子量は、ゲルパーミエーションクロマトグラフィー（ＧＰＣ）法によって測定される標準ポリエチレンオキサイド換算の値である。例えば、重量平均分子量は、溶剤として水を用い、ＧＰＣシステム（東ソ－株式会社製ＳＣ－８０１０）を使用し、市販の標準ポリエチレンオキサイドで検量線を作成して求めることができる。 The weight average molecular weight of the polymer is preferably 10,000 to 500,000, more preferably 50,000 to 200,000. The weight average molecular weight is a value in terms of standard polyethylene oxide measured by a gel permeation chromatography (GPC) method. For example, the weight average molecular weight can be obtained by preparing a calibration curve with commercially available standard polyethylene oxide using GPC system (SC-8010 manufactured by Tosoh Corporation) using water as a solvent.
　カチオン性高分子としては、例えば、ポリ（ジアリルジメチルアンモニウムクロライド）（ＰＤＤＡ）を好ましく用いることができる。本実施形態において用いることのできる他のカチオン性高分子としては、ポリエチレンイミン（ＰＥＩ）、ポリビニルアミン（ＰＶＡｍ）、ポリ（ビニルピロリドン・Ｎ，Ｎ－ジメチルアミノエチルアクリル酸）共重合体等が挙げられる。但し、カチオン性高分子としての一例であり、これに限るものではない。 As the cationic polymer, for example, poly (diallyldimethylammonium chloride) (PDDA) can be preferably used. Other cationic polymers that can be used in the present embodiment include polyethyleneimine (PEI), polyvinylamine (PVAm), poly (vinylpyrrolidone / N, N-dimethylaminoethylacrylic acid) copolymer, and the like. It is done. However, it is an example as a cationic polymer, and is not limited thereto.
　アニオン性高分子としては、例えば、ポリスチレンスルホン酸（ＰＳＳ）を好ましく用いることができる。本実施形態において用いることのできる他のアニオン性高分子としては、ポリビニル硫酸（ＰＶＳ）、ポリアクリル酸（ＰＡＡ）、ポリメタクリル酸（ＰＭＡ）等が挙げられる。但し、アニオン性高分子としての一例であり、これに限るものではない。 As the anionic polymer, for example, polystyrene sulfonic acid (PSS) can be preferably used. Other anionic polymers that can be used in the present embodiment include polyvinyl sulfate (PVS), polyacrylic acid (PAA), polymethacrylic acid (PMA), and the like. However, this is an example of an anionic polymer and is not limited thereto.
　電荷付与物質は、検知ゾーンの少なくとも一部に存在していてよく、検知ゾーンの全体にわたり存在していてよく、検知ゾーンをはみ出して存在していてもよい。 The charge imparting substance may exist in at least a part of the detection zone, may exist throughout the detection zone, or may exist outside the detection zone.
　検出物質は例えば、固相タンパク質であってよい。固相タンパク質としては、例えば、抗体が挙げられる。抗体は、被検出物質と抗原抗体反応する抗体であり、ポリクローナル抗体であってもモノクローナル抗体であってもよい。 The detection substance may be, for example, a solid phase protein. Examples of the solid phase protein include antibodies. The antibody is an antibody that reacts with a substance to be detected by antigen-antibody reaction, and may be a polyclonal antibody or a monoclonal antibody.
　検出物質は、電荷付与物質上に存在していればよく、電荷付与物質が存在する領域の一部に存在していてよく、全体にわたり存在していてもよい。 The detection substance only needs to be present on the charge imparting substance, may be present in a part of the region where the charge imparting substance is present, or may be present throughout.
　検出物質に対する電荷付与物質の質量比（電荷付与物質／検出物質）は、０．０１～１００が好ましく、０．１～１０がより好ましい。電荷付与物質／検出物質は、０．０１以上、０．１以上、０．３以上、０．５又は０．８以上であってよく、１００以下、１０以下、８以下、５以下又は３以下であってよい。電荷付与物質／検出物質が、０．０１以上である場合、検出物質３ｂを強く固着できる。電荷付与物質／検出物質が１００以下である場合、被検出物質との非特異的な相互作用が起こらない。 The mass ratio of the charge imparting substance to the detection substance (charge imparting substance / detection substance) is preferably 0.01 to 100, more preferably 0.1 to 10. The charge-providing substance / detecting substance may be 0.01 or more, 0.1 or more, 0.3 or more, 0.5 or 0.8 or more, and is 100 or less, 10 or less, 8 or less, 5 or less, or 3 or less. It may be. When the charge imparting substance / detection substance is 0.01 or more, the detection substance 3b can be strongly fixed. When the charge imparting substance / detection substance is 100 or less, non-specific interaction with the substance to be detected does not occur.
　検出物質に対する電荷付与物質の質量比（電荷付与物質／検出物質）は、電荷付与物質及び検出物質の両方が存在する領域における質量比として定義される。 The mass ratio of the charge imparting substance to the detection substance (charge imparting substance / detection substance) is defined as the mass ratio in the region where both the charge imparting substance and the detection substance exist.
　検出物質の配向制御について、固相タンパク質を例に挙げて、図６をもとにその仕組みを説明する。図６に示すように、検出物質３を構成する固相タンパク質は、固相タンパク質基部３ａと、固相タンパク質端部３ｂとを有している。電荷付与物質１は、固相タンパク質基部３ａに局在する表面電荷に対し、静電的相互作用を示す。その結果、固相タンパク質基部３ａが電荷付与物質１（流路２）の方向を向き、固相タンパク質端部３ｂが最表面に位置するため、検出物質（固相タンパク質）３の配向制御が可能となる。 The mechanism of the orientation control of the detection substance will be described with reference to FIG. 6, taking a solid phase protein as an example. As shown in FIG. 6, the solid phase protein constituting the detection substance 3 has a solid phase protein base portion 3a and a solid phase protein end portion 3b. The charge-imparting substance 1 exhibits an electrostatic interaction with the surface charge localized in the solid phase protein base 3a. As a result, since the solid phase protein base 3a faces the direction of the charge imparting substance 1 (channel 2) and the solid phase protein end 3b is located on the outermost surface, the orientation of the detection substance (solid phase protein) 3 can be controlled. It becomes.
　検出物質は、検出物質と電荷付与物質との静電的相互作用により生じる電界により配向していてよい。また、一実施形態において、検出物質は、検出物質を保持する基材（流路）表面の電位により配向しているということもできる。 The detection substance may be oriented by an electric field generated by electrostatic interaction between the detection substance and the charge imparting substance. In one embodiment, it can also be said that the detection substance is oriented by the potential of the surface of the substrate (flow path) holding the detection substance.
　液体試料検査キットにおいて、流路上の検知ゾーンは、被検出物質を検出した際に色変化を示す。検知ゾーンにおける色変化は、被検出物質が検出物質により（検出物質と反応して）検知ゾーンに保持されることによって生じる。色変化は、光学的手法で確認可能な色変化であってよい。 In the liquid sample inspection kit, the detection zone on the flow path shows a color change when a substance to be detected is detected. The color change in the detection zone occurs when the detection target substance is held in the detection zone by the detection substance (reacts with the detection substance). The color change may be a color change that can be confirmed by an optical method.
　上記光学的手法としては、主に目視による判定と蛍光強度を測定する手法の２つが挙げられる。目視によって判定する場合には、検知前と検知後の色をＣＩＥ１９７６Ｌ＊ａ＊ｂ＊色空間の表色系で測定した際の、２つの色刺激間の色差（ＪＩＳ　Ｚ８７８１－４：２０１３に記載のΔＥ）が０．５以上となるような色変化が生じることが好ましい。この色差が０．５以上であると、色の違いを目視で確認することが容易になる。蛍光強度を測定して判定する場合には、検知ゾーンでの蛍光強度（Ｆｌ１）と、検知ゾーンに隣接する上流域および下流域での蛍光強度（Ｆｌ２）との比（Ｆｌ１／Ｆｌ２）＝１０／１以上となるような色変化が生じることが好ましい。この比が１０／１以上であると、シグナルとノイズの分離が容易になる。 As the optical method, there are mainly two methods: a visual determination and a method of measuring fluorescence intensity. When judging visually, the color difference between two color stimuli (described in JIS Z8781-4: 2013) when the color before detection and after detection is measured in the color system of CIE1976L * a * b * color space. It is preferable that a color change occurs such that ΔE) is 0.5 or more. When this color difference is 0.5 or more, it becomes easy to visually confirm the color difference. When determining by measuring the fluorescence intensity, the ratio (Fl1 / Fl2) = 10 of the fluorescence intensity (Fl1) in the detection zone and the fluorescence intensity (Fl2) in the upstream region and downstream region adjacent to the detection zone. It is preferable that a color change that is 1 or more occurs. When this ratio is 10/1 or more, separation of signal and noise becomes easy.
　膜担体は、流路の少なくとも表面の一部に電荷付与物質を塗布する工程（第１の塗布工程）と、塗布された電荷付与物質上に検出物質を塗布する工程（第２の塗布工程）と、を備える方法により、製造できる。 The film carrier has a step of applying a charge-imparting substance on at least a part of the surface of the flow path (first application step), and a step of applying a detection substance on the applied charge-imparting material (second application step). It can manufacture by the method provided with these.
　第１の塗布工程では、流路の少なくとも表面の一部に電荷付与物質を塗布する。第１の塗布工程では、検知ゾーンの少なくとも一部に電荷付与物質を塗布してよく、検知ゾーンの全体にわたり電荷付与物質を塗布してよく、検知ゾーンからはみ出して塗布してもよい。 In the first application step, the charge imparting substance is applied to at least a part of the surface of the flow path. In the first application step, the charge imparting substance may be applied to at least a part of the detection zone, the charge imparting substance may be applied over the entire detection zone, or may be applied outside the detection zone.
　電荷付与物質は、電荷付与物質を含有する水溶液を電荷付与物質上に塗布することにより、実施してよい。この場合、第１の塗布工程と第２の塗布工程との間に、電荷付与物質を含有する水溶液中の溶媒を揮発させる工程（第１の乾燥工程）を備える。第１の乾燥工程は、真空乾燥機等を用いて実施できる。 The charge imparting substance may be implemented by applying an aqueous solution containing the charge imparting substance on the charge imparting substance. In this case, a step (first drying step) of volatilizing the solvent in the aqueous solution containing the charge imparting substance is provided between the first coating step and the second coating step. The first drying step can be performed using a vacuum dryer or the like.
　電荷付与物質を含有する水溶液中の電荷付与物質の濃度は、０．０１～２０質量％が好ましく、０．１～５質量％がより好ましい。 The concentration of the charge-providing substance in the aqueous solution containing the charge-providing substance is preferably 0.01 to 20% by mass, and more preferably 0.1 to 5% by mass.
　第２の塗布工程では、塗布された電荷付与物質上に検出物質を塗布する。検出物質は、電荷付与物質上の一部に塗布してよく、電荷付与物質が存在する領域の全体にわたり塗布してもよく、電荷付与物質が存在する領域からはみ出して塗布してもよい。 In the second application step, the detection substance is applied on the applied charge imparting substance. The detection substance may be applied to a part of the charge-providing substance, or may be applied over the entire region where the charge-providing substance exists, or may be applied so as to protrude from the area where the charge-providing substance exists.
　検出物質は、検出物質を含有する溶液（検出物質溶液）を電荷付与物質上に塗布することにより、電荷付与物質上に保持することができる。この場合、第２の塗布工程後に、検出物質溶液中の溶媒を揮発させる工程（第２の乾燥工程）を備える。 The detection substance can be held on the charge imparting substance by applying a solution containing the detection substance (detection substance solution) on the charge imparting substance. In this case, a step of volatilizing the solvent in the detection substance solution (second drying step) is provided after the second application step.
　検出物質溶液は、検出物質を精製水に溶解させた水溶液が好ましい。検出物質を安定に存在させるために、検出物質溶液に対し、他の化合物、例えば、塩化ナトリウム、トリスヒドロキシメチルアミノメタン等を添加してもよい。 The detection substance solution is preferably an aqueous solution in which the detection substance is dissolved in purified water. In order to allow the detection substance to exist stably, other compounds such as sodium chloride and trishydroxymethylaminomethane may be added to the detection substance solution.
　検出物質溶液中の検出物質の濃度は、０．００００１～１ｗ／ｖ％が好ましく、０．０００１～０．１ｗ／ｖ％がより好ましい。 The concentration of the detection substance in the detection substance solution is preferably 0.00001 to 1 w / v%, more preferably 0.0001 to 0.1 w / v%.
　検出物質溶液のｐＨによって、適切な電荷付与物質１は変化しうる。例えば、検出物質溶液のｐＨが７．０より大きい場合、用いる電荷付与物質１はカチオン性高分子のほうが、静電的相互作用を強める点で、好ましい。例えば、検出物質溶液のｐＨが７．０より小さい場合、用いる電荷付与物質１はアニオン性高分子のほうが、静電的相互作用を強める点で、好ましい。検出物質溶液のｐＨが７．０の場合は、カチオン性高分子及びアニオン性高分子のどちらも電荷付与物質１として使用できる。 The appropriate charge imparting substance 1 can vary depending on the pH of the detection substance solution. For example, when the pH of the detection substance solution is higher than 7.0, the charge imparting substance 1 to be used is preferably a cationic polymer in terms of enhancing electrostatic interaction. For example, when the pH of the detection substance solution is less than 7.0, the charge imparting substance 1 to be used is preferably an anionic polymer in terms of enhancing electrostatic interaction. When the pH of the detection substance solution is 7.0, either a cationic polymer or an anionic polymer can be used as the charge imparting substance 1.
　電荷付与物質を塗布する工程の前に、インプリントにより流路に凹凸構造を形成する工程を更に備えていてよい。 Prior to the step of applying the charge-imparting substance, a step of forming an uneven structure in the flow path by imprinting may be further provided.
　検出物質の塗布量に対する電荷付与物質の塗布量の比（電荷付与物質塗布量／検出物質塗布量）は、好ましくは０．０１～１００であり、より好ましくは０．１～１０である。電荷付与物質／検出物質は、０．０１以上、０．１以上、０．３以上、０．５又は０．８以上であってよく、１００以下、１０以下、８以下、５以下又は３以下であってよい。なお、電荷付与物質塗布量／検出物質塗布量は、各物質の質量（固形分）基準で算出された値である。 The ratio of the application amount of the charge-imparting substance to the application amount of the detection substance (charge-application substance application amount / detection substance application amount) is preferably 0.01 to 100, more preferably 0.1 to 10. The charge-providing substance / detecting substance may be 0.01 or more, 0.1 or more, 0.3 or more, 0.5 or 0.8 or more, and is 100 or less, 10 or less, 8 or less, 5 or less, or 3 or less. It may be. Note that the charge application amount / detection material application amount is a value calculated based on the mass (solid content) of each substance.
　本実施形態の膜担体４は、電荷付与物質が存在しない膜担体及び検出物質に対する電荷付与物質の質量比が０．０１～１００の範囲外である膜担体に比べ、目視判定可能な限界倍率が大きいため、高感度な判定を可能とする膜担体と評価できる。 The membrane carrier 4 of the present embodiment has a limit magnification that can be visually determined compared to a membrane carrier in which no charge-providing substance is present and a membrane carrier in which the mass ratio of the charge-providing substance to the detection substance is outside the range of 0.01 to 100. Since it is large, it can be evaluated as a membrane carrier capable of highly sensitive determination.
　本実施形態の膜担体４では、基材（流路２）表面に、カチオン性高分子、アニオン性高分子等の電荷付与物質を塗布することにより、基材表面に電荷を付与し、帯電した検出物質（例えば固相タンパク質）との間に生じる静電的相互作用により、電荷付与物質が検出物質を強く接着させている。この手法を用いることにより、基材の材質又は表面の形状によらず基材に電荷を付与できる。よってＰＯＣＴ試薬の基材に使用した場合にも、本実施形態は、電荷を有する検出物質と強く接着できる。本実施形態は、検出物質に存在する局在電荷を利用することにより、検出物質の配向制御が可能となる。本実施形態は、電荷付与物質により生じた電界を用いて、検出物質の配向制御が可能となる。 In the membrane carrier 4 of the present embodiment, a charge imparting substance such as a cationic polymer or an anionic polymer is applied to the surface of the base material (flow path 2), so that the base material surface is charged and charged. Due to the electrostatic interaction that occurs between the detection substance (for example, solid phase protein), the charge-providing substance strongly adheres the detection substance. By using this method, a charge can be imparted to the substrate regardless of the material of the substrate or the shape of the surface. Therefore, even when used as a base material for a POCT reagent, this embodiment can strongly adhere to a detection substance having a charge. In this embodiment, the orientation of the detection substance can be controlled by using the localized charge existing in the detection substance. In the present embodiment, the orientation of the detection substance can be controlled using the electric field generated by the charge imparting substance.
　以下、本実施形態を具体的に説明するが、本実施形態はこれらの実験例に限定されるものではない。 Hereinafter, the present embodiment will be described in detail, but the present embodiment is not limited to these experimental examples.
［実施例１］
＜電荷付与物質の塗布＞
　流路の材料（流路材料）として市販のニトロセルロースメンブレン（Ｍｉｌｌｉｐｏｒｅ社製）を用いた。膜担体の下端から１．０ｃｍの位置に、電荷付与物質として２０質量％のポリ（ジアリルジメチルアンモニウムクロライド）（ＰＤＤＡ、Ａｌｄｒｉｃｈ社製、Ｍｗ＝１０００００～２０００００）を精製水で２０倍に希釈し得られた水溶液を塗布し、真空乾燥機内でよく乾燥させた。なお、電荷付与物質塗布量は、ＰＤＤＡを含む水溶液の質量から水の質量を除いた質量（固形分）である。 [Example 1]
<Application of charge imparting substance>
A commercially available nitrocellulose membrane (manufactured by Millipore) was used as the material for the channel (channel material). At a position 1.0 cm from the lower end of the membrane carrier, 20% by mass of poly (diallyldimethylammonium chloride) (PDDA, manufactured by Aldrich, Mw = 100000-200000) as a charge-imparting substance can be diluted 20 times with purified water. The obtained aqueous solution was applied and dried well in a vacuum dryer. The charge application amount is the mass (solid content) obtained by removing the mass of water from the mass of the aqueous solution containing PDDA.
＜固相タンパク質（検出物質）の固定＞
　ニトロセルロースメンブレンにおいてＰＤＤＡを塗布した領域に、固相タンパク質として抗Ａ型インフルエンザウイルスＮＰ抗体を水に懸濁させた抗Ａ型インフルエンザウイルスＮＰ抗体浮遊液（抗Ａ型インフルエンザウイルスＮＰ抗体の濃度は０．００３３ｗ／ｖ％）を、ＰＤＤＡの塗布面積と同じ面積となるように塗布し、温風下でよく乾燥させた。なお、固相タンパク質塗布量は、抗Ａ型インフルエンザウイルスＮＰ抗体浮遊液の質量から、水の質量を除いた質量（固形分）である。 <Immobilization of solid phase protein (detection substance)>
Anti-influenza A virus NP antibody suspension in which anti-influenza A virus NP antibody is suspended in water as a solid phase protein in the area where PDDA is applied on the nitrocellulose membrane (the concentration of anti-influenza A virus NP antibody is 0 .0033 w / v%) was applied so as to have the same area as that of PDDA, and dried well under warm air. The solid phase protein coating amount is a mass (solid content) obtained by subtracting the mass of water from the mass of the anti-influenza A virus NP antibody suspension.
　実施例１では、固相タンパク質に対する電荷付与物質の質量比（電荷付与物質塗布量／固相タンパク質塗布量）（固形分換算比）は１とした。 In Example 1, the mass ratio of the charge-imparting substance to the solid phase protein (charge-giving substance coating amount / solid-phase protein coating amount) (solid content conversion ratio) was 1.
＜標識物質のセット＞
　精製抗Ａ型インフルエンザウイルスＮＰ抗体（上記と別の抗体）を使用した。精製抗Ａ型インフルエンザウイルスＮＰ抗体に粒子径０．２μｍの赤色ラテックス粒子（ＳＣ－０４２－Ｒ　ＪＳＲライフサイエンス社製）を共有結合で標識し、糖、界面活性剤及びタンパク質を含むトリス緩衝液にラテックス粒子の濃度が０．０２５ｗ／ｖ％になるように懸濁し、ソニケーションを行って充分に分散浮遊させた抗Ａ型標識体を調製した。 <Set of labeling substances>
A purified anti-influenza A virus NP antibody (an antibody different from the above) was used. The purified anti-influenza A virus NP antibody is labeled with 0.2 μm red latex particles (SC-042-R manufactured by JSR Life Sciences) with a covalent bond, and added to a Tris buffer containing sugar, surfactant and protein. An anti-A-type labeling body was prepared by suspending it so that the concentration of latex particles was 0.025 w / v%, and performing sonication to sufficiently disperse and float.
　抗Ａ型標識体を大きさが３ｃｍ×１ｃｍのガラス繊維（３３ＧＬＡＳＳ　ＮＯ．１０５３９７６６　Ｓｃｈｌｅｉｃｈｅｒ＆Ｓｃｈｕｅｌｌ製）に１平方センチメートルあたり５０μＬになる量を塗布し、温風下で良く乾燥させ、標識体パッドを作製した。その後作製した膜担体の端部２ｍｍだけを標識物質パッドを重ね、カッターで幅５ｍｍの短冊に裁断して一体化された液体試料検査キットを作製した。 An amount of 50 μL per square centimeter was applied to a 3 cm × 1 cm glass fiber (33GLASS NO.10539766, manufactured by Schleicher & Schuell), and the anti-A type labeled body was dried well under warm air to prepare a labeled body pad. After that, a labeling material pad was overlapped only on the end portion 2 mm of the prepared membrane carrier, and the integrated liquid sample inspection kit was prepared by cutting into a strip having a width of 5 mm with a cutter.
　＜検知評価＞
　上記のように作製された液体試料検査キットの端部に、液体試料を１００μＬ滴下した。液体試料は、希釈溶液としてデンカ生研社製クイックナビ―Ｆｌｕに付属している検体浮遊液を用いた。Ａ型インフルエンザウイルス　Ａ／Ｂｅｉｊｉｎｇ／３２／９２（Ｈ３Ｎ２）の希釈倍率を２×１０４から大きくしていった際に、試験開始後１０分後に着色ラインの有無を目視できなくなる希釈倍率（目視判定可能な限界倍率）を求めた。以降、流路をメンブレンとした場合の結果を表１に示す。
　＜総合評価＞
　目視判定可能な限界倍率から判定した。極めて高感度な場合をＡ、高感度な場合をＢ、高感度でない場合をＣとした。 <Detection evaluation>
100 μL of the liquid sample was dropped on the end of the liquid sample inspection kit produced as described above. As a liquid sample, a specimen suspension attached to Quick Navi-Flu manufactured by Denka Seken Co., Ltd. was used as a diluted solution. When the dilution factor of influenza A virus A / Beijing / 32/92 (H3N2) is increased from 2 × 10 4, the dilution factor (visual determination) that makes it impossible to visually check the presence of a colored line 10 minutes after the start of the test Possible limit magnification). Hereinafter, Table 1 shows the results when the flow path is a membrane.
<Comprehensive evaluation>
It judged from the limit magnification which can be visually judged. The case of extremely high sensitivity was A, the case of high sensitivity was B, and the case of not high sensitivity was C.
［実施例２］
　実施例１における固相タンパク質に対する電荷付与物質の質量比（電荷付与物質塗布量／固相タンパク質塗布量）を１００としたこと以外は、実施例１と同様の条件で実験を行った。 [Example 2]
The experiment was performed under the same conditions as in Example 1 except that the mass ratio of the charge-imparting substance to the solid-phase protein in Example 1 (charge-imparting substance application amount / solid-phase protein application amount) was 100.
［実施例３］
　実施例１における固相タンパク質に対する電荷付与物質の質量比（電荷付与物質塗布量／固相タンパク質塗布量）を０．０１としたこと以外は、実施例１と同様の条件で実験を行った。 [Example 3]
The experiment was performed under the same conditions as in Example 1 except that the mass ratio of the charge-imparting substance to the solid-phase protein in Example 1 (charged substance application amount / solid-phase protein application amount) was 0.01.
［実施例４］
　実施例１における電荷付与物質を２０質量％のポリスチレンスルホン酸（ＰＳＳ、Ａｌｄｒｉｃｈ社製、Ｍｗ＝７００００）としたこと以外は、実施例１と同様の条件で実験を行った。 [Example 4]
The experiment was performed under the same conditions as in Example 1 except that the charge-providing substance in Example 1 was 20% by mass of polystyrene sulfonic acid (PSS, manufactured by Aldrich, Mw = 70000).
［実施例５］
　実施例１における電荷付与物質をＰＳＳとし、固相タンパク質に対する電荷付与物質の質量比（電荷付与物質塗布量／固相タンパク質塗布量）を１００としたこと以外は、実施例１と同様の条件で実験を行った。 [Example 5]
The same conditions as in Example 1 except that the charge-providing substance in Example 1 is PSS and the mass ratio of the charge-providing substance to the solid-phase protein is 100 (charge-providing substance coating amount / solid-phase protein coating amount). The experiment was conducted.
［実施例６］
　実施例１における電荷付与物質をＰＳＳとし、固相タンパク質に対する電荷付与物質の質量比（電荷付与物質塗布量／固相タンパク質塗布量）を０．０１としたこと以外は、実施例１と同様の条件で実験を行った。 [Example 6]
The charge imparting substance in Example 1 is PSS, and the mass ratio of the charge imparting substance to the solid phase protein (charge imparting substance coating amount / solid phase protein coating amount) is 0.01. The experiment was conducted under conditions.
［比較例１］
　実施例１における電荷付与物質の塗布を行うことなく、固相タンパク質に対する電荷付与物質の質量比（電荷付与物質塗布量／固相タンパク質塗布量）を０としたこと以外は、実施例１と同様の条件で実験を行った。 [Comparative Example 1]
The same as Example 1 except that the mass ratio of the charge-imparting substance to the solid phase protein (charge-giving substance application amount / solid-phase protein application amount) was set to 0 without applying the charge-giving substance in Example 1. The experiment was conducted under the following conditions.
［比較例２］
　実施例１における電荷付与物質を固相タンパク質に対する電荷付与物質の質量比（電荷付与物質塗布量／固相タンパク質塗布量）を１０００としたこと以外は、実施例１と同様の条件で実験を行った。 [Comparative Example 2]
The experiment was performed under the same conditions as in Example 1 except that the charge-providing substance in Example 1 was set to a mass ratio of the charge-providing substance to the solid phase protein (charged substance coating amount / solid phase protein coating amount) of 1000. It was.
［比較例３］
　実施例１における電荷付与物質をＰＳＳとし、固相タンパク質に対する電荷付与物質の質量比（電荷付与物質塗布量／固相タンパク質塗布量）を０．００１としたこと以外は、実施例１と同様の条件で実験を行った。 [Comparative Example 3]
The charge imparting substance in Example 1 is PSS, and the mass ratio of the charge imparting substance with respect to the solid phase protein (charge imparting substance coating amount / solid phase protein coating amount) is 0.001. The experiment was conducted under conditions.
［実施例７］
＜モールドの準備＞
　モールドは、レーザー加工及び機械切削によって作製した。このモールドはアルミ合金Ａ５０５２製である。この金型の中心部には、径が２５μｍ、ピッチが３０μｍ、深さが３０μｍの円錐形状の凹部が、図７のような三角配列形式で３ｃｍ×３ｃｍの範囲に加工されている。
　上記のモールドの凹凸面に対し、転写した際のモールドと熱可塑性樹脂の剥離を容易かつ確実にするため、離型処理を施した。離型処理の手法は、ダイキン工業社製オプツールＨＤ－２１００ＴＨに約１分浸し、乾燥させたのち、一晩静置することで行った。 [Example 7]
<Mold preparation>
The mold was produced by laser processing and mechanical cutting. This mold is made of aluminum alloy A5052. A conical recess having a diameter of 25 μm, a pitch of 30 μm, and a depth of 30 μm is machined in the center of the mold in a triangular array form as shown in FIG. 7 in a range of 3 cm × 3 cm.
A release treatment was performed on the uneven surface of the mold in order to easily and reliably peel off the mold and the thermoplastic resin when transferred. The mold release treatment was performed by immersing in OPTOOL HD-2100TH manufactured by Daikin Industries, Ltd. for about 1 minute, drying, and allowing to stand overnight.
＜微細構造の転写＞
　上記のようにして得られたモールドを用いて、熱可塑性樹脂に微細構造を転写した。熱可塑性樹脂としては、ポリスチレン（ＰＳ、デンカ社製デンカスチレンシート、膜厚３００μｍ）を用いた。加工方法として熱インプリントを用い、装置はＳＣＩＶＡＸ社製Ｘ－３００を用いた。成形温度は１２０℃、印加圧力は５．５ＭＰａとし、１０分間転写を行った。転写後は、圧力を印加したまま熱可塑性プラスチックとモールドを８０℃まで冷却し、その後圧力を除くことで、表面に円錐形状の凸部を有する流路を作製した。流路２には、ピッチが３０μｍ、径が２５μｍ、高さが３０μｍの円錐形状の凸部６が、図８のような三角配列形式で３ｃｍ×３ｃｍの範囲に加工されている。 <Transfer of fine structure>
The microstructure was transferred to a thermoplastic resin using the mold obtained as described above. As the thermoplastic resin, polystyrene (PS, Denka styrene sheet manufactured by Denka Co., Ltd., film thickness: 300 μm) was used. Thermal imprinting was used as a processing method, and an X-300 manufactured by SCIVAX was used as the apparatus. The molding temperature was 120 ° C., the applied pressure was 5.5 MPa, and transfer was performed for 10 minutes. After the transfer, the thermoplastic plastic and the mold were cooled to 80 ° C. while applying pressure, and then the pressure was removed to produce a flow path having a conical convex portion on the surface. In the flow path 2, conical convex portions 6 having a pitch of 30 μm, a diameter of 25 μm, and a height of 30 μm are processed into a 3 cm × 3 cm range in a triangular arrangement form as shown in FIG.
　その後の操作は、実施例１における流路をポリスチレンとしたこと以外は、実施例１と同様の条件で実験を行った。以降、流路をポリスチレンとした場合の結果を表２に示す。 The subsequent operation was conducted under the same conditions as in Example 1 except that the flow path in Example 1 was made of polystyrene. Hereinafter, Table 2 shows the results when the flow path is made of polystyrene.
［実施例８］
　実施例７における固相タンパク質に対する電荷付与物質の質量比（電荷付与物質塗布量／固相タンパク質塗布量）を１００としたこと以外は、実施例７と同様の条件で実験を行った。 [Example 8]
The experiment was performed under the same conditions as in Example 7, except that the mass ratio of the charge-imparting substance to the solid-phase protein in Example 7 (charge-imparting substance coating amount / solid-phase protein coating amount) was 100.
［実施例９］
　実施例７における固相タンパク質に対する電荷付与物質の質量比（電荷付与物質塗布量／固相タンパク質塗布量）を０．０１としたこと以外は、実施例７と同様の条件で実験を行った。 [Example 9]
The experiment was performed under the same conditions as in Example 7 except that the mass ratio of the charge-imparting substance to the solid-phase protein in Example 7 (charged substance application amount / solid-phase protein application amount) was 0.01.
［実施例１０］
　実施例７における電荷付与物質をＰＳＳとしたこと以外は、実施例７と同様の条件で実験を行った。 [Example 10]
The experiment was performed under the same conditions as in Example 7 except that PSS was used as the charge-providing substance in Example 7.
［実施例１１］
　実施例７における電荷付与物質をＰＳＳとし、固相タンパク質に対する電荷付与物質の質量比（電荷付与物質塗布量／固相タンパク質塗布量）を１００としたこと以外は、実施例７と同様の条件で実験を行った。 [Example 11]
The same conditions as in Example 7 except that the charge-providing substance in Example 7 was PSS and the mass ratio of the charge-providing substance to the solid phase protein (charge-providing substance coating amount / solid-phase protein coating amount) was 100. The experiment was conducted.
［実施例１２］
　実施例７における電荷付与物質をＰＳＳとし、固相タンパク質に対する電荷付与物質の質量比（電荷付与物質塗布量／固相タンパク質塗布量）を０．０１としたこと以外は、実施例７と同様の条件で実験を行った。 [Example 12]
Example 7 is the same as Example 7 except that the charge-providing substance in Example 7 is PSS, and the mass ratio of the charge-providing substance to the solid-phase protein (charge-providing substance coating amount / solid-phase protein coating amount) is 0.01. The experiment was conducted under conditions.
［比較例４］
　実施例７における電荷付与物質の塗布を行うことなく、固相タンパク質に対する電荷付与物質の質量比（電荷付与物質塗布量／固相タンパク質塗布量）を０としたこと以外は、実施例７と同様の条件で実験を行った。 [Comparative Example 4]
Example 7 is the same as Example 7 except that the mass ratio of the charge-providing substance to the solid phase protein (charge-applying substance application amount / solid-phase protein application quantity) was set to 0 without applying the charge-providing substance in Example 7. The experiment was conducted under the following conditions.
［比較例５］
　実施例７における固相タンパク質に対する電荷付与物質の質量比（電荷付与物質塗布量／固相タンパク質塗布量）を０．０００１としたこと以外は、実施例７と同様の条件で実験を行った。 [Comparative Example 5]
The experiment was performed under the same conditions as in Example 7, except that the mass ratio of the charge-imparting substance to the solid-phase protein in Example 7 (charge-imparting substance coating amount / solid-phase protein coating amount) was 0.0001.
［比較例６］
　実施例７における電荷付与物質をＰＳＳとし、固相タンパク質に対する電荷付与物質の質量比（電荷付与物質塗布量／固相タンパク質塗布量）を１００００としたこと以外は、実施例７と同様の条件で実験を行った。 [Comparative Example 6]
Under the same conditions as in Example 7, except that the charge-providing substance in Example 7 was PSS and the mass ratio of the charge-providing substance to the solid phase protein (charged substance coating amount / solid phase protein coating amount) was 10,000. The experiment was conducted.
　表１及び表２より以下が認められた。電荷付与物質を塗布し、かつ、固相タンパク質に対する電荷付与物質の質量比（電荷付与物質塗布量／固相タンパク質塗布量）が０．０１～１００である場合、目視判定可能な限界倍率が増加していることが認められた。 From Table 1 and Table 2, the following was recognized. When the charge-imparting substance is applied and the mass ratio of the charge-imparting substance to the solid phase protein (the amount of the charge-imparting substance / the amount of the solid-phase protein applied) is 0.01 to 100, the limit magnification that can be visually determined increases. It was recognized that
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　本実施形態により、検出物質の配向が制御された、高感度な液体試料検査キットが得られる。本実施形態の液体試料検査キットは、例えば、本実施形態を用いていない液体試料検査キットに比べて試験時の判定スコアが１．５倍になり、高感度化を達成できる。本実施形態の液体試料検査キットを用いることにより、被検出物質の量が少ない罹患初期における検査精度の向上が見込まれる。 According to this embodiment, a highly sensitive liquid sample inspection kit in which the orientation of the detection substance is controlled can be obtained. The liquid sample test kit of this embodiment has a test score 1.5 times higher than that of a liquid sample test kit that does not use this embodiment, and can achieve high sensitivity. By using the liquid sample test kit of the present embodiment, it is expected that the test accuracy in the early stage of the disease with a small amount of the substance to be detected will be improved.
１　　電荷付与物質
２　　流路
３　　検出物質
３ａ　　固相タンパク質基部
３ｂ　　固相タンパク質端部
４，４Ａ，４Ｂ，４Ｃ　　膜担体
４ｘ　　滴下ゾーン
４ｙ　　検知ゾーン
５　　凹部
６　　凸部
７　　金型（モールド）
１０　　液体試料検査キット
１０ａ　　　筐体
１０ｂ　　第一開口部
１０ｃ　　第二開口部
１１　　ピッチ DESCRIPTION OF SYMBOLS 1 Charge provision substance 2 Flow path 3 Detection substance 3a Solid phase protein base 3b Solid phase protein edge part 4,4A, 4B, 4C Membrane carrier 4x Dropping zone 4y Detection zone 5 Recess 6 Protrusion 7 Mold (mold)
10 Liquid Sample Inspection Kit 10a Case 10b First Opening 10c Second Opening 11 Pitch

Claims (10)

  1. 　流路と電荷付与物質と検出物質とを備え、
    　前記検出物質は前記電荷付与物質を介して前記流路に保持され、
    　前記検出物質に対する前記電荷付与物質の質量比（電荷付与物質／検出物質）が、０．０１～１００である、膜担体。     A flow path, a charge-providing substance, and a detection substance;
    The detection substance is held in the flow path via the charge imparting substance,
    A membrane carrier having a mass ratio of the charge-providing substance to the detection substance (charge-providing substance / detection substance) of 0.01 to 100.
  2. 　前記検出物質が、前記検出物質と前記電荷付与物質との静電的相互作用により生じる電界によって配向している、請求項１に記載の膜担体。 The film carrier according to claim 1, wherein the detection substance is oriented by an electric field generated by an electrostatic interaction between the detection substance and the charge imparting substance.
  3. 　前記電荷付与物質が、カチオン性高分子及びアニオン性高分子からなる群より選択される１種以上である、請求項１又は２に記載の膜担体。 The membrane carrier according to claim 1 or 2, wherein the charge-providing substance is at least one selected from the group consisting of a cationic polymer and an anionic polymer.
  4. 　前記電荷付与物質が、ポリ（ジアリルジメチルアンモニウムクロライド）である、請求項１～３のいずれか一項に記載の膜担体。 The membrane carrier according to any one of claims 1 to 3, wherein the charge-imparting substance is poly (diallyldimethylammonium chloride).
  5. 　前記電荷付与物質が、ポリスチレンスルホン酸である、請求項１～４のいずれか一項に記載の膜担体。 The membrane carrier according to any one of claims 1 to 4, wherein the charge-imparting substance is polystyrene sulfonic acid.
  6. 　前記流路が、樹脂で形成されている、請求項１～５のいずれか一項に記載の膜担体。 The membrane carrier according to any one of claims 1 to 5, wherein the flow path is formed of a resin.
  7. 　前記流路が、凹凸構造を有する、請求項１～６のいずれか一項に記載の膜担体。 The membrane carrier according to any one of claims 1 to 6, wherein the flow path has an uneven structure.
  8. 　請求項１～７のいずれか一項に記載の膜担体を有する、液体試料検査キット。 A liquid sample inspection kit comprising the membrane carrier according to any one of claims 1 to 7.
  9. 　流路上に電荷付与物質を塗布する工程と、
    　塗布された前記電荷付与物質上に検出物質を塗布する工程と、を備え、
    　前記検出物質の塗布量に対する前記電荷付与物質の塗布量（電荷付与物質塗布量／検出物質塗布量）が０．０１～１００である、膜担体の製造方法。     Applying a charge imparting substance on the flow path;
    Applying a detection substance on the applied charge-imparting substance, and
    The method for producing a membrane carrier, wherein the charge application amount (charge application amount / detection material application amount) is 0.01 to 100 with respect to the detection material application amount.
  10. 　前記電荷付与物質を塗布する工程の前に、インプリントにより前記流路に凹凸構造を形成する工程を更に備える、請求項９に記載の膜担体の製造方法。 The method for manufacturing a film carrier according to claim 9, further comprising a step of forming an uneven structure in the flow path by imprinting before the step of applying the charge-imparting substance.
PCT/JP2018/016815 2017-04-25 2018-04-25 Membrane carrier, method for producing same, and liquid sample test kit WO2018199168A1 (en)

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