WO2020241871A1 - Substance detecting device - Google Patents

Substance detecting device Download PDF

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Publication number
WO2020241871A1
WO2020241871A1 PCT/JP2020/021458 JP2020021458W WO2020241871A1 WO 2020241871 A1 WO2020241871 A1 WO 2020241871A1 JP 2020021458 W JP2020021458 W JP 2020021458W WO 2020241871 A1 WO2020241871 A1 WO 2020241871A1
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WO
WIPO (PCT)
Prior art keywords
substance
sample
flow path
labeled
antibody
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Application number
PCT/JP2020/021458
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French (fr)
Japanese (ja)
Inventor
晃彦 石田
学 渡慶次
Original Assignee
国立大学法人北海道大学
日本ユニシス株式会社
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Application filed by 国立大学法人北海道大学, 日本ユニシス株式会社 filed Critical 国立大学法人北海道大学
Priority to JP2021521902A priority Critical patent/JP7350268B2/en
Publication of WO2020241871A1 publication Critical patent/WO2020241871A1/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 substance detection device using a sheet.
  • an antigen (labeled antigen) derived from the sample whose concentration is to be detected is added to the antigen (labeled antigen) not derived from the sample labeled with the enzyme, and an antibody is produced between the two.
  • an antibody is produced between the two.
  • the unreacted target antigen and the labeled antigen are removed from the reaction vessel by washing in order to measure the amount of the labeled antigen bound to the antibody by utilizing the color development by the enzymatic reaction.
  • the antibody In order to prevent the reacted labeled antigen from being washed out during this washing process, the antibody needs to be fixed to the bottom of the reaction vessel from the beginning. As described above, in the conventional competitive method, it is generally essential that the unreacted labeled antigen and the labeled antigen bound to the antibody are clearly separated.
  • a polystyrene microtiter plate As a conventional reaction vessel, a polystyrene microtiter plate is generally used.
  • Non-Patent Documents 1 to 5 which are a part of them, the antibody is immobilized on chitosan applied to paper fibers by using a cross-linking reagent (glutaraldehyde).
  • the antibody immobilization steps include application of chitosan, drying, application of glutaraldehyde, reaction of chitosan and glutaraldehyde, washing of unreacted glutaraldehyde, drying, application of antibody, reaction of antibody and glutaraldehyde, washing of unreacted antibody. It consists of removal and the like.
  • Non-Patent Documents 1 to 5 many operations and a long time are required for the antibody fixing step as described above. Further, since it is difficult to remove the cleaning liquid performed in the immobilization step by suction or shaking as in the case of a conventional polystyrene reaction vessel, it is possible to remove the cleaning liquid by sucking it on paper such as an experimental wiper. Do. As described above, in the sheet-shaped substance detection device, the complicated manufacturing process has been an issue for commercialization.
  • An object of the present invention is to provide a substance detection device that is easy to manufacture even when a sheet is used.
  • the substance detection device of the present invention is a substance detection device that detects a substance to be detected in a sample based on the intensity of color development generated due to the action of an enzyme and a substrate, and is a hydrophilic region and the hydrophilic region.
  • a sheet having a hydrophobic region defining an outer edge is provided, and a sample supply unit to which a sample solution containing the sample is supplied, a coloring unit containing the substrate, and the sample supply to the hydrophilic region.
  • An intermediate flow path connecting the portion to the color-developing portion is formed, and either the capturing substance, which is a substance that binds to the substance to be detected, or the substance to be detected, which is not derived from the sample, is labeled by the enzyme.
  • the first labeling substance which is a composite of the above
  • the second labeling substance which is a complex of one of the trapping substance and the detected substance labeled with the enzyme and the other, are the first.
  • Both or the former of the first labeling substance and the trapping substance are present so that the concentration of the labeling substance exists in the intermediate flow path while increasing in magnitude according to the concentration of the detected substance in the sample. It is contained in at least one of the sample liquid, the sample supply unit, and the intermediate flow path without being fixed, and the intermediate flow path determines the speed at which the first labeling substance moves in the intermediate flow path.
  • the second labeling substance contains a rate adjusting substance that increases the rate of movement in the intermediate flow path.
  • the first labeling substance and the second labeling substance are labeled with an enzyme, and the coloring part contains a substrate. Therefore, when these labeling substances reach the coloring part, the coloring part develops color according to the action of the enzyme and the substrate in the labeling substance.
  • the color development in the color-developing portion has an intensity corresponding to the concentration of these labeling substances that have reached the color-developing portion.
  • the concentration of the first labeling substance is evaluated by evaluating the intensity of color development generated in the coloring portion from the time when the first labeling substance reaches the coloring portion to the time when the second labeling substance reaches the coloring portion. Can be done.
  • the concentration of the first labeling substance in the intermediate flow path has a magnitude corresponding to the concentration of the substance to be detected in the sample. Therefore, the concentration of the substance to be detected in the sample can be evaluated by evaluating the color development intensity of the color-developing portion.
  • the trapping substance for binding to the substance to be detected is not fixed to the sheet. Therefore, the substance detection device of the present invention does not require a trapping substance immobilization step in the manufacturing process. Therefore, it is easier to manufacture than the conventional technique that requires such an immobilization step.
  • the "contained without being fixed” means a chemical fixing means (for example, chitosan and glutar) for fixing the substance to the sheet so that the substance does not move on the sheet when the device is used. It is shown that the conventional means for fixing the antibody (capturing substance) to the sheet using aldehyde) is not used. Further, the "substance to be detected” in the present invention may correspond to any of those derived from a sample and those not derived from a sample, unless otherwise specified. Further, in the embodiment in which both the first labeling substance and the trapping substance are contained in at least one of the sample solution, the sample supply unit and the intermediate flow path, the portion contained by the first labeling substance and the trapping substance is contained. It may be different or the same.
  • the combination of the substance to be detected and the capture substance may be, for example, a combination of an antigen and an antibody, a combination of an aptamer and its target molecule, a combination of an enzyme and a substrate, or a combination of an enzyme and its inhibitor. There may be.
  • the speed adjusting substance is a surfactant. According to this, as shown in Examples described later, it is possible to appropriately make a difference in the speed of movement in the intermediate flow path between the first labeled substance and the second labeled substance.
  • the sample solution is adjusted to an amount in which the first labeling substance reaches the coloring portion and the second labeling substance does not reach the coloring portion. According to this, since the second labeling substance does not reach the color-developing portion, the concentration of the substance to be detected in the sample can be appropriately evaluated based on the intensity at which the first labeling substance develops the color of the coloring portion.
  • the present invention it is preferable to have a laminated body in which a plurality of sheets having the hydrophilic region and the hydrophobic region are laminated. According to this, by laminating a plurality of sheets, excessive drying is suppressed in the hydrophilic region.
  • the coloring portion is formed on a sheet different from the intermediate flow path in the plurality of sheets. Assuming that both the intermediate flow path and the coloring part are formed on one sheet, the permeation of the sample liquid from the intermediate flow path to the coloring part is caused only by the permeation of the sample liquid in the direction along one sheet. Go ahead. As a result, color development in the color-developing portion progresses. Therefore, uneven color development in the direction along the sheet is likely to occur. On the other hand, if the color-developing portion is formed on a sheet different from the intermediate flow path, the color development progresses by the permeation of the sample liquid so as to straddle the sheets from the intermediate flow path to the color-developing portion. become. Therefore, uneven color development in the direction along the sheet is unlikely to occur. Therefore, the concentration of the substance to be detected in the sample can be appropriately evaluated based on the color development intensity of the color-developing portion.
  • the first labeling substance is a complex in which the substance to be detected, which is not derived from the sample, is labeled by the enzyme, and is contained in the sample solution. It is contained in at least one of the sample supply unit and the intermediate flow path, and the second labeling substance may be produced by the reaction between the first labeling substance and the trapping substance. According to this, the substance to be detected in the sample and the first labeling substance react competitively with the capturing substance. Therefore, the first labeling substance is appropriately present in the intermediate flow path at a concentration corresponding to the concentration of the substance to be detected in the sample.
  • the first labeling substance is a complex in which the trapping substance is labeled by the enzyme and is contained in the sample solution, and the second labeling substance is the first labeling. It may be produced by the reaction of the substance with the substance to be detected in the sample. According to this, the second labeling substance is generated according to the concentration of the substance to be detected in the sample. Therefore, the first labeled substance that is not bound to the substance to be detected is appropriately present in the intermediate flow path at a concentration corresponding to the concentration of the substance to be detected in the sample.
  • FIG. 2 is a sectional view taken along line II-II of the paper device of FIG. It is a flow chart which shows the usage method of the paper device of FIG. It is a graph which shows the result which concerns on one Example of the paper device of FIG. 1 which used progesterone as a target antigen.
  • the paper device 1 is a device that detects the concentration of an antigen (hereinafter referred to as a target antigen) in a sample.
  • a target antigen an antigen contained in a sample such as animal blood.
  • the target antigen may be any substance as long as it can be an antigen in the antigen-antibody reaction.
  • biotin, cortisol, estrogen, testosterone, prolactin, ANP (atrial natriuretic peptide), BNP (brain natriuretic peptide), aflatoxin, okratoxin, patulin, deoxynivalenol, nivalenol, etc. are targeted.
  • ANP atrial natriuretic peptide
  • BNP brain natriuretic peptide
  • aflatoxin okratoxin
  • patulin deoxynivalenol
  • nivalenol etc.
  • the aptamer may be either a nucleic acid aptamer or a peptide aptamer.
  • Target molecules of aptamers may include, for example, metal ions, heterocyclic molecules, amino acids, nucleosides, nucleotides, sugars, peptides, proteins, protein complexes such as ribosomes, viruses, bacteria, cancer cells and the like.
  • metal ions such as metal ions, heterocyclic molecules, amino acids, nucleosides, nucleotides, sugars, peptides, proteins, protein complexes such as ribosomes, viruses, bacteria, cancer cells and the like.
  • the color development generated by the involvement of at least two substances is utilized.
  • One of the two substances is an enzyme, and as described later, it is used in a state of being contained in a sample solution as a complex bound to an antigen.
  • the other of the two substances is the color-developing substrate 1 (chromogen), which is partially contained in the paper device 1 as described later.
  • the color-developing substrate 2 may be required for color development.
  • the color-developing substrate 2 is used, for example, in a state of being contained in the sample solution, as described later.
  • coloring substrates 1 and 2 the substances listed in Table 1 may be used.
  • any one of horseradish peroxidase (HRP), ⁇ -galactosidase ( ⁇ -GAL), etc. can be used as an enzyme candidate, and a plurality of color-developing substrates 1 are used for each enzyme candidate.
  • HRP horseradish peroxidase
  • ⁇ -GAL ⁇ -galactosidase
  • TMB 5,5'-tetramethylbenzidine
  • the color-developing substrate 2 is determined according to the combination of the enzyme and the color-developing substrate 1. For example, when HRP and 3,3'-diaminobenzidine (DAB) are used as the enzyme and coloring substrate 1, H 2 O 2 is used as the coloring substrate 2.
  • the paper device 1 includes a laminated body 2 in which sheets 10, 20 and 30 made of a water-absorbent sheet such as filter paper are laminated.
  • the direction in which the sheets are laminated is referred to as a stacking direction.
  • the laminated body 2 may be configured by laminating two sheets separated from each other, or may be configured by folding one sheet in two. In the latter case, for example, the sheets 10 to 30 are connected to each other in a row to form one sheet. Then, this one sheet is folded with the boundary lines of the sheets 10, 20 and 30 as a polygonal line to form a laminated body 2 in which the sheets 10 to 30 are laminated.
  • the laminated body 2 includes a hatching region 2a composed of diagonal lines along the direction from the upper left to the lower right, and a hatching region 2b consisting of diagonal lines along the direction from the upper right to the lower left. I'm out.
  • the region 2b is a hydrophobic region impregnated with the hydrophobic ink.
  • the region 2b is referred to as a hydrophobic region 2b.
  • the region 2a is not impregnated with the ink. Therefore, the region 2a is a region having hydrophilicity.
  • the region 2a is referred to as a hydrophilic region 2a.
  • each of the sheets 10 and 20 contains both a hydrophilic region 2a and a hydrophobic region 2b.
  • the entire sheet 30 is formed by the hydrophobic region 2b.
  • the pattern of the hydrophobic region 2b is formed by printing on a sheet using, for example, a hydrophobic solid ink and an inkjet printer for the solid ink. After printing the pattern, the sheet is heated using a dryer to melt the ink on the sheet and allow it to penetrate into the sheet. As a result, a hydrophobic region 2b impregnated with the hydrophobic ink is formed from one surface of the sheet to the other surface.
  • the hydrophobic region 2b is a state in which a dried product of hydrophobic ink is present in the sheet.
  • screen printing, photolithography, or the like may be used to form the pattern on the sheet.
  • the laminated body 2 has a flow path 40 formed by the hydrophilic region 2a.
  • the flow path 40 is formed by communicating the hydrophilic regions 2a of the sheets 10 and 20 with each other.
  • the sheet 10 is formed with introduction holes 11 and color-developing portions 12 arranged in a row along the A direction.
  • the introduction hole 11 is arranged at a position near the right end of the sheet 10 in FIG.
  • the introduction hole 11 has a disk shape as shown in FIG.
  • the sample solution is introduced into the introduction hole 11.
  • the sample solution is a mixture of a sample, which is an aqueous solution containing a target antigen, with an aqueous solution of an enzyme-labeled complex (first labeled substance in the present invention) composed of an antigen not derived from the sample.
  • this complex is referred to as a labeled antigen.
  • the antigen in the labeled antigen the same substance as the target antigen is used.
  • the color-developing substrate 2 is also required for color development, the sample solution is assumed to further contain the color-developing substrate 2.
  • a coloring portion 12 is formed on the sheet 10.
  • the coloring unit 12 is a part of the flow path 40.
  • the color-developing portion 12 is arranged at a position closer to the left end portion of the sheet 10 in FIG. As shown in FIG. 1, the color-developing portion 12 has the same disk shape as the introduction hole 11.
  • the color-developing unit 12 contains a color-developing substrate 1.
  • the sheet 20 is formed with a sample supply section 21, two intermediate sections 22, and two communication sections 24, which are a part of the flow path 40. As shown in FIG. 2, these are arranged in a line in the order of the sample supply section 21, the communication section 24, the intermediate section 22, the communication section 24, and the intermediate section 22 from the right in the A direction. Of these, the sample supply section 21 and the intermediate section 22 have the same disk shape as the introduction hole 11 and the color development section 12 of the sheet 10, as shown in FIG.
  • the sample supply unit 21 is arranged at a position that exactly overlaps with the introduction hole 11 of the sheet 10 when viewed from the stacking direction.
  • the sample supply unit 21 contains an antibody against the target antigen (hereinafter, simply referred to as an antibody) without being fixed.
  • "without fixing” means that a chemical fixing means for fixing the antibody to the sheet 20 so as not to move when the paper device 1 is used is not used.
  • the intermediate portion 22 located at the leftmost end in FIG. 2 is arranged at a position that exactly overlaps with the coloring portion 12 of the sheet 10 when viewed from the stacking direction.
  • the intermediate portion 22 and the coloring portion 12 are in contact with each other in the stacking direction.
  • each communication portion 24 extends linearly along the A direction.
  • the two communication sections 24 communicate the sample supply section 21 and the two intermediate sections 22 with each other in the A direction.
  • the sample supply section 21, the two intermediate sections 22, and the two communication sections 24 communicate with each other to form a flow path for the sample liquid from the sample supply section 21 to the coloring section 12.
  • the flow path composed of the two intermediate portions 22 and the two communication portions 24 corresponds to the intermediate flow path in the present invention.
  • the sample supply section 21, the two intermediate sections 22, and the two communication sections 24 contain a speed adjuster (corresponding to the speed control substance in the present invention) as a whole.
  • the speed adjusting agent regulates the interaction between the paper fibers constituting the flow path 40 and the above four components, and causes a difference in moving speed between the four components. Since the above four components have different molecular sizes and different hydrophilicity due to the molecular structure, the magnitude of the interaction with the paper fiber is different.
  • the speed adjuster utilizes this to cause a difference in moving speed between the components.
  • the rate regulator one that causes a difference in the migration rate of these components so that the unreacted labeled antigen migrates before the labeled antigen-antibody complex may be selected.
  • a speed difference may or may not occur.
  • a surfactant such as PBST (Phosphate buffered saline with Tween-20) may be used.
  • a sample solution is prepared (step S1).
  • an aqueous solution of the labeled antigen is prepared.
  • This aqueous solution has a predetermined concentration and a predetermined amount set in advance.
  • a predetermined amount of a sample containing the target antigen is mixed with this aqueous solution.
  • the aqueous solution of the labeled antigen and the target antigen thus obtained is further mixed with the aqueous solution of the color-developing substrate 2 as needed to prepare a sample solution.
  • the prepared sample solution is introduced into the sample supply unit 21 through the introduction hole 11 as shown in FIG. 1 (step S2).
  • the sample liquid permeates from the sample supply section 21 through the two intermediate sections 22 and the two communication sections 24 toward the coloring section 12.
  • the target antigen and the labeled antigen react with the antibody contained in the sample supply unit 21.
  • four components of the target antigen-antibody complex, the labeled antigen-antibody complex, the unreacted target antigen, and the unreacted labeled antigen are generated in the sample solution.
  • the concentration of the unreacted labeled antigen in the sample solution becomes a magnitude corresponding to the concentration of the target antigen originally contained in the sample.
  • the above four components move along with the flow of the sample liquid that permeates toward the coloring unit 12.
  • the sample supply section 21, the two intermediate sections 22, and the two communication sections 24 contain a speed adjuster.
  • this rate regulator creates a rate difference between the unreacted labeled antigens so that they migrate ahead of the labeled antigen-antibody complex. As a result, the unreacted labeled antigen reaches the color-developing portion 12 before the labeled antigen-antibody complex.
  • the above four components are distributed in different ranges in the flow path 40.
  • the distribution of the above four components in the flow path 40 does not need to form a band so narrow that they can be clearly distinguished from each other, and a wide band may be formed and a part thereof may overlap.
  • the enzyme labeled with the labeled antigen reacts with the coloring substrate 1 of the coloring part 12 (including the coloring substrate 2 in some cases), so that the coloring part 12 Color develops.
  • the intensity of color development becomes a magnitude corresponding to the concentration of the unreacted labeled antigen that reaches the color-developing portion 12.
  • the concentration of the unreacted labeled antigen becomes a magnitude corresponding to the concentration of the target antigen originally contained in the sample, as described above. Therefore, the color development of the coloring unit 12 becomes stronger as the concentration of the target antigen increases.
  • step S3 take a picture of the paper device 1 with a digital camera.
  • the photographing is carried out so that the situation in which the coloring unit 12 develops color is included in the photographing range.
  • step S4 an analysis regarding the concentration of the target antigen in the sample is executed based on the imaging result of step S3 (step S4).
  • a computer-based digital image analysis is used for the analysis in step S4. Specifically, for example, the color corresponding to the color development is obtained by calculating the pixel value (for example, red, green, and blue (RGB) values) of each pixel existing in the region corresponding to the color development unit 12 in the captured image.
  • RGB red, green, and blue
  • a color development intensity value indicating the intensity of the component (for example, the cyan (C) value of cyan, magenta, and yellow (CMY)) is derived.
  • the average value of the color development intensity values is calculated for the pixels in the region corresponding to the color development unit 12.
  • the computer derives the concentration of the target antigen in the sample by comparing the average value of the color development intensity values with the predetermined reference data.
  • the reference data is data showing the relationship between the density and the average value of the color development intensity.
  • the reference data is acquired in advance based on, for example, the result of coloring the coloring unit 12 in the same manner as in steps S1 and S2 for a plurality of samples containing target antigens having different known concentrations.
  • the concentration of the target antigen derived as described above is output by an output device such as a display connected to or mounted on the computer.
  • the unreacted labeled antigen and the labeled antigen-antibody complex are labeled with an enzyme, and the coloring portion 12 contains the coloring substrate 1. Therefore, when these labeling substances reach the coloring unit 12, the coloring unit 12 develops color according to the action of the enzyme in the labeling substance and the coloring substrate 1 (in some cases, the action including the coloring substrate 2).
  • the color development in the color development unit 12 has an intensity corresponding to the concentration of these labeling substances that have reached the color development unit 12.
  • the unreacted labeled antigen reaches the color-developing part before the labeled antigen-antibody complex due to the speed regulator contained in the flow path. Therefore, unreacted labeling is performed by evaluating the intensity of color development generated in the coloring unit 12 after the unreacted labeled antigen reaches the coloring unit 12 until the labeled antigen-antibody complex reaches the coloring unit 12.
  • the concentration of antigen can be evaluated.
  • the concentration of the unreacted labeled antigen in the flow path 40 becomes a magnitude corresponding to the concentration of the antigen in the sample. Therefore, the concentration of the antigen in the sample can be evaluated by evaluating the color development intensity of the color development unit 12.
  • the antibody is not fixed in the flow path 40 but is included in the sample supply unit 21. Therefore, the paper device 1 according to the present embodiment does not require an immobilization step in the manufacturing process. Therefore, it is easier to manufacture than the conventional technique that requires a fixing step.
  • the amount of the sample liquid is adjusted so that the flow of the sample liquid is stopped and the movement of the components is stopped when the sample liquid reaches the coloring portion 12. That is, the labeled antigen-antibody complex does not reach the coloring part 12. Therefore, the concentration of the antigen in the sample can be appropriately evaluated based on the intensity at which the unreacted labeled antigen develops the color of the coloring portion 12.
  • the flow path 40 composed of the hydrophilic region 2a is formed in the laminated body 2 in which the sheets 10 to 30 are laminated. Therefore, excessive drying of the sample solution in the flow path 40 is suppressed.
  • the sheet 30 is entirely formed by hydrophobic regions. Therefore, the drying of the sample liquid from the lower surface of the sheet 20 is suppressed by the sheet 30.
  • the color-developing portion 12 is formed on the sheet 10, while the sample supply portion 21, the two intermediate portions 22, and the two communicating portions 24 are formed on the sheet 20. That is, in the flow path 40, the color-developing portion 12 and other portions are formed on separate sheets. Assuming that the entire flow path 40 is formed on one sheet, the permeation of the sample solution into the color-developing portion 12 proceeds only by permeation in the direction along one sheet. As a result, the color development in the color development unit 12 progresses. Therefore, uneven color development in the direction along the sheet is likely to occur.
  • the color development proceeds by the permeation of the sample liquid from the intermediate portion 22 of the sheet 10 to the coloring portion 12 of the sheet 20 so as to straddle the sheets. Therefore, uneven color development in the direction along the sheet is unlikely to occur. Therefore, the concentration of the antigen in the sample can be appropriately evaluated based on the color development intensity of the color development unit 12.
  • Example 1 Hereinafter, Example 1 relating to the first embodiment will be described.
  • the target antigen was progesterone
  • the labeled antigen was a progesterone-HRP complex
  • the antibody was an anti-progesterone antibody.
  • the color-developing substrate 1 was TMB
  • the color-developing substrate 2 was H 2 O 2 .
  • the paper device 1 was manufactured as follows.
  • the pattern shown in FIG. 1 was formed on the filter paper using a solid ink type inkjet printer. Then, by heating the filter paper in which the pattern was formed, the solid ink solidified on the filter paper was melted and permeated into the filter paper. As a result, a pattern of the hydrophobic region defining the outer edge of the flow path 40 composed of the hydrophilic region was formed on the filter paper.
  • the region corresponding to the flow path 40 of the filter paper was impregnated with PBST at a predetermined concentration.
  • the antibody solution was added dropwise to the sample supply unit 21, and then the filter paper was dried for a predetermined time.
  • the TMB solution was dropped onto the region corresponding to the color-developing portion 12 of the filter paper, and then the filter paper was dried.
  • the inspection using the paper device 1 produced as described above was performed as follows. First, a sample solution containing the target antigen, labeled antigen and H 2 O 2 was prepared, and an appropriate amount thereof was dropped onto the sample supply unit 21 through the introduction hole 11. After the dropped sample solution reached the coloring unit 12, the image of the paper device 1 was taken with a digital camera after waiting for a predetermined time. Similar to step S4 above, the captured image was digitally analyzed to obtain the intensity of a specific color (cyan) as the color development intensity.
  • FIG. 4 shows the difference in color development intensity when the above test is performed using sample solutions having different concentrations of the target antigen (progesterone). According to FIG. 4, the color development intensity increases as the progesterone concentration increases. This indicates that the concentration of the target antigen can be quantitatively measured by the above test.
  • Example 2 A paper device 1 was produced in the same manner as in Example 1 except that the flow path 40 was not impregnated with PBST.
  • a plurality of paper devices 1 in which the flow path 40 was impregnated with PBST having Tween-20 at various concentrations were produced in the same manner as in Example 1.
  • two types of sample solutions having a concentration of the target antigen of 0 ng / mL that is, a sample solution containing no target antigen
  • 5 ng / mL were prepared in the same manner as in Example 1. The inspection of Example 1 was carried out.
  • the concentration of the surfactant when the concentration of the surfactant is further increased, the color development intensity becomes maximum at both the target antigen concentrations of 0 ng / mL and 5 ng / mL, and both target antigens.
  • the concentration was the same and was constant with respect to the concentration of the surfactant.
  • C1 is a lower limit value that makes it difficult to observe the color development intensity depending on the concentration of the target antigen because the color development intensity is small when the concentration of the surfactant is lower than this value (see FIG. 5).
  • C2 is an upper limit value at which the maximum color development intensity is obtained regardless of the target antigen concentration when the concentration of the surfactant is higher than this value.
  • the sample supply unit 21 does not contain an antibody.
  • a complex (first labeled substance in the present invention) composed of an antibody labeled with an enzyme (hereinafter, referred to as a labeled antibody) is used instead of the labeled antigen.
  • a labeled antibody-target antigen complex composed of an antibody labeled with an enzyme (hereinafter, referred to as a labeled antibody)
  • three components, a labeled antibody-target antigen complex, an unreacted labeled antibody, and an unreacted target antigen, which are generated by the reaction of the target antigen and the labeled antibody are generated in the sample solution.
  • the moving speed in the flow path 40 between the unreacted labeled antibody and the labeled antibody-target antigen complex due to the action of the speed regulator in the flow path 40. makes a difference.
  • the concentration of the target antigen in the sample can be derived based on the digital image analysis of the imaging result of the paper device 1.
  • the paper device 100 shown in FIG. 6 may be adopted when there is no sufficient difference between the action and the action (and therefore, the movement speed is unlikely to be different).
  • the paper device 100 includes a laminated body 102 in which filter paper sheets 110 and 120 are laminated. Similar to the paper device 1, the laminated body 102 is formed with a flow path 140 composed of a hydrophilic region whose outer edge is defined by a hydrophobic region.
  • the sheet 110 is formed with introduction holes 111 arranged in a row along the A direction and a coloring portion 112 which is a part of the flow path 140.
  • the color-developing unit 112 contains a color-developing substrate 1.
  • the sheet 120 is formed with a sample supply section 121, an antibody setting section 122, two intermediate sections 123, and three communication sections 124, which are part of the flow path 140. From one end to the other end in the A direction, the sample supply section 121, the communication section 124, the antibody installation section 122, the communication section 124, the intermediate section 123, the communication section 124, and the intermediate section 123 are arranged in this order along the A direction. They are lined up in a row.
  • the sample supply unit 121 is arranged at a position that exactly overlaps with the introduction hole 111 of the sheet 110 when viewed from the stacking direction. Further, the outermost intermediate portion 123 in the A direction is arranged at a position that exactly overlaps with the coloring portion 112 when viewed from the stacking direction. The intermediate portion 123 and the coloring portion 112 are in contact with each other in the stacking direction.
  • the sample supply unit 121, the antibody installation unit 122, the two intermediate units 123, and the three communication units 124 contain a speed adjuster as a whole.
  • a speed regulator for example, a surfactant such as PBST may be used.
  • the antibody setting unit 122 contains a second antibody against the target antigen, which is different from the antibody used for the labeled antibody, without being fixed.
  • the second antibody is an antibody that can bind not only to the target antigen that has not reacted with the labeled antibody but also to the target antigen in the labeled antibody-target antigen complex.
  • the amount of the second antibody against the substance in the sample solution is made negligibly small.
  • concentration of the rate-regulating agent by adjusting the concentration of the rate-regulating agent, a difference in the moving speed in the flow path 140 is made between the labeled antibody-target antigen-second antibody complex and the unreacted labeled antibody. Then, by setting the labeled antibody in the sample solution to a predetermined amount, the labeled antibody-target antigen-second antibody complex and the unreacted labeled antibody are transferred to the flow path 140 at a concentration corresponding to the concentration of the target antigen in the sample. It will exist.
  • the concentration of the target antigen in the sample can be determined. According to this configuration, a difference in molecular size is likely to occur between the labeled antibody-target antigen-second antibody complex and the unreacted labeled antibody. Therefore, a sufficient difference is likely to occur between the interaction between the unreacted labeled antibody and the paper fiber in the flow path 40 and the interaction between the labeled antibody-target antigen complex and the paper fiber in the flow path 40, and the migration speed is high. Is also likely to make a difference.
  • the paper device 200 shown in FIG. 7 may be used instead of the paper device 1 according to the first embodiment described above.
  • the paper device 200 includes sheets 210 and 220 made of filter paper and a laminate 202 in which the same sheets 20 as in the above-described embodiment are laminated. Similar to the paper device 1, the laminated body 202 is formed with a flow path 240 composed of a hydrophilic region whose outer edge is defined by a hydrophobic region.
  • the sheet 210 is formed with introduction holes 211 and detection windows 212 arranged in a row along the A direction.
  • the sheet 220 is formed with a substrate setting portion 221 and a coloring portion 222 arranged in a row along the A direction.
  • the substrate setting section 221 is arranged at a position where it just overlaps the introduction hole 211 and the sample supply section 21 when viewed from the stacking direction, and is in contact with the sample supply section 21 in the stacking direction.
  • the color-developing portion 222 is arranged at a position just overlapping the outermost intermediate portion 22 in the detection window 212 and the sheet 20 when viewed from the stacking direction, and is in contact with the intermediate portion 22 in the stacking direction.
  • the color-developing unit 222 contains a color-developing substrate 1.
  • the flow path 240 is configured from the substrate installation section 221 of the sheet 220 to the sample supply section 21, the two intermediate sections 22 and the two communication sections 24 of the sheet 20 to the coloring section 222 of the sheet 220. ..
  • the paper device 200 is adopted, for example, when HRP is used as an enzyme and H 2 O 2 is used as a coloring substrate 2.
  • H 2 O 2 is contained in the substrate setting section 221 instead of the sample solution.
  • H 2 O 2 is stably retained in the paper device 200 even after drying by applying an aqueous solution of hydrogen peroxide urea to the region corresponding to the substrate setting portion 221 and then drying the paper device 200.
  • an intermediate part 22 may be used instead of a substrate setting part 221. That is, the portion corresponding to the substrate installing part 221 in the paper device 200 does not include the H 2 O 2, any one or two of the intermediate portion 22 may include H 2 O 2.
  • H 2 O 2 can be installed in the intermediate portion 22 by applying an aqueous hydrogen peroxide urea solution to the portion of the intermediate portion 22 and drying the portion. Drying may be performed after applying the solution of the color-developing substrate 1 to the portion of the color-developing portion 222.
  • an antibody is installed in any one or two intermediate portions 22, or the intermediate portion 22 closer to the sample supply unit 21 and its intermediate portion 22 and the like.
  • Antibodies may be installed in the communication portions 24 on both sides.
  • FIG. 8 shows the inspection results performed using the paper device in which the configuration of the paper device 200 is changed as follows.
  • the labeled antigen is applied to the sample supply unit 221
  • the antibody is applied to the intermediate portion 22 closer to the sample supply unit 21 and the communication portions 24 on both sides thereof, and the intermediate portion 22 closer to the sample supply unit 21.
  • H 2 O 2 were installed without fixing each.
  • the color-developing substrate 1 was installed in the color-developing unit 222.
  • a predetermined volume of progesterone aqueous solution having a different concentration was supplied to the sample supply unit 221 through the introduction hole 211.
  • FIG. 8 is a plot of the color development intensity (cyan value) in the color development unit 222 at this time with respect to the concentration of progesterone in the progesterone aqueous solution. As shown in FIG. 8, the color development intensity increases as the progesterone concentration increases.
  • control flow path 70 shown in FIG. 9 may be added to the paper device 1 according to the first embodiment described above.
  • the control flow path 70 includes a flow path 60 formed on a filter paper sheet 50 and a coloring portion 63 formed on a sheet different from the sheet 50, and is composed of a hydrophilic region whose outer edge is defined by a hydrophobic region. It is a flow path.
  • the flow path 60 has the same shape and size as the flow path composed of the sample supply portion 21, the two intermediate portions 22, and the two communication portions 24 formed on the sheet 20.
  • the flow path 60 contains the same rate regulator as that contained in the flow path 40, but does not contain the antibody.
  • the sheet 50 is inserted between the sheets 10 and 20 so that one end 61 of the flow path 60 is arranged between the introduction hole 11 and the sample supply section 21. Above the other end 62 of the flow path 60, a coloring portion 63 having the same shape and size as the coloring portion 12 is provided.
  • the color-developing unit 63 contains a color-developing substrate 1.
  • the control flow path 70 is formed in the same manner as the flow path 40 except that the antibody is not contained.
  • the sample liquid permeates into the flow path 40 and also permeates into the control flow path 70. Since the control flow path 70 does not contain an antibody, the maximum amount of labeled antigen reaches the color-developing portion 63 regardless of the concentration of the antigen in the sample.
  • the color development in the color development unit 63 can be compared with the color development in the color development unit 12.
  • the concentration of the antigen in the sample may be evaluated by evaluating the color development intensity of the color development unit 12 relative to the color development intensity of the color development unit 63.
  • a surfactant is used as the speed regulator, and PBST is used as a specific example.
  • other substances may be used as long as the components in the flow path have different moving speeds.
  • various organic solvents can be mentioned as candidates.
  • the labeled antigen-antibody complex does not reach the coloring portion 12.
  • an embodiment in which the labeled antigen-antibody complex can finally reach the color-developing portion 12 may be adopted.
  • the concentration of the antigen in the sample can be evaluated by evaluating the color development intensity of the color-developing portion 12 by the unreacted labeled antigen before the labeled antigen-antibody complex reaches the color-developing portion 12.
  • the method of placing substances such as the color-developing substrate 1, the color-developing substrate 2, the antibody, and the labeled antigen on the paper device is to apply the solution and then dry it. It is done. That is, a method is adopted in which none of the substances is fixed to the paper device (without using chemical fixing means). Therefore, none of the substances are chemically bonded to the sheet (cellulose).
  • the method of applying the solution and then drying it is a specific example of a method of placing these substances on a paper device without using chemical fixing means, and other methods may be used.

Abstract

The objective of the present invention is for manufacture of a substance detecting device to be easy, even if a sheet is employed.　A paper device 1 is an apparatus for detecting a substance by employing color development caused at least due to the action of an enzyme and a color developing substrate 1. A specimen supply portion 21 to which a specimen liquid containing a sample is supplied, a color developing portion 12 containing the color developing substrate 1, and a flow path joining the specimen supply portion 21 to the color developing portion 12 are formed as a flow path 40 comprising a hydrophilic region, in a laminated body 2 consisting of sheets 10 to 30. The specimen liquid contains an antigen labeled using an enzyme. The specimen supply portion 21 contains an unfixed antibody. The flow path 40 contains a speed regulator. The speed regulator causes the speed at which the labeled antigen that has not reacted with the antibody moves through the flow path 40 to be greater than the speed at which a labeled antigen/antibody complex, formed by a reaction between the labeled antigen and the antibody, moves through the flow path 40.

Description

物質検出装置Substance detector
　本発明は、シートを使用した物質検出装置に関する。 The present invention relates to a substance detection device using a sheet.
　物質検出の一手法として、酵素免疫測定の一つである競合法がある。この手法では、反応容器（ウェル）内において、濃度の検出対象となる検体由来の抗原（目的抗原）に、酵素を標識した検体由来でない抗原（標識抗原）を加えて、この二者間で抗体との反応を競争的に行わせる。反応のあと抗体に結合した標識抗原の量を酵素反応による発色を利用して測定するために、未反応の目的抗原および標識抗原を洗浄によって反応容器から除去する。この洗浄過程で、反応した標識抗原が洗い出されないようにするため、抗体は始めから反応容器の底に固定されている必要がある。このように、従来の競合法では、一般的には未反応の標識抗原と抗体に結合した標識抗原が明確に分離されることが不可欠である。なお、従来の反応容器としては、ポリスチレン製のマイクロタイタープレートが一般的に利用されている。 As one method of substance detection, there is a competitive method which is one of enzyme immunoassays. In this method, in the reaction vessel (well), an antigen (labeled antigen) derived from the sample whose concentration is to be detected is added to the antigen (labeled antigen) not derived from the sample labeled with the enzyme, and an antibody is produced between the two. Have a competitive reaction with. After the reaction, the unreacted target antigen and the labeled antigen are removed from the reaction vessel by washing in order to measure the amount of the labeled antigen bound to the antibody by utilizing the color development by the enzymatic reaction. In order to prevent the reacted labeled antigen from being washed out during this washing process, the antibody needs to be fixed to the bottom of the reaction vessel from the beginning. As described above, in the conventional competitive method, it is generally essential that the unreacted labeled antigen and the labeled antigen bound to the antibody are clearly separated. As a conventional reaction vessel, a polystyrene microtiter plate is generally used.
　一方、濾紙のようなシートにウェルや流路を印刷することで作製された２次元的な反応容器の中で様々な反応を行う手法も報告されている。これによると酵素免疫測定を安価、簡便、迅速に実施できることから、近年、かかるシート状の酵素免疫測定法や免疫測定用紙製チップが世界中で活発に開発されている。シート状の酵素免疫測定法については、すでに多くの報告がある。そのうちの一部である非特許文献１～５では、抗体は、紙繊維に塗布されたキトサンに架橋試薬（グルタルアルデヒド）を用いて固定化されている。抗体の固定化工程は、キトサンの塗布、乾燥、グルタルアルデヒドの塗布、キトサンとグルタルアルデヒドの反応、未反応グルタルアルデヒドの洗浄、乾燥、抗体の塗布、抗体とグルタルアルデヒドの反応、未反応抗体の洗浄除去等からなる。 On the other hand, methods of performing various reactions in a two-dimensional reaction vessel made by printing wells and flow paths on a sheet such as filter paper have also been reported. According to this, since enzyme immunoassay can be performed inexpensively, easily, and quickly, such sheet-shaped enzyme immunoassays and immunoassay paper chips have been actively developed all over the world in recent years. There are already many reports on sheet-shaped enzyme immunoassays. In Non-Patent Documents 1 to 5, which are a part of them, the antibody is immobilized on chitosan applied to paper fibers by using a cross-linking reagent (glutaraldehyde). The antibody immobilization steps include application of chitosan, drying, application of glutaraldehyde, reaction of chitosan and glutaraldehyde, washing of unreacted glutaraldehyde, drying, application of antibody, reaction of antibody and glutaraldehyde, washing of unreacted antibody. It consists of removal and the like.
　非特許文献１～５では、上記のような抗体の固定工程に多くの操作と長い時間を要する。また、固定化工程において実施される洗浄液の除去は、従来のポリスチレン製の反応容器で行われていたような吸引や振り払いでは困難であるため、実験用ワイパーのような紙等に吸い取ることで行う。このように、シート状の物質検出装置では、製造工程が煩雑であることが製品化の課題となっていた。 In Non-Patent Documents 1 to 5, many operations and a long time are required for the antibody fixing step as described above. Further, since it is difficult to remove the cleaning liquid performed in the immobilization step by suction or shaking as in the case of a conventional polystyrene reaction vessel, it is possible to remove the cleaning liquid by sucking it on paper such as an experimental wiper. Do. As described above, in the sheet-shaped substance detection device, the complicated manufacturing process has been an issue for commercialization.
　本発明の目的は、シートを用いた場合でも製造が容易である物質検出装置を提供することにある。 An object of the present invention is to provide a substance detection device that is easy to manufacture even when a sheet is used.
　本発明の物質検出装置は、酵素と基質の作用に起因して発生する発色の強度に基づいて検体中の被検出物質を検出する物質検出装置であって、親水性領域と当該親水性領域の外縁を画定する疎水性領域とを有するシートを備えており、前記親水性領域に、前記検体を含んだ試料液が供給される試料供給部と、前記基質を含んだ発色部と、前記試料供給部から前記発色部までを結ぶ中間流路と、が形成されており、前記被検出物質と結合する物質である捕捉物質及び前記検体に由来しない前記被検出物質のいずれか一方が前記酵素によって標識された複合物である第１標識物質と、前記捕捉物質及び前記被検出物質のいずれか一方が前記酵素によって標識されたものと他方との複合物である第２標識物質とが、前記第１標識物質の濃度が前記検体中の前記被検出物質の濃度に応じた大きさとなりつつ前記中間流路に存在することとなるように、前記第１標識物質及び前記捕捉物質の両方又は前者が、前記試料液、前記試料供給部及び前記中間流路の少なくともいずれかに、固定されずに含まれており、前記中間流路が、前記第１標識物質が前記中間流路中を移動する速度を前記第２標識物質が前記中間流路中を移動する速度より大きくする速度調節物質を含んでいる。 The substance detection device of the present invention is a substance detection device that detects a substance to be detected in a sample based on the intensity of color development generated due to the action of an enzyme and a substrate, and is a hydrophilic region and the hydrophilic region. A sheet having a hydrophobic region defining an outer edge is provided, and a sample supply unit to which a sample solution containing the sample is supplied, a coloring unit containing the substrate, and the sample supply to the hydrophilic region. An intermediate flow path connecting the portion to the color-developing portion is formed, and either the capturing substance, which is a substance that binds to the substance to be detected, or the substance to be detected, which is not derived from the sample, is labeled by the enzyme. The first labeling substance, which is a composite of the above, and the second labeling substance, which is a complex of one of the trapping substance and the detected substance labeled with the enzyme and the other, are the first. Both or the former of the first labeling substance and the trapping substance are present so that the concentration of the labeling substance exists in the intermediate flow path while increasing in magnitude according to the concentration of the detected substance in the sample. It is contained in at least one of the sample liquid, the sample supply unit, and the intermediate flow path without being fixed, and the intermediate flow path determines the speed at which the first labeling substance moves in the intermediate flow path. The second labeling substance contains a rate adjusting substance that increases the rate of movement in the intermediate flow path.
　本発明の物質検出装置によると、第１標識物質及び第２標識物質は酵素によって標識されており、発色部は基質を含んでいる。したがって、発色部にこれらの標識物質が到達すると、標識物質中の酵素と基質との作用に応じて発色部が発色する。発色部における発色は、発色部に到達したこれらの標識物質の濃度に応じた強度となる。 According to the substance detection device of the present invention, the first labeling substance and the second labeling substance are labeled with an enzyme, and the coloring part contains a substrate. Therefore, when these labeling substances reach the coloring part, the coloring part develops color according to the action of the enzyme and the substrate in the labeling substance. The color development in the color-developing portion has an intensity corresponding to the concentration of these labeling substances that have reached the color-developing portion.
　一方、中間流路に含まれている速度調節物質により、第１標識物質は第２標識物質より早く発色部に到達する。したがって、第１標識物質が発色部に到達してから第２標識物質が発色部に到達するまでに発色部において発生する発色の強度を評価することで、第１標識物質の濃度を評価することができる。中間流路中の第１標識物質の濃度は、検体中の被検出物質の濃度に応じた大きさとなる。このため、発色部の発色の強度を評価することで、検体中の被検出物質の濃度を評価できる。 On the other hand, due to the speed adjusting substance contained in the intermediate flow path, the first labeling substance reaches the coloring part earlier than the second labeling substance. Therefore, the concentration of the first labeling substance is evaluated by evaluating the intensity of color development generated in the coloring portion from the time when the first labeling substance reaches the coloring portion to the time when the second labeling substance reaches the coloring portion. Can be done. The concentration of the first labeling substance in the intermediate flow path has a magnitude corresponding to the concentration of the substance to be detected in the sample. Therefore, the concentration of the substance to be detected in the sample can be evaluated by evaluating the color development intensity of the color-developing portion.
　さらに、本発明においては、被検出物質と結合させるための捕捉物質がシートに固定されていない。したがって、本発明の物質検出装置は、製造工程において捕捉物質の固定化工程を必要としない。よって、かかる固定化工程を必要とする従来技術と比べ、製造が容易である。 Furthermore, in the present invention, the trapping substance for binding to the substance to be detected is not fixed to the sheet. Therefore, the substance detection device of the present invention does not require a trapping substance immobilization step in the manufacturing process. Therefore, it is easier to manufacture than the conventional technique that requires such an immobilization step.
　なお、本発明における「固定されずに含まれている」とは、装置の使用時にシート上で物質が移動しないようにこれをシートに固定するための化学的な固定手段（例えば、キトサン及びグルタルアルデヒドを用いた、抗体（捕捉物質）のシートへの従来の固定手段）が用いられずに含まれていることを示す。また、本発明における「被検出物質」は、特に限定がない限り、検体由来のものとそうでないものとのいずれにも該当し得る。また、第１標識物質及び捕捉物質の両方が、試料液、試料供給部及び中間流路の少なくともいずれかに含まれている態様においては、第１標識物質と捕捉物質で含まれている箇所が異なっていても、同じであってもよい。 In the present invention, "contained without being fixed" means a chemical fixing means (for example, chitosan and glutar) for fixing the substance to the sheet so that the substance does not move on the sheet when the device is used. It is shown that the conventional means for fixing the antibody (capturing substance) to the sheet using aldehyde) is not used. Further, the "substance to be detected" in the present invention may correspond to any of those derived from a sample and those not derived from a sample, unless otherwise specified. Further, in the embodiment in which both the first labeling substance and the trapping substance are contained in at least one of the sample solution, the sample supply unit and the intermediate flow path, the portion contained by the first labeling substance and the trapping substance is contained. It may be different or the same.
　また、被検出物質と捕捉物質の組み合わせは、例えば、抗原と抗体の組み合わせであってもよいし、アプタマーとその標的分子の組み合わせ、酵素と基質の組み合わせ、あるいは、酵素とその阻害剤の組み合わせであってもよい。 Further, the combination of the substance to be detected and the capture substance may be, for example, a combination of an antigen and an antibody, a combination of an aptamer and its target molecule, a combination of an enzyme and a substrate, or a combination of an enzyme and its inhibitor. There may be.
　また、本発明においては、前記速度調節物質が界面活性剤であることが好ましい。これによると、後述の実施例に示すように、第１標識物質と第２標識物質の間で中間流路中を移動する速度に適切に差を生じることができる。 Further, in the present invention, it is preferable that the speed adjusting substance is a surfactant. According to this, as shown in Examples described later, it is possible to appropriately make a difference in the speed of movement in the intermediate flow path between the first labeled substance and the second labeled substance.
　また、本発明においては、前記試料液が、前記第１標識物質が前記発色部に到達し且つ前記第２標識物質が前記発色部に到達しない量に調整されていることが好ましい。これによると、第２標識物質が発色部に到達しないので、第１標識物質が発色部を発色させる強度に基づき、検体中の被検出物質の濃度を適切に評価できる。 Further, in the present invention, it is preferable that the sample solution is adjusted to an amount in which the first labeling substance reaches the coloring portion and the second labeling substance does not reach the coloring portion. According to this, since the second labeling substance does not reach the color-developing portion, the concentration of the substance to be detected in the sample can be appropriately evaluated based on the intensity at which the first labeling substance develops the color of the coloring portion.
　また、本発明においては、前記親水性領域及び前記疎水性領域を有する複数のシートが積層された積層体を備えていることが好ましい。これによると、複数のシートが積層されることにより、親水性領域において乾燥が過度に進むのが抑制される。 Further, in the present invention, it is preferable to have a laminated body in which a plurality of sheets having the hydrophilic region and the hydrophobic region are laminated. According to this, by laminating a plurality of sheets, excessive drying is suppressed in the hydrophilic region.
　また、本発明においては、前記発色部が、前記複数のシートにおける前記中間流路とは別のシートに形成されていることが好ましい。仮に、１つのシートに中間流路と発色部の両方が形成されているとすると、１枚のシートに沿った方向に関する試料液の浸透のみによって中間流路から発色部への試料液の浸透が進んでいく。これによって、発色部における発色が進行する。したがって、シートに沿った方向に関する発色のむらが生じやすい。これに対し、発色部が中間流路とは別のシートに形成されていると、中間流路から発色部へとシート間を跨ぐように試料液の浸透が進むことによって発色が進んでいくことになる。よって、シートに沿った方向に関する発色のむらが生じにくい。したがって、発色部の発色強度に基づいて検体中の被検出物質の濃度を適切に評価できる。 Further, in the present invention, it is preferable that the coloring portion is formed on a sheet different from the intermediate flow path in the plurality of sheets. Assuming that both the intermediate flow path and the coloring part are formed on one sheet, the permeation of the sample liquid from the intermediate flow path to the coloring part is caused only by the permeation of the sample liquid in the direction along one sheet. Go ahead. As a result, color development in the color-developing portion progresses. Therefore, uneven color development in the direction along the sheet is likely to occur. On the other hand, if the color-developing portion is formed on a sheet different from the intermediate flow path, the color development progresses by the permeation of the sample liquid so as to straddle the sheets from the intermediate flow path to the color-developing portion. become. Therefore, uneven color development in the direction along the sheet is unlikely to occur. Therefore, the concentration of the substance to be detected in the sample can be appropriately evaluated based on the color development intensity of the color-developing portion.
　また、本発明においては、前記第１標識物質が、前記検体に由来しない前記被検出物質が前記酵素によって標識された複合物であって、前記試料液に含まれており、前記捕捉物質が、前記試料供給部及び前記中間流路の少なくともいずれかに含まれており、前記第２標識物質が、前記第１標識物質と前記捕捉物質との反応により生成されてもよい。これによると、検体中の被検出物質と第１標識物質とが捕捉物質と競争的に反応する。したがって、第１標識物質が検体中の被検出物質の濃度に応じた濃度で適切に中間流路中に存在することとなる。 Further, in the present invention, the first labeling substance is a complex in which the substance to be detected, which is not derived from the sample, is labeled by the enzyme, and is contained in the sample solution. It is contained in at least one of the sample supply unit and the intermediate flow path, and the second labeling substance may be produced by the reaction between the first labeling substance and the trapping substance. According to this, the substance to be detected in the sample and the first labeling substance react competitively with the capturing substance. Therefore, the first labeling substance is appropriately present in the intermediate flow path at a concentration corresponding to the concentration of the substance to be detected in the sample.
　また、本発明においては、前記第１標識物質が、前記捕捉物質が前記酵素によって標識された複合物であって、前記試料液に含まれており、前記第２標識物質が、前記第１標識物質と前記検体中の前記被検出物質との反応により生成されてもよい。これによると、第２標識物質は検体中の被検出物質の濃度に応じて発生する。したがって、被検出物質と結合していない第１標識物質が検体中の被検出物質の濃度に応じた濃度で適切に中間流路中に存在することとなる。 Further, in the present invention, the first labeling substance is a complex in which the trapping substance is labeled by the enzyme and is contained in the sample solution, and the second labeling substance is the first labeling. It may be produced by the reaction of the substance with the substance to be detected in the sample. According to this, the second labeling substance is generated according to the concentration of the substance to be detected in the sample. Therefore, the first labeled substance that is not bound to the substance to be detected is appropriately present in the intermediate flow path at a concentration corresponding to the concentration of the substance to be detected in the sample.
本発明の一実施形態である第１の実施形態に係る紙デバイスの分解斜視図である。It is an exploded perspective view of the paper device which concerns on 1st Embodiment which is 1 Embodiment of this invention. 図１の紙デバイスのII－II線断面図である。FIG. 2 is a sectional view taken along line II-II of the paper device of FIG. 図１の紙デバイスの使用方法を示すフロー図である。It is a flow chart which shows the usage method of the paper device of FIG. 目的抗原をプロゲステロンとした図１の紙デバイスの一実施例に係る結果を示すグラフである。It is a graph which shows the result which concerns on one Example of the paper device of FIG. 1 which used progesterone as a target antigen. 目的抗原（プロゲステロン）を含むものと含まないものとの２種類の試料液を用いると共に界面活性剤の濃度を変更しつつ行った図１の紙デバイスの別の一実施例に係る結果を示すグラフである。A graph showing the results of another embodiment of the paper device of FIG. 1, which was carried out using two types of sample solutions, one containing the target antigen (progesterone) and the other containing no target antigen, and changing the concentration of the surfactant. Is. 本発明の別の一実施形態である第２の実施形態に係る紙デバイスの分解斜視図である。It is an exploded perspective view of the paper device which concerns on the 2nd Embodiment which is another Embodiment of this invention. 第１の実施形態の変形例に係る紙デバイスの分解斜視図である。It is an exploded perspective view of the paper device which concerns on the modification of 1st Embodiment. 第1の実施形態の別の変形例に係る紙デバイスを用いて行った検査結果を示すグラフである。It is a graph which shows the inspection result performed using the paper device which concerns on another modification of 1st Embodiment. 第１の実施形態の別の変形例に係る紙デバイスの分解斜視図である。It is an exploded perspective view of the paper device which concerns on another modification of 1st Embodiment.
＜第１の実施形態＞
　以下、本発明の一実施形態である第１の実施形態に係る紙デバイス１について説明する。紙デバイス１は、検体中の抗原（以下、目的抗原という。）の濃度を検出する装置である。本装置は、例えば、動物の血液等の検体に含まれるプロゲステロンを検出するために用いられる。目的抗原（本発明における被検出物質）としては、抗原抗体反応における抗原となり得る物質であれば、どのような物質であってもよい。プロゲステロンの他、例えば、ビオチン、コルチゾール、エストロゲン、テストステロン、プロラクチン、ＡＮＰ（心房性ナトリウム利尿ペプチド）、ＢＮＰ（脳性ナトリウム利尿ペプチド）、アフラトキシン、オクラトキシン、パツリン、デオキシニバレノール、ニバレノール等が対象とされてよい。なお、目的抗原と抗体（本発明における捕捉物質）の組み合わせの代わりに、アプタマーとその標的分子の組み合わせ、酵素と基質の組み合わせ、酵素とその阻害剤の組み合わせ等が用いられてもよい。アプタマーとしては、核酸アプタマー及びペプチドアプタマーのいずれでもよい。アプタマーの標的分子としては、例えば、金属イオン、複素環分子、アミノ酸、ヌクレオシド、ヌクレオチド、糖質、ペプチド、タンパク質、リボソーム等のタンパク質複合体、ウイルス、バクテリア、がん細胞等があり得る。このように、検出の対象となる物質（本発明における被検出物質）とその物質に結合する物質（本発明における捕捉物質）との組み合わせであれば、様々な物質の組み合わせが用いられてよい。 <First Embodiment>
Hereinafter, the paper device 1 according to the first embodiment, which is an embodiment of the present invention, will be described. The paper device 1 is a device that detects the concentration of an antigen (hereinafter referred to as a target antigen) in a sample. This device is used, for example, to detect progesterone contained in a sample such as animal blood. The target antigen (substance to be detected in the present invention) may be any substance as long as it can be an antigen in the antigen-antibody reaction. In addition to progesterone, for example, biotin, cortisol, estrogen, testosterone, prolactin, ANP (atrial natriuretic peptide), BNP (brain natriuretic peptide), aflatoxin, okratoxin, patulin, deoxynivalenol, nivalenol, etc. are targeted. Good. Instead of the combination of the target antigen and the antibody (capturing substance in the present invention), a combination of an aptamer and its target molecule, a combination of an enzyme and a substrate, a combination of an enzyme and its inhibitor, and the like may be used. The aptamer may be either a nucleic acid aptamer or a peptide aptamer. Target molecules of aptamers may include, for example, metal ions, heterocyclic molecules, amino acids, nucleosides, nucleotides, sugars, peptides, proteins, protein complexes such as ribosomes, viruses, bacteria, cancer cells and the like. As described above, as long as the combination of the substance to be detected (the substance to be detected in the present invention) and the substance bound to the substance (the trapping substance in the present invention), various combinations of substances may be used.
　紙デバイス１においては、少なくとも２つの物質が関わって生じる発色が利用されている。２つの物質の一方は酵素であり、後述の通り、抗原と結合した複合体として試料液に含まれた状態で用いられる。２つの物質の他方は発色基質１（色原体）であり、後述の通り、紙デバイス１に部分的に含まれている。酵素及び発色基質１の組み合わせによっては、発色のために発色基質２が必要となる場合がある。この場合、発色基質２は、後述の通り、例えば、試料液に含まれた状態で用いられる。かかる酵素、発色基質１及び２としては、表１に掲げる物質が使用されてもよい。表１に示すように、酵素の候補として西洋ワサビペルオキシダーゼ（ＨＲＰ）、β－ガラクトシダーゼ（β－ＧＡＬ）等のいずれか１つが使用可能であり、酵素の各候補に対して複数の発色基質１の候補が存在する。例えば、ＨＲＰに対しては１,２－フェニレンジアミン、３，３’，５，５’－テトラメチルベンジジン（ＴＭＢ）等のいずれか１つが使用可能である。発色基質２としては、酵素及び発色基質１の組み合わせに応じて決定される。例えば、酵素及び発色基質１としてＨＲＰ及び３，３’－ジアミノベンジジン（ＤＡＢ）が使用される場合、発色基質２としてＨ2Ｏ2が使用される。また、酵素及び発色基質１としてＨＲＰ及びＮ－エチル－Ｎ－（３－スルホプロピル）アニリンナトリウム（ＡＬＰＳ）が使用される場合、発色基質２としてＨ2Ｏ2及び４－アミノアンチピリン（４－ＡＡ）の両方が使用される。一方、例えば、酵素及び発色基質１としてアルカリホスファターゼ（ＡＬＰ）及びＤ－グルコース－６－リン酸（ＮＡＤＰ）が用いられる場合、発色基質２は不要である。 In the paper device 1, the color development generated by the involvement of at least two substances is utilized. One of the two substances is an enzyme, and as described later, it is used in a state of being contained in a sample solution as a complex bound to an antigen. The other of the two substances is the color-developing substrate 1 (chromogen), which is partially contained in the paper device 1 as described later. Depending on the combination of the enzyme and the color-developing substrate 1, the color-developing substrate 2 may be required for color development. In this case, the color-developing substrate 2 is used, for example, in a state of being contained in the sample solution, as described later. As such enzymes, coloring substrates 1 and 2, the substances listed in Table 1 may be used. As shown in Table 1, any one of horseradish peroxidase (HRP), β-galactosidase (β-GAL), etc. can be used as an enzyme candidate, and a plurality of color-developing substrates 1 are used for each enzyme candidate. There are candidates. For example, for HRP, any one of 1,2-phenylenediamine, 3,3', 5,5'-tetramethylbenzidine (TMB) and the like can be used. The color-developing substrate 2 is determined according to the combination of the enzyme and the color-developing substrate 1. For example, when HRP and 3,3'-diaminobenzidine (DAB) are used as the enzyme and coloring substrate 1, H 2 O 2 is used as the coloring substrate 2. In addition, when HRP and N-ethyl-N- (3-sulfopropyl) aniline sodium (ALPS) are used as the enzyme and the coloring substrate 1, H 2 O 2 and 4-aminoantipyrine (4-AA) are used as the coloring substrate 2. ) Are both used. On the other hand, for example, when alkaline phosphatase (ALP) and D-glucose-6-phosphate (NADP) are used as the enzyme and the color-developing substrate 1, the color-developing substrate 2 is unnecessary.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　次に、紙デバイス１の構成について、図１及び図２を参照しつつ説明する。以下、図２の左右方向をＡ方向とする。紙デバイス１は、濾紙等の吸水性を有するシートからなるシート１０、２０及び３０が積層した積層体２を備えている。以下、シートが積層された方向を積層方向という。積層体２は、互いに分離した２枚のシートが積層することで構成されてもよいし、１つのシートが２つに折り畳まれることで構成されていてもよい。後者の場合、例えば、シート１０～３０が互いに一列に繋がって１枚のシートを構成する。そして、この１枚のシートが、シート１０、２０及び３０の互いの境界線を折れ線として折り畳まれることで、シート１０～３０が積層した積層体２が形成される。 Next, the configuration of the paper device 1 will be described with reference to FIGS. 1 and 2. Hereinafter, the left-right direction in FIG. 2 is referred to as the A direction. The paper device 1 includes a laminated body 2 in which sheets 10, 20 and 30 made of a water-absorbent sheet such as filter paper are laminated. Hereinafter, the direction in which the sheets are laminated is referred to as a stacking direction. The laminated body 2 may be configured by laminating two sheets separated from each other, or may be configured by folding one sheet in two. In the latter case, for example, the sheets 10 to 30 are connected to each other in a row to form one sheet. Then, this one sheet is folded with the boundary lines of the sheets 10, 20 and 30 as a polygonal line to form a laminated body 2 in which the sheets 10 to 30 are laminated.
　積層体２は、図２に示すように、左上から右下に向かう方向に沿った斜線からなるハッチングの領域２ａと、右上から左下に向かう方向に沿った斜線からなるハッチングの領域２ｂとを含んでいる。領域２ｂは、疎水性のインクを含侵させた疎水性を有する領域である。以下、領域２ｂを疎水性領域２ｂという。これに対し、領域２ａにはかかるインクを含侵させていない。このため、領域２ａは親水性を有する領域である。以下、領域２ａを親水性領域２ａという。親水性領域２ａの一か所に水や水溶液からなる液体を含ませると、その液体は、時間の経過に伴ってその一か所から周囲へと浸透していくことによって広がっていく。液体の広がりは、親水性領域２ａと疎水性領域２ｂとの境界に到達するまで継続する。シート１０及び２０のそれぞれは親水性領域２ａ及び疎水性領域２ｂの両方を含んでいる。シート３０は全体が疎水性領域２ｂによって形成されている。 As shown in FIG. 2, the laminated body 2 includes a hatching region 2a composed of diagonal lines along the direction from the upper left to the lower right, and a hatching region 2b consisting of diagonal lines along the direction from the upper right to the lower left. I'm out. The region 2b is a hydrophobic region impregnated with the hydrophobic ink. Hereinafter, the region 2b is referred to as a hydrophobic region 2b. On the other hand, the region 2a is not impregnated with the ink. Therefore, the region 2a is a region having hydrophilicity. Hereinafter, the region 2a is referred to as a hydrophilic region 2a. When a liquid composed of water or an aqueous solution is contained in one place of the hydrophilic region 2a, the liquid spreads by permeating from the one place to the surroundings with the passage of time. The spread of the liquid continues until it reaches the boundary between the hydrophilic region 2a and the hydrophobic region 2b. Each of the sheets 10 and 20 contains both a hydrophilic region 2a and a hydrophobic region 2b. The entire sheet 30 is formed by the hydrophobic region 2b.
　疎水性領域２ｂのパターンは、例えば、疎水性のソリッドインクを用い、ソリッドインク用のインクジェットプリンターを使用してシート上に印刷することで形成する。パターンの印刷後、乾燥機を用いてシートを加熱することにより、シート上のインクを融解させつつシート中に浸透させる。これにより、シートの一方の表面から他方の表面まで疎水性のインクを含浸した疎水性領域２ｂが形成される。疎水性領域２ｂは、疎水性のインクの乾燥物がシート中に存在した状態となる。なお、シート上へのパターンの形成には、ソリッドインク式以外のインクジェットプリンターの他、スクリーン印刷、フォトリソグラフィー等が使用されてもよい。 The pattern of the hydrophobic region 2b is formed by printing on a sheet using, for example, a hydrophobic solid ink and an inkjet printer for the solid ink. After printing the pattern, the sheet is heated using a dryer to melt the ink on the sheet and allow it to penetrate into the sheet. As a result, a hydrophobic region 2b impregnated with the hydrophobic ink is formed from one surface of the sheet to the other surface. The hydrophobic region 2b is a state in which a dried product of hydrophobic ink is present in the sheet. In addition to an inkjet printer other than the solid ink type, screen printing, photolithography, or the like may be used to form the pattern on the sheet.
　積層体２は、図１及び図２に示すように、親水性領域２ａによって形成された流路４０を有している。流路４０は、シート１０及び２０のそれぞれの親水性領域２ａが互いに連通することで形成されている。シート１０には、Ａ方向に沿って一列に並んだ導入孔１１及び発色部１２が形成されている。導入孔１１は、図２においてシート１０の右端部寄りの位置に配置されている。導入孔１１は、図１に示すように円板形状を有している。導入孔１１には試料液が導入される。試料液は、目的抗原を含んだ水溶液である検体に、酵素によって標識された、検体由来でない抗原からなる複合体（本発明における第１標識物質）の水溶液を混合させたものである。以下、この複合体を標識抗原という。標識抗原における抗原としては、目的抗原と同じ物質が用いられる。また、発色のために発色基質２も必要となる場合、試料液はさらに発色基質２を含んだものとする。また、シート１０には発色部１２が形成されている。発色部１２は流路４０の一部である。発色部１２は、図２においてシート１０の左端部寄りの位置に配置されている。発色部１２は、図１に示すように、導入孔１１と同じ円板形状を有している。発色部１２には発色基質１が含まれている。 As shown in FIGS. 1 and 2, the laminated body 2 has a flow path 40 formed by the hydrophilic region 2a. The flow path 40 is formed by communicating the hydrophilic regions 2a of the sheets 10 and 20 with each other. The sheet 10 is formed with introduction holes 11 and color-developing portions 12 arranged in a row along the A direction. The introduction hole 11 is arranged at a position near the right end of the sheet 10 in FIG. The introduction hole 11 has a disk shape as shown in FIG. The sample solution is introduced into the introduction hole 11. The sample solution is a mixture of a sample, which is an aqueous solution containing a target antigen, with an aqueous solution of an enzyme-labeled complex (first labeled substance in the present invention) composed of an antigen not derived from the sample. Hereinafter, this complex is referred to as a labeled antigen. As the antigen in the labeled antigen, the same substance as the target antigen is used. If the color-developing substrate 2 is also required for color development, the sample solution is assumed to further contain the color-developing substrate 2. Further, a coloring portion 12 is formed on the sheet 10. The coloring unit 12 is a part of the flow path 40. The color-developing portion 12 is arranged at a position closer to the left end portion of the sheet 10 in FIG. As shown in FIG. 1, the color-developing portion 12 has the same disk shape as the introduction hole 11. The color-developing unit 12 contains a color-developing substrate 1.
　シート２０には、流路４０の一部である試料供給部２１、２つの中間部２２及び２つの連通部２４が形成されている。これらは、図２に示すように、右から試料供給部２１、連通部２４、中間部２２、連通部２４及び中間部２２の順に、Ａ方向に沿って一列に並んでいる。これらのうち、試料供給部２１及び中間部２２は、図１に示すように、シート１０の導入孔１１及び発色部１２と同じ円板形状を有している。試料供給部２１は、積層方向から見てシート１０の導入孔１１とちょうど重なる位置に配置されている。試料供給部２１には、目的抗原に対する抗体（以下、単に抗体という。）が固定されずに含まれている。なお、「固定されずに」とは、紙デバイス１の使用時に抗体が移動しないようにこれをシート２０に固定するための化学的な固定手段が用いられていないことをいう。試料供給部２１に試料液が供給されると、試料液中の目的抗原及び標識抗原が試料供給部２１に含まれた抗体と反応する。これにより、試料液中には、目的抗原－抗体複合体、標識抗原－抗体複合体（第２標識物質）、未反応の目的抗原及び未反応の標識抗原の４成分が生じる。 The sheet 20 is formed with a sample supply section 21, two intermediate sections 22, and two communication sections 24, which are a part of the flow path 40. As shown in FIG. 2, these are arranged in a line in the order of the sample supply section 21, the communication section 24, the intermediate section 22, the communication section 24, and the intermediate section 22 from the right in the A direction. Of these, the sample supply section 21 and the intermediate section 22 have the same disk shape as the introduction hole 11 and the color development section 12 of the sheet 10, as shown in FIG. The sample supply unit 21 is arranged at a position that exactly overlaps with the introduction hole 11 of the sheet 10 when viewed from the stacking direction. The sample supply unit 21 contains an antibody against the target antigen (hereinafter, simply referred to as an antibody) without being fixed. In addition, "without fixing" means that a chemical fixing means for fixing the antibody to the sheet 20 so as not to move when the paper device 1 is used is not used. When the sample solution is supplied to the sample supply unit 21, the target antigen and the labeled antigen in the sample solution react with the antibody contained in the sample supply unit 21. As a result, four components of the target antigen-antibody complex, the labeled antigen-antibody complex (second labeled substance), the unreacted target antigen, and the unreacted labeled antigen are generated in the sample solution.
　図２において最も左端に位置した中間部２２は、積層方向から見てシート１０の発色部１２とちょうど重なる位置に配置されている。この中間部２２と発色部１２は、積層方向に関して互いに接触している。各連通部２４は、図１に示すようにＡ方向に沿って直線状に延びている。２つの連通部２４は、試料供給部２１及び２つの中間部２２同士をＡ方向に連通させている。試料供給部２１、２つの中間部２２及び２つの連通部２４が互いに連通することにより、試料供給部２１から発色部１２に至る試料液の流路が形成されている。なお、２つの中間部２２及び２つの連通部２４からなる流路は、本発明における中間流路に対応する。 The intermediate portion 22 located at the leftmost end in FIG. 2 is arranged at a position that exactly overlaps with the coloring portion 12 of the sheet 10 when viewed from the stacking direction. The intermediate portion 22 and the coloring portion 12 are in contact with each other in the stacking direction. As shown in FIG. 1, each communication portion 24 extends linearly along the A direction. The two communication sections 24 communicate the sample supply section 21 and the two intermediate sections 22 with each other in the A direction. The sample supply section 21, the two intermediate sections 22, and the two communication sections 24 communicate with each other to form a flow path for the sample liquid from the sample supply section 21 to the coloring section 12. The flow path composed of the two intermediate portions 22 and the two communication portions 24 corresponds to the intermediate flow path in the present invention.
　試料供給部２１、２つの中間部２２及び２つの連通部２４には、全体に速度調節剤（本発明における速度調節物質に対応）が含まれている。速度調節剤は、流路４０を構成する紙繊維と上記４成分との相互作用を調節し、４成分の間に移動速度の差を生じさせるものである。上記４成分は、互いに分子サイズが異なると共に分子構造に起因する親水性が異なることから、紙繊維との間に作用する相互作用の大きさが異なる。速度調節剤は、これを利用し、成分同士で移動速度に差を生じさせるものである。速度調節剤には、未反応の標識抗原が標識抗原－抗体複合体より先に移動していくようにこれらの成分の移動速度に差を生じさせるものが選択されればよい。４成分中のその他の成分については速度差が生じても生じなくてもよい。速度調節剤として、例えば、ＰＢＳＴ（Ｐｈｏｓｐｈａｔｅ　ｂｕｆｆｅｒｅｄ　ｓａｌｉｎｅ　ｗｉｔｈ　Ｔｗｅｅｎ－２０）等の界面活性剤が用いられてよい。 The sample supply section 21, the two intermediate sections 22, and the two communication sections 24 contain a speed adjuster (corresponding to the speed control substance in the present invention) as a whole. The speed adjusting agent regulates the interaction between the paper fibers constituting the flow path 40 and the above four components, and causes a difference in moving speed between the four components. Since the above four components have different molecular sizes and different hydrophilicity due to the molecular structure, the magnitude of the interaction with the paper fiber is different. The speed adjuster utilizes this to cause a difference in moving speed between the components. As the rate regulator, one that causes a difference in the migration rate of these components so that the unreacted labeled antigen migrates before the labeled antigen-antibody complex may be selected. For the other components in the four components, a speed difference may or may not occur. As the speed regulator, for example, a surfactant such as PBST (Phosphate buffered saline with Tween-20) may be used.
［紙デバイス１の使用方法］
　以下、紙デバイス１の使用方法について図３を参照しつつ説明する。まず、試料液を調製する（ステップＳ１）。試料液の調製においては、標識抗原の水溶液を準備する。この水溶液は、あらかじめ設定された所定の濃度及び所定量とする。そして、この水溶液に、目的抗原を含んだ検体を所定量、混合する。このように取得した標識抗原及び目的抗原の水溶液を、必要に応じて発色基質２の水溶液をさらに混合して試料液とする。 [How to use the paper device 1]
Hereinafter, how to use the paper device 1 will be described with reference to FIG. First, a sample solution is prepared (step S1). In the preparation of the sample solution, an aqueous solution of the labeled antigen is prepared. This aqueous solution has a predetermined concentration and a predetermined amount set in advance. Then, a predetermined amount of a sample containing the target antigen is mixed with this aqueous solution. The aqueous solution of the labeled antigen and the target antigen thus obtained is further mixed with the aqueous solution of the color-developing substrate 2 as needed to prepare a sample solution.
　次に、調製した試料液を、図１に示すように導入孔１１から試料供給部２１に導入する（ステップＳ２）。試料液は、試料供給部２１から、２つの中間部２２及び２つの連通部２４を経て、発色部１２に向かって浸透していく。試料液中では、目的抗原及び標識抗原は、試料供給部２１に含まれた抗体と反応する。これにより、試料液中には、上記の通り、目的抗原－抗体複合体、標識抗原－抗体複合体、未反応の目的抗原及び未反応の標識抗原の４成分が生じる。目的抗原及び標識抗原は、互いに競争的に抗体と反応するので、試料液中、未反応の標識抗原の濃度は、検体中に元々含まれていた目的抗原の濃度に応じた大きさとなる。上記４成分は、発色部１２に向かって浸透していく試料液の流れに乗って移動する。一方、試料供給部２１、２つの中間部２２及び２つの連通部２４には速度調節剤が含まれている。この速度調節剤は、上記の通り、未反応の標識抗原が標識抗原－抗体複合体より先に移動していくようにこれらの間に速度差を生じさせる。これにより、未反応の標識抗原が標識抗原－抗体複合体より先に発色部１２に到達する。 Next, the prepared sample solution is introduced into the sample supply unit 21 through the introduction hole 11 as shown in FIG. 1 (step S2). The sample liquid permeates from the sample supply section 21 through the two intermediate sections 22 and the two communication sections 24 toward the coloring section 12. In the sample solution, the target antigen and the labeled antigen react with the antibody contained in the sample supply unit 21. As a result, as described above, four components of the target antigen-antibody complex, the labeled antigen-antibody complex, the unreacted target antigen, and the unreacted labeled antigen are generated in the sample solution. Since the target antigen and the labeled antigen react competitively with the antibody, the concentration of the unreacted labeled antigen in the sample solution becomes a magnitude corresponding to the concentration of the target antigen originally contained in the sample. The above four components move along with the flow of the sample liquid that permeates toward the coloring unit 12. On the other hand, the sample supply section 21, the two intermediate sections 22, and the two communication sections 24 contain a speed adjuster. As described above, this rate regulator creates a rate difference between the unreacted labeled antigens so that they migrate ahead of the labeled antigen-antibody complex. As a result, the unreacted labeled antigen reaches the color-developing portion 12 before the labeled antigen-antibody complex.
　なお、速度調節剤により速度差が生じた結果、上記４成分は、流路４０中で互いに異なる範囲に分布することになる。この上記４成分の流路４０での分布は、互いに明瞭に区別がつくほど狭いバンドを形成している必要はなく、広いバンドを形成してその一部が重なっていてもよい。試料供給部２１に滴下する試料液の体積は、流路４０全体をちょうど濡らす程度にすることにより、試料液が発色部１２に到達したときに試料液の流れが停止するとともに成分の移動も停止する。さらに、流路４０の乾燥が進むことにより、異なる速度で移動した成分が、流路４０内の各領域に互いに分離されることになる。未反応の標識抗原が発色部１２に到達すると、その標識抗原に標識された酵素と発色部１２の発色基質１とが（場合によって発色基質２も含めて）反応することで、発色部１２が発色する。発色の強度は、発色部１２に到達する未反応の標識抗原の濃度に応じた大きさとなる。一方、未反応の標識抗原の濃度は、上記の通り、検体中に元々含まれていた目的抗原の濃度に応じた大きさとなる。したがって、発色部１２の発色は、目的抗原の濃度が高いほど強くなる。 As a result of the speed difference caused by the speed adjusting agent, the above four components are distributed in different ranges in the flow path 40. The distribution of the above four components in the flow path 40 does not need to form a band so narrow that they can be clearly distinguished from each other, and a wide band may be formed and a part thereof may overlap. By setting the volume of the sample liquid dropped onto the sample supply unit 21 to just wet the entire flow path 40, the flow of the sample liquid is stopped and the movement of the components is also stopped when the sample liquid reaches the coloring unit 12. To do. Further, as the drying of the flow path 40 progresses, the components moved at different speeds are separated from each other into each region in the flow path 40. When the unreacted labeled antigen reaches the coloring part 12, the enzyme labeled with the labeled antigen reacts with the coloring substrate 1 of the coloring part 12 (including the coloring substrate 2 in some cases), so that the coloring part 12 Color develops. The intensity of color development becomes a magnitude corresponding to the concentration of the unreacted labeled antigen that reaches the color-developing portion 12. On the other hand, the concentration of the unreacted labeled antigen becomes a magnitude corresponding to the concentration of the target antigen originally contained in the sample, as described above. Therefore, the color development of the coloring unit 12 becomes stronger as the concentration of the target antigen increases.
　次に、紙デバイス１をデジタルカメラで撮影する（ステップＳ３）。撮影は、発色部１２が発色した状況が撮影範囲に含まれるように実施される。次に、ステップＳ３の撮影結果に基づいて、検体中の目的抗原の濃度に関する分析が実行される（ステップＳ４）。ステップＳ４の分析には、コンピュータによるデジタル画像解析が用いられる。具体的には、例えば、撮影画像における発色部１２に対応する領域内に存在する各画素の画素値（例えば、レッド、グリーン及びブルー（ＲＧＢ）値）を演算した値として、発色に対応する色成分の強度を示す発色強度値（例えば、シアン、マゼンタ及びイエロー（ＣＭＹ）のうちのシアン（Ｃ）値）が導出される。次に、発色部１２に対応する領域内の画素に関して発色強度値の平均値が算出される。次に、コンピュータは、発色強度値の平均値を所定の参照データと照らし合わせることで検体中の目的抗原の濃度を導出する。参照データは、濃度と発色強度の平均値との関係を示すデータである。参照データは、例えば、互いに異なる既知の濃度の目的抗原を含んだ複数の検体に関してステップＳ１及びＳ２と同様に発色部１２を発色させた結果に基づいてあらかじめ取得される。上記のように導出された目的抗原の濃度は、コンピュータに接続された、又はコンピュータに搭載されたディスプレイ等の出力装置によって出力される。 Next, take a picture of the paper device 1 with a digital camera (step S3). The photographing is carried out so that the situation in which the coloring unit 12 develops color is included in the photographing range. Next, an analysis regarding the concentration of the target antigen in the sample is executed based on the imaging result of step S3 (step S4). A computer-based digital image analysis is used for the analysis in step S4. Specifically, for example, the color corresponding to the color development is obtained by calculating the pixel value (for example, red, green, and blue (RGB) values) of each pixel existing in the region corresponding to the color development unit 12 in the captured image. A color development intensity value indicating the intensity of the component (for example, the cyan (C) value of cyan, magenta, and yellow (CMY)) is derived. Next, the average value of the color development intensity values is calculated for the pixels in the region corresponding to the color development unit 12. Next, the computer derives the concentration of the target antigen in the sample by comparing the average value of the color development intensity values with the predetermined reference data. The reference data is data showing the relationship between the density and the average value of the color development intensity. The reference data is acquired in advance based on, for example, the result of coloring the coloring unit 12 in the same manner as in steps S1 and S2 for a plurality of samples containing target antigens having different known concentrations. The concentration of the target antigen derived as described above is output by an output device such as a display connected to or mounted on the computer.
［本実施形態の概要］
　以上説明した第１の実施形態によると、未反応の標識抗原及び標識抗原－抗体複合体は酵素によって標識されており、発色部１２には発色基質１が含まれている。したがって、発色部１２にこれらの標識物質が到達すると、標識物質中の酵素と発色基質１との作用（場合によって発色基質２も含めた作用）に応じて発色部１２が発色する。発色部１２における発色は、発色部１２に到達したこれらの標識物質の濃度に応じた強度となる。 [Outline of the present embodiment]
According to the first embodiment described above, the unreacted labeled antigen and the labeled antigen-antibody complex are labeled with an enzyme, and the coloring portion 12 contains the coloring substrate 1. Therefore, when these labeling substances reach the coloring unit 12, the coloring unit 12 develops color according to the action of the enzyme in the labeling substance and the coloring substrate 1 (in some cases, the action including the coloring substrate 2). The color development in the color development unit 12 has an intensity corresponding to the concentration of these labeling substances that have reached the color development unit 12.
　一方、流路に含まれている速度調節剤により、未反応の標識抗原は標識抗原－抗体複合体より前に発色部に到達する。したがって、未反応の標識抗原が発色部１２に到達してから標識抗原－抗体複合体が発色部１２に到達するまでに発色部１２において発生する発色の強度を評価することで、未反応の標識抗原の濃度を評価することができる。流路４０中の未反応の標識抗原の濃度は、検体中の抗原の濃度に応じた大きさとなる。このため、発色部１２の発色の強度を評価することで、検体中の抗原の濃度を評価できる。 On the other hand, the unreacted labeled antigen reaches the color-developing part before the labeled antigen-antibody complex due to the speed regulator contained in the flow path. Therefore, unreacted labeling is performed by evaluating the intensity of color development generated in the coloring unit 12 after the unreacted labeled antigen reaches the coloring unit 12 until the labeled antigen-antibody complex reaches the coloring unit 12. The concentration of antigen can be evaluated. The concentration of the unreacted labeled antigen in the flow path 40 becomes a magnitude corresponding to the concentration of the antigen in the sample. Therefore, the concentration of the antigen in the sample can be evaluated by evaluating the color development intensity of the color development unit 12.
　さらに、本実施形態においては、抗体が流路４０に固定されず、試料供給部２１に含まれている。したがって、本実施形態に係る紙デバイス１は、製造工程において固定化工程を必要としない。よって、固定化工程を必要とする従来技術と比べ、製造が容易である。 Further, in the present embodiment, the antibody is not fixed in the flow path 40 but is included in the sample supply unit 21. Therefore, the paper device 1 according to the present embodiment does not require an immobilization step in the manufacturing process. Therefore, it is easier to manufacture than the conventional technique that requires a fixing step.
　また、本実施形態においては、試料液が発色部１２に到達したときに試料液の流れが停止するとともに成分の移動が停止するように試料液の量が調整されている。つまり、標識抗原－抗体複合体が発色部１２に到達しない。よって、未反応の標識抗原が発色部１２を発色させる強度に基づき、検体中の抗原の濃度を適切に評価できる。 Further, in the present embodiment, the amount of the sample liquid is adjusted so that the flow of the sample liquid is stopped and the movement of the components is stopped when the sample liquid reaches the coloring portion 12. That is, the labeled antigen-antibody complex does not reach the coloring part 12. Therefore, the concentration of the antigen in the sample can be appropriately evaluated based on the intensity at which the unreacted labeled antigen develops the color of the coloring portion 12.
　また、本実施形態においては、シート１０～３０が積層された積層体２中に親水性領域２ａからなる流路４０が形成されている。このため、流路４０において試料液の乾燥が過度に進むのが抑制される。特に、シート３０は全体が疎水性領域によって形成されている。このため、シート２０の下面からの試料液の乾燥がシート３０によって抑制されている。 Further, in the present embodiment, the flow path 40 composed of the hydrophilic region 2a is formed in the laminated body 2 in which the sheets 10 to 30 are laminated. Therefore, excessive drying of the sample solution in the flow path 40 is suppressed. In particular, the sheet 30 is entirely formed by hydrophobic regions. Therefore, the drying of the sample liquid from the lower surface of the sheet 20 is suppressed by the sheet 30.
　また、本実施形態においては、発色部１２はシート１０に形成されている一方で、試料供給部２１、２つの中間部２２及び２つの連通部２４はシート２０に形成されている。つまり、流路４０は、発色部１２と他の部分とが互いに別のシートに形成されている。仮に、流路４０全体が１つのシートに形成されているとすると、１枚のシートに沿った方向に関する浸透のみによって発色部１２への試料液の浸透が進んでいく。これによって、発色部１２における発色が進行する。したがって、シートに沿った方向に関する発色のむらが生じやすい。これに対し、本実施形態においては、シート１０の中間部２２からシート２０の発色部１２へと、シート間を跨ぐように試料液の浸透が進むことによって発色が進んでいく。よって、シートに沿った方向に関する発色のむらが生じにくい。したがって、発色部１２の発色強度に基づいて検体中の抗原の濃度を適切に評価できる。 Further, in the present embodiment, the color-developing portion 12 is formed on the sheet 10, while the sample supply portion 21, the two intermediate portions 22, and the two communicating portions 24 are formed on the sheet 20. That is, in the flow path 40, the color-developing portion 12 and other portions are formed on separate sheets. Assuming that the entire flow path 40 is formed on one sheet, the permeation of the sample solution into the color-developing portion 12 proceeds only by permeation in the direction along one sheet. As a result, the color development in the color development unit 12 progresses. Therefore, uneven color development in the direction along the sheet is likely to occur. On the other hand, in the present embodiment, the color development proceeds by the permeation of the sample liquid from the intermediate portion 22 of the sheet 10 to the coloring portion 12 of the sheet 20 so as to straddle the sheets. Therefore, uneven color development in the direction along the sheet is unlikely to occur. Therefore, the concentration of the antigen in the sample can be appropriately evaluated based on the color development intensity of the color development unit 12.
［実施例１］
　以下、第１の実施形態に関する実施例１について説明する。実施例１では、目的抗原をプロゲステロン、標識抗原をプロゲステロン－ＨＲＰ複合体、抗体を抗プロゲステロン抗体とした。発色基質１はＴＭＢ、発色基質２はＨ2Ｏ2とした。 [Example 1]
Hereinafter, Example 1 relating to the first embodiment will be described. In Example 1, the target antigen was progesterone, the labeled antigen was a progesterone-HRP complex, and the antibody was an anti-progesterone antibody. The color-developing substrate 1 was TMB, and the color-developing substrate 2 was H 2 O 2 .
　まず、紙デバイス１を以下の通りに作製した。ソリッドインク式のインクジェットプリンターを用い、図１に示すパターンを濾紙に形成した。その後、パターンを形成した濾紙を加熱することで、濾紙上で固化したソリッドインクを融解し、濾紙中に浸透させた。これにより、親水性領域からなる流路４０の外縁を画定する疎水性領域のパターンを濾紙上に形成した。次に、濾紙の流路４０に対応する領域に所定の濃度のＰＢＳＴを含侵させた。次に、抗体溶液を試料供給部２１に滴下した後、濾紙を所定時間乾燥させた。次に、濾紙の発色部１２に対応する領域にＴＭＢ溶液を滴下した後、濾紙を乾燥させた。 First, the paper device 1 was manufactured as follows. The pattern shown in FIG. 1 was formed on the filter paper using a solid ink type inkjet printer. Then, by heating the filter paper in which the pattern was formed, the solid ink solidified on the filter paper was melted and permeated into the filter paper. As a result, a pattern of the hydrophobic region defining the outer edge of the flow path 40 composed of the hydrophilic region was formed on the filter paper. Next, the region corresponding to the flow path 40 of the filter paper was impregnated with PBST at a predetermined concentration. Next, the antibody solution was added dropwise to the sample supply unit 21, and then the filter paper was dried for a predetermined time. Next, the TMB solution was dropped onto the region corresponding to the color-developing portion 12 of the filter paper, and then the filter paper was dried.
　以上のように作製された紙デバイス１を用いた検査を次の通りに行った。まず、目的抗原、標識抗原及びＨ2Ｏ2を含む試料液を調製し、その適量を、導入孔１１を通じて試料供給部２１に滴下した。滴下した試料液が発色部１２に到達した後に所定時間待って紙デバイス１の画像をデジタルカメラで撮影した。上記ステップＳ４と同様、撮影画像をデジタル解析することにより、特定の色み（シアン）の濃さを発色強度として取得した。図４は、目的抗原（プロゲステロン）の濃度が異なる試料液を使用して上記検査を行った場合の発色強度の違いを表している。図４によると、プロゲステロンの濃度が高くなるのに応じて発色強度も高くなっている。これは、目的抗原の濃度を上記検査により定量的に測定できることを示している。 The inspection using the paper device 1 produced as described above was performed as follows. First, a sample solution containing the target antigen, labeled antigen and H 2 O 2 was prepared, and an appropriate amount thereof was dropped onto the sample supply unit 21 through the introduction hole 11. After the dropped sample solution reached the coloring unit 12, the image of the paper device 1 was taken with a digital camera after waiting for a predetermined time. Similar to step S4 above, the captured image was digitally analyzed to obtain the intensity of a specific color (cyan) as the color development intensity. FIG. 4 shows the difference in color development intensity when the above test is performed using sample solutions having different concentrations of the target antigen (progesterone). According to FIG. 4, the color development intensity increases as the progesterone concentration increases. This indicates that the concentration of the target antigen can be quantitatively measured by the above test.
［実施例２］
　流路４０にＰＢＳＴを含侵させないこと以外、実施例１と同様に紙デバイス１を作製した。また、Ｔｗｅｅｎ－２０を様々な濃度としたＰＢＳＴを流路４０に含侵させた複数の紙デバイス１を実施例１と同様に作製した。これらの紙デバイス１に関して、目的抗原の濃度を０ｎｇ／ｍＬとしたもの（つまり、目的抗原を含まない試料液）及び５ｎｇ／ｍＬとしたものの２種類の試料液を実施例１と同様に調製して実施例１の検査を行った。図５の白丸（〇）は、濃度を０ｎｇ／ｍＬとした試料液に関して各紙デバイス１を用いた検査の結果（４回の検査結果の平均値）を示している。図５の黒丸（●）は、濃度を５ｎｇ／ｍＬとした試料液に関して各紙デバイス１を用いた検査の結果（４回の検査結果の平均値）を示している。図５の「未処理」は、流路４０に何も含侵させない紙デバイス１に対応する。図５の界面活性剤の濃度０％は、界面活性剤を含まないＰＢＳを含浸させた紙デバイス１に対応する。図５の〇に示すように、界面活性剤の濃度が大きくなると発色強度が増加する。これは、界面活性剤の濃度が低いときに発色部１２に到達するのは、未反応の標識抗原がほとんどであるのに対し、界面活性剤の濃度が高くなると、抗体と反応した標識抗原も発色部１２に到達するようになるためである。この結果は、界面活性剤の濃度を調節することによって発色部１２まで到達できる未反応の標識抗原の量を調節できることを表している。また、図５の〇及び●を比較すると、界面活性剤の濃度０．０１％において発色強度に差異が発生している。これは、界面活性剤の濃度が大きくなるのに伴って目的抗原の濃度０ｎｇ／ｍＬ及び５ｎｇ／ｍＬの間で発色強度の差異が生じることを示している。なお、図５において、界面活性剤の濃度が０．００１％のときに、目的抗原の濃度０ｎｇ／ｍＬの発色強度が目的抗原の濃度５ｎｇ／ｍＬの濃度より大きくなっている。これは、目的抗原の濃度０ｎｇ／ｍＬに関する４回の検査のうちの１回において高い発色強度が検出された結果である。残り３回の検査においては、目的抗原の濃度５ｎｇ／ｍＬにおける結果とほぼ同じ値となった。 [Example 2]
A paper device 1 was produced in the same manner as in Example 1 except that the flow path 40 was not impregnated with PBST. In addition, a plurality of paper devices 1 in which the flow path 40 was impregnated with PBST having Tween-20 at various concentrations were produced in the same manner as in Example 1. With respect to these paper devices 1, two types of sample solutions having a concentration of the target antigen of 0 ng / mL (that is, a sample solution containing no target antigen) and 5 ng / mL were prepared in the same manner as in Example 1. The inspection of Example 1 was carried out. The white circles (◯) in FIG. 5 indicate the results of inspections using each paper device 1 (the average value of the results of four inspections) for the sample liquid having a concentration of 0 ng / mL. The black circles (●) in FIG. 5 indicate the results of inspections using each paper device 1 (the average value of the results of four inspections) for the sample solution having a concentration of 5 ng / mL. “Untreated” in FIG. 5 corresponds to the paper device 1 in which the flow path 40 is not impregnated with anything. The surfactant concentration of 0% in FIG. 5 corresponds to the surfactant-free PBS-impregnated paper device 1. As shown by ◯ in FIG. 5, the color development intensity increases as the concentration of the surfactant increases. This is because most of the unreacted labeled antigens reach the color-developing portion 12 when the concentration of the surfactant is low, whereas the labeled antigens that have reacted with the antibody also reach the colored portion 12 when the concentration of the surfactant is high. This is because the color-developing portion 12 is reached. This result indicates that the amount of unreacted labeled antigen that can reach the color-developing portion 12 can be adjusted by adjusting the concentration of the surfactant. Further, when comparing 〇 and ● in FIG. 5, there is a difference in color development intensity at a concentration of the surfactant of 0.01%. This indicates that as the concentration of the surfactant increases, a difference in color development intensity occurs between the concentration of the target antigen of 0 ng / mL and 5 ng / mL. In FIG. 5, when the concentration of the surfactant is 0.001%, the color development intensity of the target antigen concentration of 0 ng / mL is larger than the concentration of the target antigen concentration of 5 ng / mL. This is a result of the detection of high color development intensity in one of the four tests for the concentration of the target antigen of 0 ng / mL. In the remaining three tests, the values were almost the same as the results at a concentration of the target antigen of 5 ng / mL.
　なお、図５には示していないが、さらに界面活性剤の濃度を増加させると、発色強度は、目的抗原の濃度０ｎｇ／ｍＬ及び５ｎｇ／ｍＬのいずれにおいても最大となると共に、両方の目的抗原濃度に関して同じ大きさ且つ界面活性剤の濃度に対して一定値となった。これらのことは、ある範囲の濃度の界面活性剤で処理することにより、目的抗原の濃度に応じた発色強度が得られることを示している。つまり、目的抗原の濃度に依存した発色強度の差が生じるためには、界面活性剤の濃度がＣ１以上且つＣ２（＞Ｃ１）以下である必要がある。ここで、Ｃ１は、界面活性剤の濃度がこの値より低いと発色強度が小さいため目的抗原の濃度に依存した発色強度が観測しにくくなるような下限値である（図５参照）。また、Ｃ２は、上記の通り、界面活性剤の濃度がこの値より高いと、目的抗原濃度に関わらず最大の発色強度となるような上限値である。 Although not shown in FIG. 5, when the concentration of the surfactant is further increased, the color development intensity becomes maximum at both the target antigen concentrations of 0 ng / mL and 5 ng / mL, and both target antigens. The concentration was the same and was constant with respect to the concentration of the surfactant. These facts indicate that the color development intensity corresponding to the concentration of the target antigen can be obtained by treating with a surfactant having a concentration in a certain range. That is, in order for the difference in color development intensity depending on the concentration of the target antigen to occur, the concentration of the surfactant must be C1 or more and C2 (> C1) or less. Here, C1 is a lower limit value that makes it difficult to observe the color development intensity depending on the concentration of the target antigen because the color development intensity is small when the concentration of the surfactant is lower than this value (see FIG. 5). Further, as described above, C2 is an upper limit value at which the maximum color development intensity is obtained regardless of the target antigen concentration when the concentration of the surfactant is higher than this value.
＜第２の実施形態＞
　以下、本発明の一実施形態である第２の実施形態に関して説明する。第２の実施形態は、第１の実施形態に係る紙デバイス１において、試料供給部２１に抗体が含まれていないものが用いられる。また、試料液の調製には、標識抗原の代わりに、酵素によって標識された抗体（以下、標識抗体という。）からなる複合体（本発明における第１標識物質）が用いられる。この場合、試料液中には、目的抗原と標識抗体との反応によって生じる標識抗体－目的抗原複合体、未反応の標識抗体及び未反応の目的抗原の３成分が生じることになる。かかる試料液が試料供給部２１に供給されると、流路４０中の速度調節剤の作用により、未反応の標識抗体と標識抗体－目的抗原複合体との間で流路４０中の移動速度に差が生じる。これによって、未反応の標識抗体のみが発色部１２に到達することで、第１の実施形態と同様、検体中の目的抗原の濃度に応じた強度で発色部１２が発色することになる。したがって、上記ステップＳ３及びＳ４と同様に、紙デバイス１の撮影結果のデジタル画像解析に基づき、検体中の目的抗原の濃度が導出可能である。 <Second embodiment>
Hereinafter, a second embodiment, which is an embodiment of the present invention, will be described. In the second embodiment, in the paper device 1 according to the first embodiment, the sample supply unit 21 does not contain an antibody. Further, in the preparation of the sample solution, a complex (first labeled substance in the present invention) composed of an antibody labeled with an enzyme (hereinafter, referred to as a labeled antibody) is used instead of the labeled antigen. In this case, three components, a labeled antibody-target antigen complex, an unreacted labeled antibody, and an unreacted target antigen, which are generated by the reaction of the target antigen and the labeled antibody, are generated in the sample solution. When such a sample solution is supplied to the sample supply unit 21, the moving speed in the flow path 40 between the unreacted labeled antibody and the labeled antibody-target antigen complex due to the action of the speed regulator in the flow path 40. Makes a difference. As a result, only the unreacted labeled antibody reaches the color-developing portion 12, so that the color-developing portion 12 develops color with an intensity corresponding to the concentration of the target antigen in the sample as in the first embodiment. Therefore, similarly to steps S3 and S4, the concentration of the target antigen in the sample can be derived based on the digital image analysis of the imaging result of the paper device 1.
　第２の実施形態において、目的抗原の分子サイズが小さいために、未反応の標識抗体と流路４０の紙繊維の相互作用と、標識抗体－目的抗原複合体と流路４０の紙繊維の相互作用との間に十分な差が生じない（よって、移動速度にも差が生じにくい）場合には、図６に示す紙デバイス１００が採用されてもよい。紙デバイス１００は、濾紙製のシート１１０及び１２０が積層した積層体１０２を備えている。積層体１０２には、紙デバイス１と同様、疎水性領域によって外縁が画定された、親水性領域からなる流路１４０が形成されている。シート１１０には、Ａ方向に沿って一列に並んだ導入孔１１１及び流路１４０の一部である発色部１１２が形成されている。発色部１１２には発色基質１が含まれている。シート１２０には、流路１４０の一部である試料供給部１２１、抗体設置部１２２、２つの中間部１２３及び３つの連通部１２４が形成されている。これらは、Ａ方向に関する一端から他端に向かって、試料供給部１２１、連通部１２４、抗体設置部１２２、連通部１２４、中間部１２３、連通部１２４及び中間部１２３の順にＡ方向に沿って一列に並んでいる。試料供給部１２１は、積層方向から見てシート１１０の導入孔１１１とちょうど重なる位置に配置されている。また、Ａ方向に関して最も外側の中間部１２３は、積層方向から見て発色部１１２とちょうど重なる位置に配置されている。この中間部１２３と発色部１１２は、積層方向に関して互いに接触している。 In the second embodiment, due to the small molecular size of the target antigen, the interaction between the unreacted labeled antibody and the paper fiber in the flow path 40 and the mutual interaction between the labeled antibody-target antigen complex and the paper fiber in the flow path 40. The paper device 100 shown in FIG. 6 may be adopted when there is no sufficient difference between the action and the action (and therefore, the movement speed is unlikely to be different). The paper device 100 includes a laminated body 102 in which filter paper sheets 110 and 120 are laminated. Similar to the paper device 1, the laminated body 102 is formed with a flow path 140 composed of a hydrophilic region whose outer edge is defined by a hydrophobic region. The sheet 110 is formed with introduction holes 111 arranged in a row along the A direction and a coloring portion 112 which is a part of the flow path 140. The color-developing unit 112 contains a color-developing substrate 1. The sheet 120 is formed with a sample supply section 121, an antibody setting section 122, two intermediate sections 123, and three communication sections 124, which are part of the flow path 140. From one end to the other end in the A direction, the sample supply section 121, the communication section 124, the antibody installation section 122, the communication section 124, the intermediate section 123, the communication section 124, and the intermediate section 123 are arranged in this order along the A direction. They are lined up in a row. The sample supply unit 121 is arranged at a position that exactly overlaps with the introduction hole 111 of the sheet 110 when viewed from the stacking direction. Further, the outermost intermediate portion 123 in the A direction is arranged at a position that exactly overlaps with the coloring portion 112 when viewed from the stacking direction. The intermediate portion 123 and the coloring portion 112 are in contact with each other in the stacking direction.
　試料供給部１２１、抗体設置部１２２、２つの中間部１２３及び３つの連通部１２４には、速度調節剤が全体に含まれている。速度調節剤として、例えば、ＰＢＳＴ等の界面活性剤が用いられてよい。抗体設置部１２２には、標識抗体に使用される抗体とは異なる、目的抗原に対する第２抗体が固定されずに含まれている。第２抗体とは、標識抗体と未反応の目的抗原のみならず、標識抗体－目的抗原複合体中の目的抗原とも結合可能な抗体である。 The sample supply unit 121, the antibody installation unit 122, the two intermediate units 123, and the three communication units 124 contain a speed adjuster as a whole. As the speed regulator, for example, a surfactant such as PBST may be used. The antibody setting unit 122 contains a second antibody against the target antigen, which is different from the antibody used for the labeled antibody, without being fixed. The second antibody is an antibody that can bind not only to the target antigen that has not reacted with the labeled antibody but also to the target antigen in the labeled antibody-target antigen complex.
　標識抗体－目的抗原複合体、未反応の標識抗体及び未反応の目的抗原の３成分を含んだ試料液が、導入孔１１１を通じて試料供給部１２１に滴下されると、試料液が抗体設置部１２２に到達した際、試料液中の物質と第２抗体とが反応する。これにより、流路１４０には、標識抗体－目的抗原－第２抗体複合体、標識抗体－目的抗原複合体、目的抗原－第２抗体複合体、未反応の標識抗体、未反応の第２抗体及び未反応の目的抗原の６成分が生じることになる。ここで、試料液中の物質に対する第２抗体の量を調整することにより、標識抗体－目的抗原複合体の量を無視できるほど小さくなるようにする。また、速度調節剤の濃度を調節することで、標識抗体－目的抗原－第２抗体複合体と未反応の標識抗体の間で流路１４０中の移動速度に差が生じるようにする。そして、試料液中の標識抗体を所定量とすることで、検体中の目的抗原の濃度に応じた濃度で標識抗体－目的抗原－第２抗体複合体と未反応の標識抗体が流路１４０に存在することになる。したがって、発色部１１２に到達した未反応の標識抗体の量を第１の実施形態と同様に測定すれば、検体中の目的抗原の濃度を求めることができる。本構成によれば、標識抗体－目的抗原－第２抗体複合体と未反応の標識抗体の間で分子サイズの差が生じやすい。このため、未反応の標識抗体と流路４０の紙繊維の相互作用と、標識抗体－目的抗原複合体と流路４０の紙繊維の相互作用との間で十分な差が生じやすく、移動速度にも差が生じやすい。 When a sample solution containing the labeled antibody-target antigen complex, unreacted labeled antibody, and unreacted target antigen is dropped onto the sample supply section 121 through the introduction hole 111, the sample solution is dropped into the antibody setting section 122. When it reaches, the substance in the sample solution reacts with the second antibody. As a result, in the flow path 140, a labeled antibody-target antigen-second antibody complex, a labeled antibody-target antigen complex, a target antigen-second antibody complex, an unreacted labeled antibody, and an unreacted second antibody And 6 components of the unreacted target antigen will be produced. Here, by adjusting the amount of the second antibody against the substance in the sample solution, the amount of the labeled antibody-target antigen complex is made negligibly small. In addition, by adjusting the concentration of the rate-regulating agent, a difference in the moving speed in the flow path 140 is made between the labeled antibody-target antigen-second antibody complex and the unreacted labeled antibody. Then, by setting the labeled antibody in the sample solution to a predetermined amount, the labeled antibody-target antigen-second antibody complex and the unreacted labeled antibody are transferred to the flow path 140 at a concentration corresponding to the concentration of the target antigen in the sample. It will exist. Therefore, if the amount of the unreacted labeled antibody that has reached the color-developing portion 112 is measured in the same manner as in the first embodiment, the concentration of the target antigen in the sample can be determined. According to this configuration, a difference in molecular size is likely to occur between the labeled antibody-target antigen-second antibody complex and the unreacted labeled antibody. Therefore, a sufficient difference is likely to occur between the interaction between the unreacted labeled antibody and the paper fiber in the flow path 40 and the interaction between the labeled antibody-target antigen complex and the paper fiber in the flow path 40, and the migration speed is high. Is also likely to make a difference.
＜変形例＞
　以上は、本発明の好適な実施形態についての説明であるが、本発明は上述の実施形態に限られるものではなく、課題を解決するための手段に記載された範囲の限りにおいて様々な変更が可能なものである。 <Modification example>
The above is a description of a preferred embodiment of the present invention, but the present invention is not limited to the above-described embodiment, and various changes can be made as long as it is described in the means for solving the problem. It is possible.
　例えば、上述の第１の実施形態に係る紙デバイス１の代わりに、図７に示す紙デバイス２００が用いられてもよい。紙デバイス２００は、濾紙製のシート２１０及び２２０並びに上述の実施形態と同じシート２０が積層した積層体２０２を備えている。積層体２０２には、紙デバイス１と同様、疎水性領域によって外縁が画定された、親水性領域からなる流路２４０が形成されている。シート２１０には、Ａ方向に沿って一列に並んだ導入孔２１１及び検出窓２１２が形成されている。シート２２０には、Ａ方向に沿って一列に並んだ基質設置部２２１及び発色部２２２が形成されている。基質設置部２２１は、積層方向から見て導入孔２１１及び試料供給部２１とちょうど重なる位置に配置されており、試料供給部２１と積層方向に関して接触している。発色部２２２は、積層方向から見て検出窓２１２及びシート２０において最も外側の中間部２２とちょうど重なる位置に配置されており、中間部２２と積層方向に関して接触している。発色部２２２には発色基質１が含まれている。以上により、シート２２０の基質設置部２２１から、シート２０の試料供給部２１、２つの中間部２２及び２つの連通部２４を経て、シート２２０の発色部２２２に至る流路２４０が構成されている。 For example, the paper device 200 shown in FIG. 7 may be used instead of the paper device 1 according to the first embodiment described above. The paper device 200 includes sheets 210 and 220 made of filter paper and a laminate 202 in which the same sheets 20 as in the above-described embodiment are laminated. Similar to the paper device 1, the laminated body 202 is formed with a flow path 240 composed of a hydrophilic region whose outer edge is defined by a hydrophobic region. The sheet 210 is formed with introduction holes 211 and detection windows 212 arranged in a row along the A direction. The sheet 220 is formed with a substrate setting portion 221 and a coloring portion 222 arranged in a row along the A direction. The substrate setting section 221 is arranged at a position where it just overlaps the introduction hole 211 and the sample supply section 21 when viewed from the stacking direction, and is in contact with the sample supply section 21 in the stacking direction. The color-developing portion 222 is arranged at a position just overlapping the outermost intermediate portion 22 in the detection window 212 and the sheet 20 when viewed from the stacking direction, and is in contact with the intermediate portion 22 in the stacking direction. The color-developing unit 222 contains a color-developing substrate 1. As described above, the flow path 240 is configured from the substrate installation section 221 of the sheet 220 to the sample supply section 21, the two intermediate sections 22 and the two communication sections 24 of the sheet 20 to the coloring section 222 of the sheet 220. ..
　紙デバイス２００は、例えば、酵素としてＨＲＰを、発色基質２としてＨ2Ｏ2を使用する場合に採用される。この場合、Ｈ2Ｏ2は、試料液ではなく基質設置部２２１に含まれている。紙デバイス２００の調製時、過酸化水素尿素水溶液を基質設置部２２１に対応する領域に塗布した後、乾燥させることにより、Ｈ2Ｏ2が乾燥後も安定に紙デバイス２００に保持される。 The paper device 200 is adopted, for example, when HRP is used as an enzyme and H 2 O 2 is used as a coloring substrate 2. In this case, H 2 O 2 is contained in the substrate setting section 221 instead of the sample solution. When preparing the paper device 200, H 2 O 2 is stably retained in the paper device 200 even after drying by applying an aqueous solution of hydrogen peroxide urea to the region corresponding to the substrate setting portion 221 and then drying the paper device 200.
　なお、基質設置部として、基質設置部２２１の代わりに中間部２２が用いられてもよい。つまり、紙デバイス２００における基質設置部２２１に対応する箇所にはＨ2Ｏ2が含まれておらず、いずれか１つ又は２つの中間部２２にＨ2Ｏ2が含まれていてもよい。例えば、紙デバイス２００の調製過程において、いずれかの中間部２２の部位に過酸化水素尿素水溶液を塗布して乾燥させることで、中間部２２にＨ2Ｏ2を設置することができる。乾燥は、発色基質１の溶液を発色部２２２の部位に塗布した後に行ってもよい。また、試料供給部２１に抗体を設置することに代えて、又は加えて、いずれか１つ又は２つの中間部２２に抗体を設置したり、試料供給部２１に近い方の中間部２２及びその両側の連通部２４に抗体を設置したりしてもよい。 In addition, as a substrate setting part, an intermediate part 22 may be used instead of a substrate setting part 221. That is, the portion corresponding to the substrate installing part 221 in the paper device 200 does not include the H 2 O 2, any one or two of the intermediate portion 22 may include H 2 O 2. For example, in the process of preparing the paper device 200, H 2 O 2 can be installed in the intermediate portion 22 by applying an aqueous hydrogen peroxide urea solution to the portion of the intermediate portion 22 and drying the portion. Drying may be performed after applying the solution of the color-developing substrate 1 to the portion of the color-developing portion 222. Further, instead of or in addition to installing the antibody in the sample supply unit 21, an antibody is installed in any one or two intermediate portions 22, or the intermediate portion 22 closer to the sample supply unit 21 and its intermediate portion 22 and the like. Antibodies may be installed in the communication portions 24 on both sides.
　図８は、紙デバイス２００の構成を次の通りに変更した紙デバイスを用いて行った検査結果を示す。本検査に係る紙デバイスでは、試料供給部２２１に標識抗原を、試料供給部２１に近い方の中間部２２及びその両側の連通部２４に抗体を、試料供給部２１に近い方の中間部２２にＨ2Ｏ2を、それぞれ固定せずに設置した。また、発色部２２２に発色基質１を設置した。そして、導入孔２１１を通じて試料供給部２２１に、濃度の異なる所定の体積のプロゲステロン水溶液を供給した。このときの発色部２２２における発色強度（シアン値）をプロゲステロン水溶液中のプロゲステロンの濃度に対してプロットしたものが図８のグラフである。図８に示す通り、プロゲステロンの濃度が高くなるのに応じて発色強度も高くなっている。 FIG. 8 shows the inspection results performed using the paper device in which the configuration of the paper device 200 is changed as follows. In the paper device related to this test, the labeled antigen is applied to the sample supply unit 221, the antibody is applied to the intermediate portion 22 closer to the sample supply unit 21 and the communication portions 24 on both sides thereof, and the intermediate portion 22 closer to the sample supply unit 21. H 2 O 2 were installed without fixing each. Further, the color-developing substrate 1 was installed in the color-developing unit 222. Then, a predetermined volume of progesterone aqueous solution having a different concentration was supplied to the sample supply unit 221 through the introduction hole 211. The graph of FIG. 8 is a plot of the color development intensity (cyan value) in the color development unit 222 at this time with respect to the concentration of progesterone in the progesterone aqueous solution. As shown in FIG. 8, the color development intensity increases as the progesterone concentration increases.
　また、上述の第１の実施形態に係る紙デバイス１に、図９に示すコントロール流路７０が追加されてもよい。コントロール流路７０は、濾紙製のシート５０に形成された流路６０及びシート５０とは別のシートに形成された発色部６３からなり、疎水性領域によって外縁が画定された親水性領域からなる流路である。流路６０は、シート２０に形成された試料供給部２１、２つの中間部２２及び２つの連通部２４からなる流路と同じ形状及び同じ大きさを有している。流路６０には、流路４０に含まれているものと同じ速度調節剤が含まれているが、抗体は含まれていない。シート５０は、流路６０の一端部６１が導入孔１１と試料供給部２１の間に配置されるようにシート１０及び２０の間に挿入されている。流路６０の他端部６２の上方には、発色部１２と同じ形状及び同じ大きさを有する発色部６３が設けられる。発色部６３には発色基質１が含まれている。以上のように、コントロール流路７０は、抗体が含まれていないこと以外、流路４０と同様に形成されている。導入孔１１を通じて試料液が供給されると、試料液が流路４０へと浸透すると共にコントロール流路７０にも浸透していく。コントロール流路７０には抗体が含まれていないため、発色部６３には、検体中の抗原の濃度に関わらず最大量の標識抗原が到達する。したがって、発色部６３における発色を発色部１２の発色に対する比較対象とすることができる。例えば、発色部６３における発色強度に対する発色部１２の相対的な発色強度を評価することにより、検体中の抗原の濃度を評価してもよい。 Further, the control flow path 70 shown in FIG. 9 may be added to the paper device 1 according to the first embodiment described above. The control flow path 70 includes a flow path 60 formed on a filter paper sheet 50 and a coloring portion 63 formed on a sheet different from the sheet 50, and is composed of a hydrophilic region whose outer edge is defined by a hydrophobic region. It is a flow path. The flow path 60 has the same shape and size as the flow path composed of the sample supply portion 21, the two intermediate portions 22, and the two communication portions 24 formed on the sheet 20. The flow path 60 contains the same rate regulator as that contained in the flow path 40, but does not contain the antibody. The sheet 50 is inserted between the sheets 10 and 20 so that one end 61 of the flow path 60 is arranged between the introduction hole 11 and the sample supply section 21. Above the other end 62 of the flow path 60, a coloring portion 63 having the same shape and size as the coloring portion 12 is provided. The color-developing unit 63 contains a color-developing substrate 1. As described above, the control flow path 70 is formed in the same manner as the flow path 40 except that the antibody is not contained. When the sample liquid is supplied through the introduction hole 11, the sample liquid permeates into the flow path 40 and also permeates into the control flow path 70. Since the control flow path 70 does not contain an antibody, the maximum amount of labeled antigen reaches the color-developing portion 63 regardless of the concentration of the antigen in the sample. Therefore, the color development in the color development unit 63 can be compared with the color development in the color development unit 12. For example, the concentration of the antigen in the sample may be evaluated by evaluating the color development intensity of the color development unit 12 relative to the color development intensity of the color development unit 63.
　また、上述の実施形態では、速度調節剤として界面活性剤、具体例としてＰＢＳＴが用いられている。しかし、流路中の成分同士で移動速度に差を生じるようなものであればその他の物質が用いられてもよい。例えば、各種の有機溶媒が候補として挙げられる。ただし、蒸発が早くないものや、抗体・抗原を変性させにくいものが用いられることが好ましい。 Further, in the above-described embodiment, a surfactant is used as the speed regulator, and PBST is used as a specific example. However, other substances may be used as long as the components in the flow path have different moving speeds. For example, various organic solvents can be mentioned as candidates. However, it is preferable to use one that does not evaporate quickly or one that does not easily denature antibodies / antigens.
　また、上述の第１の実施形態では、標識抗原－抗体複合体が発色部１２に到達しない態様が想定されている。しかし、最終的に標識抗原－抗体複合体も発色部１２に到達し得る態様が採用されてもよい。この場合においても、標識抗原－抗体複合体が発色部１２に到達する前に未反応の標識抗原による発色部１２の発色の強度を評価することで検体中の抗原の濃度を評価できる。 Further, in the above-mentioned first embodiment, it is assumed that the labeled antigen-antibody complex does not reach the coloring portion 12. However, an embodiment in which the labeled antigen-antibody complex can finally reach the color-developing portion 12 may be adopted. Also in this case, the concentration of the antigen in the sample can be evaluated by evaluating the color development intensity of the color-developing portion 12 by the unreacted labeled antigen before the labeled antigen-antibody complex reaches the color-developing portion 12.
　なお、上述の実施形態、実施例及び変形例のいずれにおいても、発色基質１、発色基質２、抗体、標識抗原等の物質を紙デバイスに設置する方法は、溶液を塗布後、乾燥することにより行われている。つまり、いずれの物質についても紙デバイスに固定しない（化学的な固定手段を用いない）方法が採用されている。したがって、どの物質もシート（セルロース）と化学結合していない。なお、溶液を塗布後、乾燥するという方法は、化学的な固定手段を用いずにこれらの物質を紙デバイスに設置する方法の一具体例であり、その他の方法が用いられてもよい。 In any of the above-described embodiments, examples and modifications, the method of placing substances such as the color-developing substrate 1, the color-developing substrate 2, the antibody, and the labeled antigen on the paper device is to apply the solution and then dry it. It is done. That is, a method is adopted in which none of the substances is fixed to the paper device (without using chemical fixing means). Therefore, none of the substances are chemically bonded to the sheet (cellulose). The method of applying the solution and then drying it is a specific example of a method of placing these substances on a paper device without using chemical fixing means, and other methods may be used.
１、１００、２００　紙デバイス
２ａ　親水性領域
２ｂ　疎水性領域
２、１０２、２０２　積層体
１０、２０、５０、１１０、１２０、２１０、２２０　シート
１２、１１２、２２２　発色部
２１、１２１　試料供給部
１２２　抗体設置部
２２１　基質設置部 1,100,200 Paper device 2a Hydrophilic region 2b Hydrophobic region 2,102,202 Laminated body 10, 20, 50, 110, 120, 210, 220 Sheet 12, 112, 222 Coloring unit 21, 121 Sample supply unit 122 Antibody installation unit 221 Substrate installation unit

Claims (7)

  1. 　酵素と基質の作用に起因して発生する発色の強度に基づいて検体中の被検出物質を検出する物質検出装置であって、
    　親水性領域と当該親水性領域の外縁を画定する疎水性領域とを有するシートを備えており、
    　前記親水性領域に、
    　前記検体を含んだ試料液が供給される試料供給部と、
    　前記基質を含んだ発色部と、
    　前記試料供給部から前記発色部までを結ぶ中間流路と、が形成されており、
    　前記被検出物質と結合する物質である捕捉物質及び前記検体に由来しない前記被検出物質のいずれか一方が前記酵素によって標識された複合物である第１標識物質と、前記捕捉物質及び前記被検出物質のいずれか一方が前記酵素によって標識されたものと他方との複合物である第２標識物質とが、前記第１標識物質の濃度が前記検体中の前記被検出物質の濃度に応じた大きさとなりつつ前記中間流路に存在することとなるように、前記第１標識物質及び前記捕捉物質の両方又は前者が、前記試料液、前記試料供給部及び前記中間流路の少なくともいずれかに、固定されずに含まれており、
    　前記中間流路が、前記第１標識物質が前記中間流路中を移動する速度を前記第２標識物質が前記中間流路中を移動する速度より大きくする速度調節物質を含んでいることを特徴とする物質検出装置。     A substance detection device that detects a substance to be detected in a sample based on the intensity of color development generated by the action of an enzyme and a substrate.
    It comprises a sheet having a hydrophilic region and a hydrophobic region defining the outer edge of the hydrophilic region.
    In the hydrophilic region,
    A sample supply unit to which a sample solution containing the sample is supplied, and
    The coloring part containing the substrate and
    An intermediate flow path connecting the sample supply section to the color development section is formed.
    The first labeled substance, which is a complex in which one of the capture substance which is a substance that binds to the substance to be detected and the substance to be detected that is not derived from the sample is labeled by the enzyme, the capture substance and the substance to be detected The concentration of the first labeled substance in the second labeled substance, which is a complex of one of the substances labeled by the enzyme and the other, is large according to the concentration of the detected substance in the sample. Both the first labeling substance and the trapping substance, or the former, are placed in at least one of the sample solution, the sample supply unit, and the intermediate flow path so that the substance is present in the intermediate flow path. Included without being fixed,
    The intermediate flow path is characterized by containing a speed adjusting substance that makes the speed at which the first labeling substance moves in the intermediate flow path larger than the speed at which the second labeling substance moves in the intermediate flow path. Substance detection device.
  2. 　前記速度調節物質が界面活性剤であることを特徴とする請求項１に記載の物質検出装置。 The substance detection device according to claim 1, wherein the speed adjusting substance is a surfactant.
  3. 　前記試料液が、前記第１標識物質が前記発色部に到達し且つ前記第２標識物質が前記発色部に到達しない量に調整されていることを特徴とする請求項１又は２に記載の物質検出装置。 The substance according to claim 1 or 2, wherein the sample solution is adjusted to an amount in which the first labeling substance reaches the coloring portion and the second labeling substance does not reach the coloring portion. Detection device.
  4. 　前記親水性領域及び前記疎水性領域を有する複数のシートが積層された積層体を備えていることを特徴とする請求項１～３のいずれか1項に記載の物質検出装置。 The substance detection device according to any one of claims 1 to 3, further comprising a laminated body in which a plurality of sheets having the hydrophilic region and the hydrophobic region are laminated.
  5. 　前記発色部が、前記複数のシートにおける前記中間流路とは別のシートに形成されていることを特徴とする請求項４に記載の物質検出装置。 The substance detection device according to claim 4, wherein the color-developing portion is formed on a sheet different from the intermediate flow path in the plurality of sheets.
  6. 　前記第１標識物質が、前記検体に由来しない前記被検出物質が前記酵素によって標識された複合物であって、前記試料液に含まれており、
    　前記捕捉物質が、前記試料供給部及び前記中間流路の少なくともいずれかに含まれており、
    　前記第２標識物質が、前記第１標識物質と前記捕捉物質との反応により生成されることを特徴とする請求項１～５のいずれか１項に記載の物質検出装置。     The first labeled substance is a complex in which the substance to be detected, which is not derived from the sample, is labeled with the enzyme, and is contained in the sample solution.
    The trapping substance is contained in at least one of the sample supply section and the intermediate flow path.
    The substance detection device according to any one of claims 1 to 5, wherein the second labeling substance is produced by a reaction between the first labeling substance and the trapping substance.
  7. 　前記第１標識物質が、前記捕捉物質が前記酵素によって標識された複合物であって、前記試料液に含まれており、
    　前記第２標識物質が、前記第１標識物質と前記検体中の前記被検出物質との反応により生成されることを特徴とする請求項１～５のいずれか１項に記載の物質検出装置。     The first labeling substance is a complex in which the capturing substance is labeled with the enzyme, and is contained in the sample solution.
    The substance detection device according to any one of claims 1 to 5, wherein the second labeled substance is produced by a reaction between the first labeled substance and the detected substance in the sample.
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