JP2020020601A - Substance detector - Google Patents

Abstract

To enhance an ability to distinguish a difference in the concentration of a substance to be detected in an aqueous solution.SOLUTION: A paper device 1 is a device that detects a substance using a color generated due to at least an action of an enzyme and a chromogenic substrate 1. In a laminate substrate 2 composed of sheets 10 to 30, there are formed a sample supply unit 32, as a channel composed of a hydrophilic region, provided with a sample liquid containing a complex in which the same substance as a detection target substance to be detected is bound to an enzyme and the substance to be detected, coloring units 24 and 25 to which the chromogenic substrate 1 is fixed, a capturing unit 33 to which the capturing substance for capturing the target substance and the complex are fixed, a test-side channel 52 connecting from the sample supplying unit 32 to the coloring unit 24 and a control-side channel 53 connecting from the sample supply unit 32 to the coloring unit 25. The test-side channel 52 and the control-side channel 53 have the same shape as each other. A communication section 22 formed of a hydrophilic region is stacked on the capturing unit 33, and the communication section 23 formed of the hydrophilic region is stacked on a pseudo-capturing unit 34.SELECTED DRAWING: Figure 2

Description

  The present invention relates to a substance detection device using a sheet.

  As a conventional technique relating to a substance detection device using a sheet such as paper, there are techniques disclosed in Patent Literature 1 and Non-Patent Literature 1. In each of the documents, a paper sheet is used, and a hydrophilic channel is formed on the sheet. At one end of the channel on the sheet, a sample supply unit to which an aqueous solution in which a substance to be detected is dissolved is supplied is set. A display portion for displaying a detection result of the substance to be detected is set at the other end of the flow path or in the middle thereof. When the aqueous solution is supplied to the sample supply unit, the aqueous solution moves from there toward the other end of the channel by capillary action. Then, when the aqueous solution reaches the display portion of the detection result, a detection result such as whether or not the substance to be detected is included in the aqueous solution is displayed.

  In particular, Non-Patent Literature 1 uses the coloring effect of a certain substance to display a detection result as described below. At least two substances (horseradish peroxidase (hereinafter, referred to as HRP)) and 3,3 ', 5,5'-tetramethylbenzidine (hereinafter, referred to as TMBZ) are involved in this coloring action. The channel in Non-Patent Document 1 is branched into two from the sample supply unit toward the other end. At the other end of each of the branched flow paths, a coloring portion to which one of the two substances (TMBZ) is fixed is provided. One of these two coloring portions is a test region, and the other is a control region. The other of the two substances (HRP) is bound to an antibody that targets the substance to be detected (eg, biotin). An aqueous solution in which the antibody and the substance to be detected are dissolved is supplied to the sample supply unit. In the aqueous solution, a part of the antibody is bound to the substance to be detected. In the flow path from the sample supply section to the color forming section, which is a test area, a capture section in which the same substance as the substance to be detected is immobilized for capturing antibodies is provided.

  The device of Non-Patent Document 1 is used as follows. When the aqueous solution is supplied to the sample supply unit, the aqueous solution moves from the sample supply unit toward the two color forming portions at the other end of the flow path by capillary action. In one flow path, the antibody in the aqueous solution that is not bound to the substance to be detected is captured by the substance fixed to the capturing section in the middle. The antibody in the aqueous solution bound to the substance to be detected reaches the colored portion on the test side. In the other flow path, both the antibody bound to the substance to be detected and the antibody not bound to the substance reach the colored portion on the control side. Then, by the action between one of the two substances fixed to each color-developing part and the other of the two substances bound to the antibody, an intensity corresponding to the concentration of the antibody in the aqueous solution that has reached each color-developing part is obtained. Then, coloring occurs. The concentration of the antibody in the aqueous solution that reaches the color forming part on the test side varies depending on the amount of the antibody captured by the capturing part. The amount of the antibody captured by the capturing unit varies depending on the concentration of the substance to be detected in the aqueous solution. Therefore, the concentration of the substance to be detected in the aqueous solution is detected by comparing the intensity of color development in the color forming part on the test side with the intensity of color development in the color forming part on the control side.

Japanese Patent No. 2930426

LSA Busa et al., "A competitive immunoassay system for microfluidic paper-based analytical detection of small size. molecules) ", Analyst, 2016, 141, p. 6598-6603

  However, according to the above-described conventional technique, there is not so much difference in the coloring intensity between the test-side coloring portion and the control-side coloring portion, and the detection result tends to vary. If there is little difference in color intensity between the test part and the control part, or if the detection results tend to vary, the ability of the device to discriminate the difference in the concentration of the target substance is low. Become. That is, for example, when there are two aqueous solutions having different concentrations of the substance to be detected, the minimum value that can be detected by the apparatus for the difference between the concentrations increases.

  An object of the present invention is to provide a substance detection device having an enhanced ability to distinguish a difference in the concentration of a substance to be detected in an aqueous solution.

  The substance detection device of the present invention is a substance detection device that detects the concentration of a substance in an aqueous solution based on the intensity of color generated due to the action of the first color-related substance and the second color-related substance, Having a hydrophilic region and a hydrophobic region defining the outer edge of the hydrophilic region, comprising a laminate in which a plurality of sheets are laminated, the hydrophilic region of the laminate, the target substance to be detected is a detection target A sample solution containing the first complex in which the same substance as described above is bound to the first chromogenic substance and the substance to be detected, or an antibody targeting the substance to be detected is bound to the first chromogenic substance. A sample supply section to which a sample liquid containing the second complex and the substance to be detected is supplied; a test-side coloring section and a control-side coloring section to which the second coloring-related substance is fixed; First capture for capturing the first complex A test section for forming a sample or a second capture substance for capturing the second complex, the capture section being fixed to the sample supply section; and a test connecting the sample supply section to the test-side coloring section. A side flow path and a control side flow path connecting the sample supply section to the control side color forming section are formed in a hydrophilic region of a first sheet which is one of the plurality of sheets, The shape of the test-side channel and the shape of the control-side channel are the same as each other or are in a mirror image relationship, and are one of the plurality of sheets and are stacked on the first sheet. A second hydrophilic portion formed of a hydrophilic region in contact with the first hydrophilic portion in the test-side flow path is formed on another sheet, and one of the plurality of sheets is formed on the first sheet. Another sheet laminated on the A fourth hydrophilic portion formed of a hydrophilic region in contact with the third hydrophilic portion corresponding to the first hydrophilic portion in the roll-side flow path is formed, and one of the first hydrophilic portion and the second hydrophilic portion is formed. Is the capturing unit.

  According to the substance detection device of the present invention, in the test channel, the substance to be detected and the first complex or the second complex in the sample liquid are captured by the capturing unit, and the remaining substance that is not captured is removed by the capturing unit. The contained aqueous solution reaches the test-side coloring portion. The first complex and the second complex contain the first coloring-related substance, and the second coloring-related substance is fixed to the test-side coloring section. Therefore, in the test-side coloring portion, coloring occurs according to the action of the first coloring-related substance and the second coloring-related substance in the first complex or the second complex. The concentration of the first complex or the second complex that reaches the test-side coloring portion varies depending on the concentration of the substance to be detected when the sample solution is prepared. Therefore, the coloring intensity in the test-side coloring portion varies depending on the concentration of the substance to be detected at the time of preparing the sample liquid. On the other hand, in the control-side flow path, the sample solution reaches the control-side coloring section without the first complex or the second complex being caught in the middle of the flow path. Therefore, when the color intensity in the test color portion and the color intensity in the control color portion are compared, the former color intensity is larger than the latter color intensity because the first complex or the second complex is captured in the capture portion. Lower.

  In the present invention, the color intensity in the test-side color portion can be appropriately evaluated by comparison with the color intensity in the control-side color portion as described above. That is, the coloring intensity of the test-side coloring portion can be appropriately evaluated based on the coloring intensity of the control-side flow path. As described above, the color intensity of the control color portion can be used as an appropriate reference because the test channel and the control channel of the present invention have the same shape as each other or have a mirror image relationship with each other. This is because it is configured to have a certain shape. If these shapes are not the same as each other and are not in a mirror image relationship, there is a possibility that the flow of the sample liquid may differ due to the difference in shape between the flow paths. If there is a difference in the flow of the sample liquid, it is difficult to discriminate whether a difference has occurred in color development due to the difference in flow or a difference due to other factors. On the other hand, in the present invention, the shape of the test-side flow path and the shape of the control-side flow path are the same, or are in a mirror image relationship with each other. For this reason, a difference in coloring intensity due to a difference in the shape of the flow channel is unlikely to occur. Therefore, it is easy to appropriately evaluate a factor other than the shape of the flow path, that is, a difference in the coloring intensity due to the capture of the first complex or the second complex in the capture unit.

  Further, in the present invention, a second hydrophilic portion comprising a hydrophilic region in contact with the first hydrophilic portion, which is a part of the test-side flow path, is provided, and any one of the first hydrophilic portion and the second hydrophilic portion is provided. One of them is a capturing unit. And the 4th hydrophilic part which consists of the hydrophilic area | region which contacted the 3rd hydrophilic part corresponding to the 1st hydrophilic part in a control side flow path is provided. As a result, the following two effects are secured as shown in a third embodiment described later. First, since the trapping efficiency of the substance in the trapping portion is improved, a difference in the coloring intensity of the test-side coloring portion from the coloring intensity of the control-side coloring portion is likely to occur. Therefore, it is easy to evaluate the coloring intensity of the test-side coloring portion based on comparison with the coloring intensity of the control-side coloring portion. Second, there is little variation in the relative magnitude of the color intensity of the test-side color portion to the color intensity of the control-side color portion (hereinafter referred to as relative color intensity). If there is little variation in the relative coloring intensity, for example, when there are two sample liquids having different concentrations of the substance to be detected, the minimum value that can be detected by the apparatus for the difference between the concentrations decreases. Based on the above two effects, the present invention distinguishes the difference in the concentration of the substance to be detected in the sample liquid in the detection of the concentration of the substance to be detected based on the comparison of the coloring intensity between the test-side coloring part and the control-side coloring part. Ability has been enhanced. The sample solution of the present invention may be prepared in any manner as long as it contains the substance to be detected and the first complex or the second complex and the substance to be detected. For example, after preparing an aqueous solution of the substance to be detected and an aqueous solution of the first complex or the second complex, these aqueous solutions may be mixed with each other. After preparing an aqueous solution of the substance to be detected, the first complex or the second complex may be dissolved in the aqueous solution. Further, an aqueous solution of the first complex or the second complex may be prepared in advance, and the substance to be detected may be dissolved in the aqueous solution.

  The “another sheet stacked on the first sheet” may be a sheet that is integrally connected to the first sheet and is stacked on the first sheet by being folded. A sheet separated from one sheet may be laminated on the first sheet. The “another sheet” on which the second hydrophilic portion is formed and the “another sheet” on which the fourth hydrophilic portion is formed may be a common sheet or different sheets. Is also good.

  Further, in the present invention, one of the plurality of sheets and another sheet stacked on the first sheet may include an end portion of the test-side channel opposite to the sample supply unit. A fifth hydrophilic portion composed of a contacted hydrophilic region is formed, and the sample in the control-side flow path is provided on another sheet, which is one of the plurality of sheets and stacked on the first sheet. A sixth hydrophilic portion composed of a hydrophilic region in contact with the end opposite to the supply portion is formed, the fifth hydrophilic portion is the test-side coloring portion, and the sixth hydrophilic portion is the control-side coloring portion. It is preferably a coloring portion. According to the related art (for example, Non-Patent Document 1), a coloring portion is provided at an end of a test-side channel formed on one sheet. For this reason, in the related art, the coloring in the coloring portion proceeds only by the permeation of the sample liquid in the direction along one sheet. Therefore, uneven coloring is likely to occur in the direction along the sheet. When color unevenness occurs, the color intensity of the color portion cannot be appropriately evaluated, and the concentration of the substance to be detected based on the color intensity of the color portion may not be detected properly. On the other hand, according to the above configuration of the present invention, the sample liquid that has reached the end of the test-side flow path from the sample supply unit penetrates along the first sheet to the inside of the end of the test-side flow path. Then, the liquid permeates from the first sheet to the fourth sheet toward the fifth hydrophilic portion, which is the test-side coloring portion. That is, unlike the related art in which color development proceeds only by permeation of the aqueous solution along one sheet, in the present invention, not only the permeation of the sample solution in one sheet but also the first to The color development also advances as the permeation of the sample solution proceeds so as to straddle the four sheets. For this reason, uneven color development in the direction along the sheet unlike the prior art is unlikely to occur. Similarly, also at the end of the control-side flow path, the control-side color-forming portion is provided in the sixth hydrophilic portion in contact with the control-side flow channel, so that color unevenness is less likely to occur. As described above, the substance to be detected can be appropriately detected based on the coloring intensities of the test-side coloring portion and the control-side coloring portion. In addition, "another sheet" in which the second hydrophilic portion is formed, "another sheet" in which the fourth hydrophilic portion is formed, "another sheet" in which the fifth hydrophilic portion is formed, and a sixth hydrophilic portion are formed. The “separate sheets” may be common sheets or different sheets.

  Further, in the present invention, it is preferable that the second hydrophilic portion, the fourth hydrophilic portion, the fifth hydrophilic portion, and the sixth hydrophilic portion are formed on a second sheet, which is a single sheet. According to this, since the second hydrophilic portion to the sixth hydrophilic portion are combined into one sheet, the device of the present invention can be realized with a relatively simple structure.

  Further, in the present invention, a third sheet, which is one of the plurality of sheets and is laminated on the second sheet, is formed at a position overlapping the test-side coloring section and the control-side coloring section. An observation hole and a hydrophobic region covering the second hydrophilic portion and the fourth hydrophilic portion are formed, and the second sheet and the third sheet introduce the sample liquid into the sample supply portion. It is preferable that an introduction channel composed of a hydrophilic region is formed. According to this, since the hydrophobic region of the third sheet laminated on the second sheet covers the second hydrophilic portion and the fourth hydrophilic portion, evaporation of the sample liquid from these portions is suppressed. In addition, a passage for introducing the sample liquid into the sample supply unit is secured by the introduction channels formed in the second sheet and the third sheet. Further, an observation hole for detecting color development in the test-side coloring section and the control-side coloring section is secured in the third sheet.

  Further, in the present invention, it is preferable that the first sheet, the second sheet, and the third sheet correspond to three regions divided by a folding line in a single folded three sheet. According to this, since the first sheet to the third sheet are combined into one sheet, the apparatus of the present invention is realized with a simple structure.

  Further, in the invention, at least a part of the test-side flow path and the control-side flow path is one of the plurality of sheets and is different from the first sheet stacked on the first sheet. Is preferably covered by the hydrophobic region of the sheet. According to this, the evaporation of the sample liquid from the test-side flow path and the control-side flow path is suppressed.

  Further, in the invention, it is preferable that the hydrophilic region forming each of the test-side coloring portion, the control-side coloring portion, and the capturing portion has a disk shape. In the process of manufacturing the device, the liquid may be dropped on a portion of the first sheet that is to be a test-side coloring portion, a control-side coloring portion, or a capturing portion. For example, there is a case where these substances are fixed by dropping a solution of the second coloring-related substance or the first capturing substance or the second supplementary substance onto these portions. In such a case, the outer edge of the region to be dropped is clearly defined by each part having a disk shape as in the above configuration of the present invention. Therefore, when the liquid is dropped onto the region corresponding to each of these portions, it is easy to define the amount of the liquid that just fills the region.

  Further, in the present invention, it is preferable that the hydrophobic region of the laminate contains a dried product of the hydrophobic ink inside. According to this, by forming a pattern with hydrophobic ink using a printer or the like, a hydrophobic region that defines a flow path composed of a hydrophilic region can be easily formed on a sheet.

1 is an exploded perspective view of a paper device according to an embodiment of the present invention. FIG. 2 is a sectional view taken along line II-II of the paper device of FIG. 1. FIG. 2 is a development view of the paper device of FIG. 1. FIG. 2 is a schematic view of a flow path formed in the paper device of FIG. 1. FIG. 2 is a flowchart illustrating a method of using the paper device of FIG. 1. FIG. 2 is a plan view of the paper device in a state where a color forming unit in FIG. 3 is a graph showing the results of Example 1, which is a test using the paper device of FIG. 1. 3 is a graph showing the results of Example 2 which is a test using the paper device of FIG. 1. 9 is a graph showing the result of a comparative example (conventional example) with respect to Example 2 according to FIG. 3 is a graph showing the results of Example 3 which is a test using the paper device of FIG. 1 and the results of Comparative Examples. It is a figure which shows the modification of a flow path. It is a figure showing another modification of a channel. It is a figure which shows the modification of a paper device.

[Paper device 1]
Hereinafter, a paper device 1 according to an embodiment of the present invention will be described. The paper device 1 is a device that detects the concentration of a substance to be detected in a sample liquid. The present device is used, for example, to detect a specific substance contained in a specimen such as animal blood. The substance to be detected may be any substance as long as it can form a complex with the antibody. For example, in addition to progesterone and biotin used in Examples described later, cortisol, estrogen, testosterone, prolactin, ANP (atrial natriuretic peptide), BNP (brain natriuretic peptide), aflatoxin, ochratoxin, patulin, deoxynivalenol, Nivalenol and the like may be targeted.

In the paper device 1, a color generated by at least two substances is used. One of the two substances (the first color-forming substance in the present invention) is an enzyme, and as described below, as a complex bound to the analyte or a complex bound to an antibody targeting the analyte. Used in the state contained in the sample solution. The other of the two substances (the second color-forming substance in the present invention) is a chromogenic substrate 1 (chromogen), which is partially fixed to the paper device 1 and used as described later. Depending on the combination of the enzyme and the chromogenic substrate 1, the chromogenic substrate 2 may be required for color development. In this case, the chromogenic substrate 2 is used, for example, in a state contained in a sample solution, as described later. The substances listed in Table 1 may be used as such enzymes and chromogenic substrates 1 and 2. As shown in Table 1, any one of HRP, β-GAL, and the like can be used as a candidate for the enzyme, and there are a plurality of candidates for the chromogenic substrate 1 for each candidate for the enzyme. For example, for HRP, any one of 1,2-phenylenediamine, TMBZ and the like can be used. The chromogenic substrate 2 is determined according to the combination of the enzyme and the chromogenic substrate 1. For example, when HRP and TPM-PS are used as the enzyme and the chromogenic substrate 1, H ₂ O ₂ is used as the chromogenic substrate 2. When HRP and ALPS are used as the enzyme and the chromogenic substrate 1, both H ₂ O ₂ and 4-AA are used as the chromogenic substrate 2. On the other hand, for example, when ALP and NADP are used as the enzyme and the chromogenic substrate 1, the chromogenic substrate 2 is unnecessary.

[Table 1]

  Next, the configuration of the paper device 1 will be described with reference to FIGS. As shown in FIGS. 1 and 2, the paper device 1 includes a filter paper sheet 10 (third sheet in the present invention), a sheet 20 (second sheet in the present invention), and a sheet 30 (first sheet in the present invention). ) Is provided. Hereinafter, the direction in which the sheets are stacked is referred to as a stacking direction. The sheets 10 to 30 are such that one sheet shown in FIG. 3 is folded in a Z-shape along the mountain fold line 1a and the valley fold line 1b into three shapes, so that the sheets 10, 20, and 30 are arranged in this order in the stacking direction. It is configured to be a laminate 2 composed of three layers of sheets. The sheets 10 to 30 correspond to three regions divided by the mountain fold line 1a and the valley fold line 1b. That is, in one sheet shown in FIG. 3, the sheet 10 is located on the left side of the mountain fold line 1a, the sheet 20 is located on the area from the mountain fold line 1a to the valley fold line 1b, and the sheet 30 is located on the valley fold line 1b. Occupies the area on the right.

  As shown in FIG. 2, the stacked body 2 includes a hatched area 2 a formed by oblique lines extending from upper left to lower right and a hatched area 2 b formed by oblique lines extending from upper right to lower left. In. The area 2b is an area having hydrophobicity impregnated with hydrophobic ink. Hereinafter, the region 2b is referred to as a hydrophobic region 2b. On the other hand, the area 2a is not impregnated with such 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 included in one location of the hydrophilic region 2a, the liquid spreads from the one location to the surroundings by a capillary phenomenon over time. The spreading of the liquid continues until it reaches the boundary between the hydrophilic region 2a and the hydrophobic region 2b.

  The pattern of the hydrophobic region 2b is formed, for example, by printing on a sheet using an inkjet printer for solid ink using hydrophobic solid ink. After the printing of the pattern, the sheet is heated by using a dryer, so that the ink on the sheet is melted and permeated into the sheet. As a result, a hydrophobic area 2b impregnated with hydrophobic ink is formed from one surface of the sheet to the other surface. The hydrophobic region 2b is in a state where a dried product of the hydrophobic ink is present in the sheet. In addition, screen printing, photolithography, or the like may be used for forming the pattern on the sheet, in addition to inkjet printing other than the solid ink type.

  The laminate 2 has a flow path formed by the hydrophilic region 2a. This channel is formed by the respective hydrophilic regions 2a of the sheets 10 to 30 communicating with each other. An introduction portion 11 is formed in the sheet 10 as a part of the flow path. The introduction unit 11 is disposed substantially at the center of the seat 10 in the left-right direction in FIG. Hereinafter, the horizontal direction in FIG. 2 is referred to as a horizontal direction, and a direction orthogonal to both the laminating direction and the horizontal direction of the sheets is referred to as a vertical direction. The introduction part 11 has a disk shape as shown in FIG. The sample liquid is introduced into the introduction unit 11. The sample liquid is one of two aqueous solutions. One of the two aqueous solutions is obtained by mixing an aqueous solution of a substance to be detected (for example, a specimen containing the substance to be detected) with an aqueous solution of a complex (first complex) containing an enzyme. This sample liquid corresponds to that used in Example 1 described below concerning progesterone. The complex is obtained by combining the same substance as the substance to be detected prepared separately from the detection target with the enzyme. Hereinafter, this complex is referred to as an analyte-enzyme complex. The other of the two aqueous solutions is obtained by mixing an aqueous solution of a substance to be detected (for example, a specimen containing the substance to be detected) with an aqueous solution of a complex (second complex) containing an enzyme. This sample liquid corresponds to that used in Example 2 described below concerning biotin. The complex is obtained by binding an antibody to the substance to be detected with an enzyme. Hereinafter, this complex is referred to as an antibody-enzyme complex. Further, when the chromogenic substrate 2 is also required for color development, the sample solution is assumed to further include the chromogenic substrate 2. The sheet 10 has observation holes 12 and 13 which are circular in plan view. The observation holes 12 and 13 are arranged in a row along the horizontal direction together with the introduction portion 11. The observation holes 12 and 13 and the introduction part 11 are arranged at equal intervals from each other.

  In the sheet 20, communication portions 21 to 23 and coloring portions 24 and 25 are formed as a part of the flow path. The communication portions 21 to 23 and the coloring portions 24 and 25 are arranged in a line at equal intervals along the horizontal direction as shown in FIG. Among these, the communication part 21 is formed in the same position as the introduction part 11 in the horizontal direction and the vertical direction at the same position as the introduction part 11. The coloring portions 24 and 25 are arranged at the same positions as the observation holes 12 and 13 in the horizontal and vertical directions. The communication portion 22 (the second hydrophilic portion in the present invention) is disposed between the communication portion 21 and the color forming portion 24, and the communication portion 23 (the fourth hydrophilic portion in the present invention) is provided between the communication portion 21 and the color forming portion 25. Are located in The communicating portions 21 to 23 and the coloring portions 24 and 25 have the same disk shape as shown in FIG. The coloring substrate 1 is fixed to the coloring portions 24 and 25, respectively. In addition, the coloring part 24 corresponds to the fifth hydrophilic part and the test-side coloring part in the present invention. Further, the coloring portion 25 corresponds to the sixth hydrophilic portion and the control-side coloring portion in the present invention.

  The sheet 30 includes a sample supply unit 32, a capture unit 33 (a first hydrophilic portion in the present invention), a pseudo capture unit 34 (a third hydrophilic portion in the present invention), and communication portions 35 to 37 are formed. Among them, the sample supply unit 32, the capture unit 33, the pseudo capture unit 34, and the communication units 35 and 36 have the same disk shape as shown in FIG. Further, these have the same shape as the communication portion 21 of the sheet 20 and the like. The communication portion 37 extends linearly along the lateral direction as shown in FIG. As shown in FIG. 1, the sample supply unit 32, the capture unit 33, the pseudo capture unit 34, and the communication units 35 and 36 are connected to the communication units 21 to 23 and the color forming units 24 and 25 of the sheet 20 in the horizontal and vertical directions, respectively. With respect to the same position. The communication section 37 allows the sample supply section 32, the capture section 33, the pseudo capture section 34, and the communication sections 35 and 36 to communicate with each other in the lateral direction. A substance that captures a substance in the sample solution (hereinafter, referred to as a capture substance) is fixed to the capture unit 33. As the capture substance according to the present embodiment, any of the following substances to be detected and antibody capture substances is used. The target substance capture substance is a substance that captures the target substance and the target substance-enzyme complex in the sample liquid. As the target substance capturing substance, for example, an antibody against the target substance may be used. As this antibody, only an antibody directly binding to the substance to be detected may be used, or a primary antibody and a secondary antibody may be used. The antibody capturing substance is a substance that captures the antibody-enzyme complex in the sample solution. As the antibody capture substance, for example, the same substance as the substance to be detected may be used.

  In the above configuration, the respective portions of the flow passages formed in the sheets 10 to 30 are in communication with each other by making contact in the stacking direction. Thereby, as shown in FIGS. 2 and 4, three flow paths of the introduction flow path 51, the test-side flow path 52, and the control-side flow path 53 are formed in the laminate 2. As shown in FIGS. 2 and 4, the introduction channel 51 is configured by the introduction unit 11 and the communication unit 21, and is a channel for introducing the sample liquid to the sample supply unit 32. The test side channel 52 and the control side channel 53 have the same shape as each other. That is, the test-side flow path 52 has a shape that overlaps with the control-side flow path 53 when rotated 180 degrees around the central axis C of the introduction flow path 51 shown in FIG. The test-side flow path 52 and the control-side flow path 53 also have a symmetrical relationship with each other with respect to a plane passing through the central axis C and parallel to the vertical direction.

  As shown in FIGS. 2 and 4, the test-side flow path 52 is configured by the capturing section 33 and the communication sections 37 and 35, and is a flow path that connects the sample supply section 32 to the coloring section 24. The communication part 22 is laminated on the capturing part 33 which is a part of the test-side channel 52 from the sheet 20 side. In addition, the coloring section 24 is laminated from the sheet 20 side to the communication section 35 which is the end of the test-side channel 52 opposite to the sample supply section 32. As shown in FIGS. 2 and 4, the control-side flow path 53 is configured by the pseudo capture section 34 and the communication sections 37 and 36, and is a flow path that connects the sample supply section 32 to the coloring section 25. The pseudo capturing part 34, which is a part of the control-side channel 53, is a part corresponding to the capturing part 33, but unlike the capturing part 33, the capturing substance is not fixed. The communication part 23 is laminated on the false capturing part 34 from the sheet 20 side. In addition, a coloring portion 25 is laminated from the sheet 20 side to a communication portion 36 which is an end of the control side channel 53 opposite to the sample supply portion 32.

  The introduction flow path 51, the test-side flow path 52, and the control-side flow path 53 communicate with each other to form a flow path of the sample liquid from the introduction section 21 to the coloring sections 24 and 25. As shown in FIG. 1, four flow paths for such a sample liquid are provided in one paper device 1.

[How to use paper device 1 (substance to be detected)]
Hereinafter, a method of using the paper device 1 in which the substance to be detected is used will be described with reference to FIG. First, a sample liquid is prepared (Step S1). In preparing a sample solution for the paper device 1 in which the substance to be detected is used, an aqueous solution of the substance to be detected and the enzyme complex is prepared. This aqueous solution has a predetermined concentration and a predetermined amount set in advance. Then, a predetermined amount of an aqueous solution of the substance to be detected (for example, a specimen containing the substance to be detected) is mixed with the aqueous solution. The aqueous solution of the substance-enzyme complex and the substance to be detected thus obtained is further mixed with an aqueous solution of the chromogenic substrate 2 as needed to prepare a sample liquid. Table 2 below shows, in the method of using the paper device 1 in which the detection target substance capturing substance was used, the solute of the sample solution, the substance fixed to the capturing section 33, and the substance fixed to the coloring sections 24 and 25, respectively. And

[Table 2]

  Next, the prepared sample solution is introduced into the introduction unit 11 (Step S2). The sample liquid introduced into the introduction part 11 permeates through the flow path composed of the hydrophilic region 2a as described below. First, the sample liquid reaches the sample supply unit 32 of the sheet 30 from the introduction unit 11 through the introduction flow path 51 of the sheets 10 and 20.

From the sample supply unit 32, the sample liquid is divided into two parts, a test-side channel 52 and a control-side channel 53. First, in the control channel 53, the sample liquid flows from the sample supply unit 32 to the pseudo capture unit 34 via the communication unit 37. The sample liquid that has reached the pseudo-capturing unit 34 penetrates into the communication unit 23 on the sheet 20 side stacked on the pseudo-capturing unit 34 and travels in the pseudo-capturing unit 34 to the communication unit 36 side. Unlike the capturing section 33, the capturing substance is not fixed to the pseudo capturing section 34. For this reason, the substance to be detected and the substance-to-be-detected-enzyme complex in the sample liquid flow to the communicating section 36 via the communicating section 37 without being captured by the pseudo capturing section 34. The sample liquid that has reached the communication part 36 penetrates the communication part 36 and further permeates from the communication part 36 to the coloring part 25. In the coloring part 25, for example, due to the action of the enzyme in the analyte-enzyme complex in the sample liquid and the coloring substrate 1 fixed to the coloring part 25, in some cases, the coloring substrate 2 in the sample liquid was also involved. The action produces a product which develops a color. The intensity of coloring in the coloring section 25 is higher as the concentration of the target substance-enzyme complex in the sample liquid is higher, without depending on the concentration of the target substance. Incidentally, chromogenic substrate 2 may be H ₂ O ₂ and 4-AA, acts enzyme in chromogenic substrate 1 and 4-AA and a chromogenic substrate 2 can products, the product is colored.

  On the other hand, in the test-side channel 52, the sample liquid flows from the sample supply unit 32 to the capture unit 33 via the communication unit 37. The sample liquid that has reached the capturing section 33 permeates the communicating section 22 on the sheet 20 side stacked on the capturing section 33 and moves toward the communicating section 35 in the capturing section 33. In the capturing section 33, the target substance and the target substance / enzyme complex in the sample liquid are captured by the capturing substance fixed to the capturing section 33. The target substance and the target substance / enzyme complex are captured by the capture substance of the capture unit 33 at a ratio corresponding to the concentration ratio of these in the sample solution. That is, as the concentration of the target substance is higher than the concentration of the target substance / enzyme complex, the target substance-enzyme complex captured by the capturing substance of the capturing unit 33 is smaller. Further, the smaller the concentration of the target substance is lower than the concentration of the target substance / enzyme complex, the more the target substance-enzyme complex trapped by the trapping portion 33 is. The concentration of the substance to be detected in the capturing section 33 is determined according to the concentration of the substance to be detected immediately after the preparation of the sample liquid (hereinafter, referred to as the initial concentration of the substance to be detected). The amount of the target substance-enzyme complex captured by 33 is small. Then, the sample liquid that has exited the capturing section 33 travels to the communicating section 35 via the communicating section 37. The sample liquid that has reached the communication section 35 permeates the communication section 35 and further permeates from the communication section 35 to the color forming section 24. In the coloring portion 24, the action of the enzyme in the analyte-enzyme complex remaining in the sample solution without being captured by the capturing portion 33 and the chromogenic substrate 1 fixed on the coloring portion 24 may cause The color of the product generated by the action is developed through the action involving the chromogenic substrate 2 in the sample solution. The intensity of coloring in the coloring portion 24 is higher as the concentration of the analyte-enzyme complex remaining in the sample liquid is higher. The intensity of coloring in the coloring portion 24 is lower than the intensity of coloring in the coloring portion 25 as the number of the substance-enzyme complex detected by the capturing portion 33 increases. As described above, the amount of the target substance-enzyme complex captured by the capture unit 33 is smaller as the concentration of the target substance is initially higher, and is larger as the concentration of the target substance is initially lower. Therefore, the magnitude of the concentration of the substance to be detected can be initially evaluated based on how much the intensity of the coloring in the coloring portion 24 is lower than the intensity of the coloring in the coloring portion 25. More specifically, the lower the intensity of the coloring in the coloring portion 24 with respect to the intensity of the coloring in the coloring portion 25, the lower the initially detected substance concentration, and the intensity of the coloring in the coloring portion 24 with respect to the intensity of the coloring in the coloring portion 25. Is higher, the concentration of the substance to be detected is initially higher. FIG. 6 schematically shows the paper device 1 in a state where the coloring portions 24 and 25 are colored.

  Next, the paper device 1 is photographed with a digital camera (step S3). The photographing is performed such that the state in which the coloring portions 24 and 25 develop color through the observation holes 12 and 13 formed in the sheet 10 is included in the photographing range.

  Next, an analysis relating to the concentration of the substance to be detected initially is performed based on the imaging result of step S3 (step S4). Digital image analysis by a computer is used for the analysis in step S4. In the digital image analysis, the computer compares the difference in the coloring intensity between the coloring portion 24 and the coloring portion 25 based on the photographing result in step S3. Specifically, for example, as a value obtained by calculating a pixel value (for example, an RGB value) of each pixel existing in an area corresponding to each coloring section 24 in a captured image, a color development indicating the intensity of a color component corresponding to the color development An intensity value (eg, the C value of CMY) is derived. Then, an average value of the coloring intensity values is calculated for the pixels in the area corresponding to each coloring section 24. Similarly, a color intensity value indicating the intensity of the color component corresponding to the color included in the color component indicated by the pixel value is derived from the pixel value of each pixel existing in the area corresponding to each color portion 25 in the captured image. You. Then, the average of the coloring intensity values is calculated for the pixels in the area corresponding to each coloring section 25. Further, a ratio of the average value of the color intensity of the color portion 24 to the average value of the color intensity of the color portion 25 (hereinafter referred to as relative color intensity) is obtained.

  Next, the computer initially evaluates the concentration of the substance to be detected based on the relative coloring intensity. As described above, the lower the intensity of the coloring in the coloring portion 24 with respect to the intensity of the coloring in the coloring portion 25, the lower the initially detected substance concentration, and the intensity of the coloring in the coloring portion 24 with respect to the intensity of the coloring in the coloring portion 25. The higher the higher, the higher the initial concentration of the substance to be detected. Therefore, the computer evaluates that the larger the relative coloring intensity, the higher the initially detected substance concentration. For example, the computer initially holds data (data corresponding to the graphs in FIGS. 7 and 8) indicating the relationship between the concentration of the substance to be detected and the relative color intensity, and the computer calculates as described above based on the data. The initial target substance concentration corresponding to the relative coloring intensity may be specifically derived. This evaluation result is output by an output device such as a display connected to the computer or mounted on the computer.

[How to use paper device 1 (antibody capturing substance)]
Hereinafter, a method of using the paper device 1 when an antibody capturing substance is used will be described. This method of use has a lot in common with the method of use when a substance to be detected is used. In this method of use, steps S1 to S4 are executed, but there are differences in the specific contents of each step. The following mainly describes differences from the method of using the paper device 1 when a substance to be detected is used, and omits common points as appropriate.

  First, in the preparation of the sample solution for the paper device 1 using the antibody capturing substance in step S1, an aqueous solution of the antibody-enzyme complex is prepared. This aqueous solution has a predetermined concentration and a predetermined amount set in advance. A predetermined amount of an aqueous solution of a substance to be detected (for example, a specimen containing the substance to be detected) is mixed with the aqueous solution. In the sample solution, the substance to be detected and the antibody-enzyme complex bind to each other to form a complex (hereinafter, referred to as a substance to be detected-antibody-enzyme complex). In the preparation of the sample solution, the antibody-enzyme complex in the aqueous solution is mixed so that the antibody-enzyme complex not bound to the analyte and the analyte-antibody-enzyme complex are mixed in the prepared sample solution. The body concentration is set high enough. The aqueous solution of the antibody-enzyme complex and the analyte-antibody-enzyme complex thus obtained is further mixed with an aqueous solution of the chromogenic substrate 2 as needed to prepare a sample solution. Table 3 below shows, in the method of using the paper device 1 using the antibody capturing substance, the solute of the sample solution, the substance fixed to the capturing section 33, and the substance fixed to the coloring sections 24 and 25, respectively. Show.

[Table 3]

  After the introduction of the sample solution in step S2, when the sample solution reaches the capture unit 33 in the test-side channel 52, the antibodies in the antibody-enzyme complex are removed by the antibody capture substance (the target substance and the analyte) fixed to the capture unit 33. All the antibody-enzyme complexes are captured by the capture unit 33. Thereby, the antibody-enzyme complex does not exist in the sample liquid when exiting the capturing section 33. On the other hand, the analyte-antibody-enzyme complex already bound to the analyte is not captured by the capture unit 33 and remains in the sample liquid as it is. Therefore, in the coloring portion 24, the action of the enzyme in the analyte-antibody-enzyme complex remaining in the sample solution and the coloring substrate 1 immobilized on the coloring portion 24 may cause the coloring substrate 2 in the sample solution in some cases. Through the action involved, the product produced by the action develops a color. The color intensity increases as the concentration of the analyte-antibody-enzyme complex remaining in the sample liquid increases. The concentration of the substance-to-be-detected-antibody-enzyme complex in the sample solution that reaches the color forming section 24 is higher as the concentration of the substance to be detected is initially higher, and is lower as the concentration of the substance to be detected is initially lower. For this reason, the concentration of the substance to be detected is initially higher as the color forming portion 24 develops a stronger color. Here, the initial concentration of the target substance is the total target concentration of the target substance in the target substance-antibody-enzyme complex in the sample solution immediately after preparation and the target substance not bound to the antibody. Corresponds to the concentration of the substance to be detected.

  On the other hand, in the control side channel 53, the sample solution reaches the coloring portion 25 without the antibody-enzyme complex in the sample solution being captured by the pseudo capture portion 34. Therefore, the color forming section 25 develops a color with an intensity corresponding to the concentration of both the antibody-enzyme complex and the analyte-antibody-enzyme complex in the sample solution that has reached the color forming section 25.

  In step S4, the computer initially evaluates the concentration of the substance to be detected based on the relative coloring intensity. As described above, the coloring of the coloring portion 24 becomes stronger as the concentration of the substance to be detected is initially higher. Therefore, the computer evaluates that the larger the relative coloring intensity, the higher the initially detected substance concentration.

[Example 1]
Hereinafter, Example 1 of the above embodiment will be described. In Example 1, progesterone (Fuji Film Wako Pure Chemical Industries, Ltd.) was used as the substance to be detected. As a substance to be detected, an anti-progesterone-3 (E) -CMO-BSA antibody (Cosmo Bio) was used as a primary antibody against the substance to be detected. Rabbit anti-progesterone antibody (Bio-Rad Laboratories) was used as a secondary antibody. HRP was used as the enzyme, TMBZ was used as the chromogenic substrate 1, and H ₂ O ₂ was used as the chromogenic substrate 2. First, the paper device 1 was produced as follows. Using a solid ink type ink jet printer (ColorQube 8580, Xerox Corporation), a plurality of patterns shown in FIG. 3 are formed on filter paper (GE Healthcare, Whatman (registered trademark) ashless quantitative filter paper grade: 41 1441-917). Thereafter, the filter paper on which the pattern is formed is put into a stationary dryer set at 100 ° C. for 1 minute and 30 seconds, so that the solid ink solidified on the filter paper is melted and permeated into the filter paper. Thereby, the pattern of the hydrophobic region that defines the outer edges of the flow path (introduction flow path 51, sample supply unit 32, test-side flow path 52, control-side flow path 53, and coloring sections 24 and 25) composed of the hydrophilic area is formed. Form on filter paper. Hereinafter, the filter paper on which the pattern of the hydrophobic region is formed is referred to as a pattern-formed filter paper.

  Next, 0.3 μL of a 0.25 mg / mL chitosan solution obtained by dissolving chitosan in a 50 mM HCl aqueous solution and then adjusting the pH to 3.5 to 5.0 by adding an NaOH aqueous solution was used as the capturing portion of the pattern-forming filter paper. The solution is dropped onto a region corresponding to No. 33 (hereinafter, referred to as a capturing portion corresponding region), and air-dried for about 5 minutes. Note that the capturing section 33 has a disk shape as described above. For this reason, since the outer edge of the region corresponding to the capturing unit is clearly defined, it is easy to define the amount of the solution that just fills the region corresponding to the capturing unit. Next, 0.3 μL of a 2.5% glutaraldehyde solution obtained by dissolving glutaraldehyde in 1% (v / v) Tween 20-PBS is dropped on the region corresponding to the capturing section, and left to stand for 2 hours. The above-described dropping of the chitosan solution and the glutaraldehyde solution is performed to fix the secondary antibody in the region corresponding to the capturing section.

  Next, cleaning of the capturing unit corresponding area is performed. In this cleaning, the filter paper on which the pattern is formed is placed upside down on an absorbent sheet made of paper (hereinafter, referred to as an absorbent sheet), and PBST (1% (v / v) Tween 20-99%) is placed on the back surface of the area corresponding to the capturing section. (V / v) PBS) 0.3 μL is added dropwise. Immediately overlay another absorbent sheet on the patterned filter paper and blot the PBST from both sides of the patterned filter paper into the absorbent sheet. After such washing of the capturing section corresponding area is repeated three times, the capturing section corresponding area is dried.

  Next, the patterned filter paper is turned face-up, and 0.3 μL of a 2 mg / mL aqueous solution of the secondary antibody is dropped on the region corresponding to the capturing portion, and then dried. This is repeated eight times, and then allowed to stand for 20 minutes. As a result, the secondary antibody is fixed to the region corresponding to the capture portion. Next, the area corresponding to the capturing section is washed three times with PBST in the same procedure as the above washing. Then, the area corresponding to the capturing unit is dried. Note that since a small amount of liquid in the region corresponding to the capturing unit only needs to be dried by the next step, this drying step is not essential if drying is performed immediately after washing. Next, 0.3 μL of an aqueous solution of a 2 mg / mL primary antibody is dropped into the region corresponding to the capturing portion after drying, and then dried. After repeating this 5 times, it is left still for 20 minutes. As a result, the primary antibody binds to the secondary antibody immobilized on the capture portion corresponding region. Next, the area corresponding to the capturing section is washed three times with PBST in the same procedure as the above washing. Next, 30 μL of a 1% BSA-PBS solution is dropped into the entire flow path of the third layer (sheet 30). Specifically, 10 μL is dropped onto both ends (regions corresponding to the communication portions 35 and 36) and the center (region corresponding to the sample supply portion 32) of the flow channel. After dripping, it is left still for 20 minutes. Next, by discharging 20 μL of PBST in a straight line from one end of the flow path to the other end of the flow path, the entire flow path corresponding to the sample supply unit 32, the test-side flow path 52, and the control-side flow path 53 is discharged. On the other hand, washing with PBST is performed three times. After washing, the channel is dried for 20 to 30 minutes. Next, 15.7 μL of a 35.7 mM TMBZ acetonitrile solution is dropped into a region corresponding to the coloring portion 24 and a region corresponding to the coloring portion 25 of the second layer (sheet 20), and dried for about 15 minutes. The coloring portions 24 and 25 have a disk shape as described above. For this reason, since the outer edges of the regions corresponding to these are clearly defined, it is easy to define the amount of the solution that just fills these regions. Next, the pattern-formed filter paper is folded into a Z-shape, cut into the size of the paper device 1, and a total of four places near the center of the four sides of each of the cut laminates 2 are stapled to form three layers (sheet 10). To 30) are fixed to each other.

After producing the paper device 1 as described above, a test for deriving the relative coloring intensity of the progesterone sample solution prepared while changing the concentration using the paper device 1 was performed as follows. First, sample solutions of various concentrations of progesterone are prepared. Each sample solution is a mixture of an aqueous solution of progesterone corresponding to the substance to be detected, an aqueous solution of progesterone to which HRP is bound, and an aqueous solution of H ₂ O ₂ . Next, 16 μL of the sample liquid is dropped on each of the four introduction portions 11 formed in one paper device 1. Then, after confirming through the observation hole 12 that the sample liquid has reached the coloring portion 24 and the coloring portion 24 has begun to change color, an image of the paper device 1 is taken with a digital camera 5 minutes later. The photographing is performed with the entire sheet 10 of the paper device 1, each of the four coloring portions 24 exposed through the observation hole 12, and each of the coloring portions 25 exposed through the observation hole 13 as a photographing range. The settings of the digital camera are as follows: ISO sensitivity: 1600, F value: 5.6, shutter speed: 1/200 second. FIG. 7 shows the result of causing the computer to calculate the relative coloring intensities in the four flow paths based on the photographing results. In FIG. 7, the horizontal axis represents the initial concentration of the progesterone to be detected in the substance to be detected, and the vertical axis represents the value calculated by the computer. Each point in the graph indicates the average value of the relative coloring intensity calculated for each of the four sets of coloring portions 24 and 25 at each initial detection target substance concentration. The error bar indicates the variation (± 1 * standard deviation) of the relative color intensity calculated for each of the four sets of color portions 24 and 25.

[Example 2]
Hereinafter, Example 2 of the above embodiment will be described. In Example 2, biotin (Sigma-Aldrich) was used as the substance to be detected. Biotin was used as the same substance as the substance to be detected as the antibody capturing substance. HRP was used as the enzyme, TMBZ was used as the chromogenic substrate 1, and H ₂ O ₂ was used as the chromogenic substrate 2. First, the paper device 1 was produced as follows. Since the method of manufacturing the paper device 1 in the second embodiment has many points in common with the method of manufacturing the paper device 1 in the first embodiment, differences from the method in the first embodiment will be mainly described below. In Example 2, BSA-bound biotin (hereinafter, referred to as a BSA-biotin complex) is immobilized on the region corresponding to the capture unit. By fixing the BSA to the region corresponding to the capturing unit, biotin is fixed to the region corresponding to the capturing unit via the BSA. Specifically, instead of the dropping of the secondary antibody to the region corresponding to the capture unit in Example 1, 0.3 μL of a 10 mg / mL aqueous solution of the biotin-BSA complex is dropped to the region corresponding to the capture unit 10 times in total. . In addition, the primary antibody is not dropped onto the region corresponding to the capture portion, and the subsequent washing and standing are not performed. Otherwise, a paper device 1 was produced in the same manner as in Example 1. Next, using the paper device 1, a test for deriving the relative coloring intensity of the biotin sample solution prepared while changing the concentration was performed as follows. First, sample solutions of various concentrations of biotin are prepared. Each sample solution is a mixture of an aqueous solution of biotin corresponding to the substance to be detected, an aqueous solution of an anti-biotin antibody bound to HRP (Sigma-Aldrich), and an aqueous solution of H ₂ O ₂ . Thereafter, in the same manner as in Example 1, a test for deriving the relative coloring intensity of each sample solution was performed. FIG. 8 shows the result of causing the computer to calculate the relative coloring intensities in the four sets of coloring portions 24 and 25 formed on one paper device 1 based on the photographing results of the digital camera obtained in this test. . In FIG. 8, the horizontal axis represents the initial concentration of the substance to be detected of biotin, and the vertical axis represents the value calculated by the computer. Each point in the graph indicates the average value of the relative coloring intensity calculated for each of the four sets of coloring portions 24 and 25 at each initial detection target substance concentration. The error bar indicates the variation (± 1 * standard deviation) of the relative color intensity calculated for each of the four sets of color portions 24 and 25. As a comparative example, a test result of biotin in Non-Patent Document 1 is shown in FIG. In this comparative example, a test was performed under the same conditions as in Example 2 except that the sheet described in Non-Patent Document 1 was used instead of the paper device 1. In FIG. 8, it can be seen that the change in the relative coloring intensity with respect to the change in the concentration of the substance to be detected is initially larger than in FIG. Further, the variation in the relative coloring intensity is small.

[Example 3]
Hereinafter, Example 3 of the above embodiment will be described. In the third embodiment, a test using the paper device 1 was performed in the same manner as in the second embodiment. In addition, as a comparative example, a test according to Example 2 was performed using a paper device 1 ′ partially different from the paper device 1. The difference between the paper device 1 ′ and the paper device 1 is that the communication units 22 and 23 are not provided, and the other configuration of the paper device 1 ′ is the same as the paper device 1. That is, for the sheet corresponding to the sheet 20 in the paper device 1 ′, the entire area corresponding to the communication portions 22 and 23 is not a hydrophilic area but a hydrophobic area. In Example 3 and the comparative example, the test was performed in the same manner as in Example 2, except that the initial concentration of the substance to be detected of biotin was 0 ng / mL. The result is shown in FIG. In FIG. 10, the vertical axis indicates the average value of the relative coloring intensity for the four sets of coloring portions 24 and 25. The error bar indicates the variation (± 1 * standard deviation) of the relative color intensity calculated for each of the four sets of color portions 24 and 25. As shown in the bar graph of FIG. 10, in the case of Example 3 in which the communicating portions 22 and 23 were provided, the relative coloring intensity was significantly lower than that of the comparative example. Therefore, when the communication portion 22 is provided, the capturing efficiency of the detection target substance and the detection target substance-enzyme complex in the capture section 33 is increased, and the amount of the detection target substance-enzyme complex reaching the coloring section 24 is reduced. It turns out that it falls. In addition, as shown by the error bars in FIG. 10, when the communicating portions 22 and 23 are provided, it can be seen that the relative coloring intensity hardly varies.

  It is considered that the variation in the relative coloring intensity in the comparative example is caused by a difference in hydrophilic property between the test-side flow path 52 and the control-side flow path 53 depending on the presence or absence of the capturing unit 33. That is, the hydrophilicity of the test channel 52 is lower than that of the control channel 53 due to the presence of the capturing portion 33 in the test channel 52. For this reason, the sample liquid easily permeates into the test-side flow path 52 and the sample liquid permeates easily into the control-side flow path 53. As a result, the relative coloring intensity tends to vary. On the other hand, in Example 3, since the communicating portions 22 and 23 are provided, the hydrophilicity of each of the test-side flow path 52 and the control-side flow path 53 is higher than that of the comparative example. Thereby, a difference in hydrophilic property between the test-side flow path 52 and the control-side flow path 53 is suppressed. As a result, variations in the relative coloring intensity are suppressed.

[Effects of Embodiment]
According to the present embodiment described above, the coloring intensity in the coloring unit 24 is evaluated using the relative coloring intensity indicating the relative magnitude to the coloring intensity in the coloring unit 25. The reason why the coloring intensity of the coloring portion 25 can be used as an appropriate reference is that the test channel 52 and the control channel 53 have the same shape. If these shapes are not the same as each other, there is a possibility that the flow of the sample liquid may differ due to the difference in shape between the flow paths. If there is a difference in the flow of the sample liquid, it is difficult to discriminate whether a difference has occurred in color development due to the difference in flow or a difference due to other factors. On the other hand, in the present embodiment, the shape of the test-side channel 52 and the shape of the control-side channel 53 are the same. For this reason, a difference in coloring intensity due to a difference in the shape of the flow channel is unlikely to occur. Therefore, a factor other than the shape of the flow path, that is, a difference in coloring intensity generated between the coloring portion 24 and the coloring portion 25 due to the capture of the target substance-enzyme complex or the antibody-enzyme complex in the capturing portion 33 is appropriately determined. Easy to evaluate. Note that the difference in the coloring intensity due to the difference in the shape of the channel is unlikely to occur not only when the test-side channel and the control-side channel have the same shape, but also when these channels are mirror images of each other. This also applies to cases where there is a relationship.

  Further, in the present embodiment, the communicating portion 22 formed of a hydrophilic region in contact with the capturing portion 33 is provided. And the communication part 23 which consists of a hydrophilic area | region which contacted the pseudo capture | acquisition part 34 is provided. As a result, the following two effects are secured as shown in the test results of Example 3 and Comparative Example. First, the relative coloring intensity is reduced by improving the efficiency of capturing the substance in the capturing unit 33. That is, a difference between the coloring intensity of the coloring portion 24 and the coloring intensity of the coloring portion 25 tends to occur. Therefore, it is easy to evaluate the coloring intensity of the coloring portion 24 based on the relative coloring intensity. Second, there is little variation in the relative coloring intensity. If there is little variation in the relative coloring intensity, for example, when there are two aqueous solutions having different concentrations of the substance to be detected, the minimum value that can be detected by the apparatus for the difference in the concentrations decreases. Due to the above two effects, in the present embodiment, the ability to discriminate the difference in concentration in the detection of the concentration of the initially detected substance based on the relative coloring intensity is enhanced.

  Further, in the present embodiment, the coloring portion 24 is stacked on the communicating portion 35 which is the end of the test channel 52 on the side opposite to the sample supply portion 32. On the other hand, according to the related art (for example, Non-Patent Document 1), the end itself of the test-side flow path formed on one sheet corresponds to the coloring portion. For this reason, in the related art, the coloring in the coloring portion proceeds only by the permeation of the sample liquid in the direction along one sheet. Therefore, uneven coloring is likely to occur in the direction along the sheet. When uneven coloring occurs, the coloring intensity of the coloring portion cannot be appropriately evaluated, and the detection of the substance to be detected based on the coloring intensity may not be performed properly. On the other hand, according to the present embodiment, the sample liquid that has reached from the sample supply unit 32 to the communication unit 35 penetrates into the communication unit 35 along the sheet 30 as shown by the two-dot chain line in FIG. Meanwhile, the ink further permeates from the sheet 30 to the sheet 20 toward the coloring portion 24. That is, unlike the related art in which the color development proceeds only by the permeation of the sample liquid along one sheet, in the present embodiment, not only the permeation of the aqueous solution in one sheet but also the communication of the sheet 30 is performed. The color development also proceeds by the penetration of the sample liquid from the section 35 to the coloring section 24 of the sheet 20 so as to straddle the sheets. For this reason, uneven color development in the direction along the sheet unlike the prior art is unlikely to occur. Similarly, in the communication portion 36 which is the end of the control-side flow path 53, the coloring portion 25 is laminated on the communication portion 36, so that uneven coloring is less likely to occur. As described above, the concentration of the substance to be detected can be appropriately detected based on the coloring intensity of the coloring portions 24 and 25.

  Further, in the present embodiment, the sheets 10 to 30 are formed as one integrated sheet. Thereby, the paper device 1 is realized with a simple structure, and all the flow paths of the sheets 10 to 30 can be collectively formed on one sheet. It has become. The communicating portions 22 and 23 and the coloring portions 24 and 25 are formed on one integrated sheet 20. In this way, the paper device 1 is realized with a relatively simple structure by combining these parts into one sheet.

  Further, in the present embodiment, the sheet 20 is laminated on the sheet 30 on which the test channel 52 and the control channel 53 are formed, and the sheet 10 is laminated on the sheet 20 on which the communication portions 22 and 23 are formed. . Thereby, at least a part of the flow path of the sample liquid is covered by the hydrophobic region of the sheet laminated thereon. Therefore, evaporation of the sample liquid from the flow path is suppressed. By configuring the paper device 1 to have such a multilayer structure, the sample supply section 32 and the coloring sections 24 and 25 are arranged relatively lower. In order to secure such a path for introducing the sample liquid into the lower layer sample supply section 32 and a path for observing the color development in the lower layer color forming sections 24 and 25, the introduction channel 51 and the observation holes 12 and 13 are provided with the paper device 1. It is provided in.

<Other modifications>
The above is a description of a preferred embodiment of the present invention. However, the present invention is not limited to the above-described embodiment, and various changes may be made within the scope described in the means for solving the problem. It is possible.

  For example, a paper device 1 'shown in FIG. 13 may be used instead of the paper device 1 according to the above-described embodiment. The paper device 1 'includes a laminate 2' in which a sheet 10 ', sheets 41 to 45, and a sheet 30' are laminated. The sheets 10 ', the sheets 41 to 45, and the sheet 30' are different from the sheets 10 to 30, and are separated from each other. The sheet 10 ′ is formed with a set of an introduction part 11 and observation holes 12 and 13 similar to the sheet 10. In the sheet 30 ′, a set of a sample supply unit 32, a test channel 52, and a control channel 53 similar to the sheet 30 are formed. The communication portion 21 is provided on the sheet 41, the communication portion 22 is provided on the sheet 42, the communication portion 23 is provided on the sheet 43, the coloring portion 24 is provided on the sheet 44, and the coloring portion 25 is provided on the sheet 45, respectively. ing. In the sheets 41 to 45, the communicating part 21 is a sample supply part 32, the communicating part 22 is a capturing part 33, the communicating part 23 is a false capturing part 34, the coloring part 24 is a communicating part 35, and the coloring part 25 is a communicating part. 36 are stacked on the sheet 30 ′ so as to be in contact with each other. Then, the sheet 10 ′ is stacked on the sheets 41 to 45 so that the introduction portion 11 comes into contact with the communication portion 21 and the coloring portions 24 and 25 can be observed from above through the observation holes 12 and 13.

  The laminate according to the present invention may be configured by folding a single sheet that is integrally connected like the laminate 2 according to the above-described embodiment, or may be configured like the laminate 2 ′ according to the present modification. It may be configured by laminating a plurality of sheets separated from each other. Also, the communication portions 21 to 23 and a part of the coloring portions 24 and 25 are configured as one sheet integrally connected, and the other portions are individually formed on a plurality of sheets separated from other sheets. Is also good. In addition, at least one of the introduction unit 11, the sample supply unit 32, the capture unit 33, the pseudo capture unit 34, and the communication units 35 to 37 may be formed on a sheet separated from other sheets.

  For example, instead of the sample supply unit 32, the test-side channel 52, and the control-side channel 53 in the above-described embodiment, a sample supply unit 61, a test-side channel 66, and a control-side channel 67 shown in FIG. 11 are used. You may. The sample supply unit 61, the test-side flow path 66, and the control-side flow path 67 are formed of hydrophilic regions formed on filter paper in the same manner as in the above-described embodiment, and are formed by forming a pattern of hydrophobic regions on the filter paper. Is formed. The test-side flow path 66 extends from the sample supply unit 61 to an end 64 where the color-forming units are stacked. In addition, a capturing section 62 similar to the capturing section 33 is provided in the middle of the test-side channel 66. A communication section made of the same hydrophilic region as the communication section 22 is laminated on the capturing section 62. The control-side channel 67 extends from the sample supply unit 61 to an end 65 where the color-forming units are stacked. Further, a pseudo trapping section 63 similar to the pseudo trapping section 34 is provided in the middle of the control-side flow path 67. In the pseudo capture portion 63, a communication portion formed of a hydrophilic region similar to the communication portion 23 is laminated. The test side channel 66 and the control side channel 67 have the same shape as each other. That is, the test-side flow path 66 has a shape that just overlaps the control-side flow path 67 when rotated by a predetermined angle around the center O of the sample supply unit 61 in FIG.

  Further, instead of the sample supply unit 32, the test-side channel 52, and the control-side channel 53 in the above-described embodiment, a sample supply unit 71, a test-side channel 76, and a control-side channel 77 shown in FIG. 12 are used. You may. The sample supply unit 71, the test-side flow path 76, and the control-side flow path 77 are formed of hydrophilic regions formed on filter paper in the same manner as in the above-described embodiment, and are formed by forming a pattern of hydrophobic regions on the filter paper. Is formed. The test-side channel 76 extends from the sample supply unit 71 to an end 74 where the coloring units are stacked. A capturing section 72 similar to the capturing section 33 is provided in the middle of the test-side channel 76. A communication section made of the same hydrophilic region as the communication section 22 is stacked on the capturing section 72. The control-side flow channel 77 extends from the sample supply unit 71 to an end 75 where the color-forming units are stacked. Further, a pseudo capturing section 73 similar to the pseudo capturing section 34 is provided in the middle of the control-side flow path 77. A communication section made of the same hydrophilic region as the communication section 23 is laminated on the pseudo capturing section 73. The test side flow path 76 and the control side flow path 77 have a mirror image relationship with each other. That is, the test-side flow path 76 and the control-side flow path 77 have shapes that are symmetric with respect to the axis X in FIG.

  In the above-described embodiment, the capturing unit 33 is formed on the sheet 30 side, and the communication unit 22 stacked on the capturing unit 33 is formed on the sheet 20 side. However, the arrangement of the capturing unit and the communication unit may be reversed. That is, a communication part may be formed as a part of the test-side channel 52 of the sheet 30, and the capturing part stacked on the communication part may be formed in the sheet 20. Also in this case, as in Example 3, the trapping efficiency of the target substance and the target substance-enzyme complex in the trapping part is increased, and the amount of the target substance-enzyme complex reaching the coloring part 24 is reduced. The effect and the effect of making the relative coloring intensity less likely to vary are ensured.

1, 1 'Paper device 2a Hydrophilic area 2b Hydrophobic area 2 Laminate 10, 20, 30 Sheet 11 Introducing section 12, 13 Observation hole 21 to 23 Communication section 24, 25 Coloring section 32, 61, 71 Sample supply section 33 , 62, 72 Capture units 34, 63, 73 Pseudo capture units 35-37 Communication unit 51 Introductory channels 52, 66, 76 Test-side channels 53, 67, 77 Control-side channels

Claims (8)

A substance detection device for detecting the concentration of a substance in an aqueous solution based on the intensity of color generated due to the action of a first color-related substance and a second color-related substance,
Having a hydrophilic region and a hydrophobic region defining the outer edge of the hydrophilic region, comprising a laminate in which a plurality of sheets are laminated,
In the hydrophilic region of the laminate,
A sample solution containing the first complex in which the same substance as the target substance to be detected is bound to the first chromogenic substance and the target substance, or an antibody targeting the target substance is the second target. A sample supply unit to which a sample liquid containing the second complex and the substance to be detected, which is bonded to one of the coloring-related substances, is supplied;
A test-side coloring portion and a control-side coloring portion to which the second coloring-related substance is fixed;
A first capturing substance for capturing the substance to be detected and the first complex, or a capturing section to which a second capturing substance for capturing the second complex is fixed,
The sample supply unit, a test-side flow path that connects the sample supply unit to the test-side coloring unit, and a control-side flow path that connects the sample supply unit to the control-side coloring unit, the plurality of sheets One of which is formed in the hydrophilic region of the first sheet,
The shape of the test-side flow path and the shape of the control-side flow path are the same as each other or in a mirror image relationship,
A second hydrophilic portion formed of a hydrophilic region in contact with the first hydrophilic portion in the test-side channel is formed on another sheet that is one of the plurality of sheets and is stacked on the first sheet. Has been
Another sheet that is one of the plurality of sheets and is stacked on the first sheet, from the hydrophilic region in contact with the third hydrophilic portion corresponding to the first hydrophilic portion in the control-side channel. A fourth hydrophilic portion is formed,
A substance detection device, wherein one of the first hydrophilic part and the second hydrophilic part is the capturing part. Another sheet, which is one of the plurality of sheets and is stacked on the first sheet, includes a hydrophilic region in contact with an end of the test-side channel opposite to the sample supply unit. A fifth hydrophilic portion is formed,
Another sheet, which is one of the plurality of sheets and is stacked on the first sheet, includes a hydrophilic region in contact with an end of the control-side channel opposite to the sample supply unit. A sixth hydrophilic portion is formed,
The fifth hydrophilic portion is the test-side coloring portion,
2. The substance detection device according to claim 1, wherein the sixth hydrophilic portion is the control-side coloring portion.   The said 2nd hydrophilic part, the said 4th hydrophilic part, the said 5th hydrophilic part, and the said 6th hydrophilic part are formed in the 2nd sheet | seat which is one sheet of Claim, The Claim 2 characterized by the above-mentioned. Substance detector. An observation hole formed in a third sheet, which is one of the plurality of sheets and stacked on the second sheet, at a position overlapping with the test-side coloring section and the control-side coloring section; And a hydrophobic region covering the hydrophilic portion and the fourth hydrophilic portion,
4. The substance detection device according to claim 3, wherein the second sheet and the third sheet have an introduction flow path formed of a hydrophilic region for introducing the sample liquid into the sample supply unit. 5.   The said 1st sheet | seat, the said 2nd sheet | seat, and the said 3rd sheet | seat correspond to three area | regions divided by the folding line in one sheet | seat folded into three, The said sheet | seat of Claim 4 characterized by the above-mentioned. Substance detector.   At least a part of the test-side flow path and the control-side flow path is one of the plurality of sheets, and is covered by a hydrophobic region of another sheet stacked on the first sheet. The substance detection device according to claim 1, wherein:   7. The hydrophilic region forming each of the test-side coloring portion, the control-side coloring portion, and the capturing portion has a disk shape. 8. Substance detection device.   The substance detection device according to any one of claims 1 to 7, wherein the hydrophobic region of the laminate includes a dried product of a hydrophobic ink therein.

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