Target substance detection method and reagent

Abstract

To provide a technique that allows for detecting a target substance in a sample with higher sensitivity at a lower cost compared to conventional techniques.SOLUTION: A target substance detection method is provided, comprising forming a complex by sandwiching a target substance between a first capture substance immobilized on a solid phase and a second capture substance labeled with a labeling substance in a first reaction field, isolating a moiety containing the labeled substance from the complex, and moving the isolated moiety through a fluid filling a tubular channel by a centrifugal force to detect the moiety containing the labeled substance using scattered light scattered by the moiety containing the labeled substance.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a detection method and a reagent for detecting a target substance in a sample, and more particularly to a detection method and a reagent for detecting a target substance with high sensitivity and at low cost. [Background technology]

[0002] Conventionally, immunoassays have been known that detect specific antigens or antibodies associated with a disease as biomarkers to qualitatively or quantitatively analyze disease detection, treatment effects, and the like. In recent years, there has been an increasing demand for highly sensitive detection that detects trace amounts of biomarkers in order to detect more effective biomarkers. Enzyme-Linked Immuno Sorbent Assay (hereinafter also referred to as "ELISA"), which is one of the immunoassays, is a method in which a target antigen, antibody, or nucleic acid contained in a sample solution is captured with a specific antibody, an antigen that binds to the target antibody, or a complementary nucleic acid, and detected using an enzyme reaction, and is widely used due to its cost and other advantages.

[0003] The ELISA method is a measurement method that allows for quantitative detection of target substances, but most of the currently popular ELISA methods are performed on 96-well microplates, which requires many steps and complicated operations. In addition, since multiple reaction steps are required, it takes a lot of time and effort to obtain measurement results. For this reason, there is a demand for shortening the operation time and reaction time. Furthermore, there is a demand for even higher sensitivity detection.

[0004] To achieve high-sensitivity detection, it is considered to improve the reactivity between the enzyme and the substrate. For this purpose, for example, a technique has been proposed in which an immune complex (labeled compound) to which a reporter has been added is released from a solid phase and reacted in a free liquid medium (Patent Document 1). In this Patent Document 1, components of the immune complex are designed so that biotins or functionalized azo dyes and avidins are introduced into the immune complex. For example, a labeled antibody is prepared to which a reporter group is bound via a streptavidin-biotin bond. When an immune complex is formed on the solid phase, a reporter group is introduced into the immune complex via a streptavidin-biotin bond. After that, when an excess of streptavidin is added, the streptavidin in the complex is replaced by the added streptavidin, and the reporter molecule in the immune complex is released. Therefore, in this technique, the reporter molecule can be detected in a free liquid medium and related to the concentration of the analyte in the sample.

[0005] Also, in order to detect immune complexes with high sensitivity, microreactors that perform reactions in microcontainers on the μm scale have been developed using microfabrication technology. It is expected that the reaction rate can be improved by using a microreactor in which the reaction field is a microspace. The realization of experimental operations such as extraction and separation on a microreactor using a microchannel or reaction chamber is also called lab on a chip. It has been proposed to form a microreactor on a circular disk, and to distribute a sample liquid by centrifugal force caused by rotation to isolate biological materials (Patent Document 2). It has also been proposed to form multiple chambers, channels, reservoirs, etc. on a circular disk, and to perform the delivery of reagents and waste liquid by rotating the disk, thereby performing the immunoassay procedure that was previously performed manually in a well with a microreactor (Patent Document 3).

[0006] In the immunoassay in the microreactor, a method using labeled beads has been proposed for the purpose of detecting a low concentration of a target substance (Patent Document 4). For example, Patent Document 4 shows a circular disk provided with a bead-filled section and a detection area, in which labeled beads whose surfaces are modified with antibodies are filled. An antigen in a sample solution injected into the disk is captured by the antibody on the labeled beads, and a labeled bead complex in which the antigen is bound to the labeled beads is sent to the detection area by the rotation of the disk and captured by the antibody fixed to the detection area. It is disclosed that the labeled bead complex fixed to the detection area is thus counted by an optical reading means of an optical disk device to detect a target substance in a sample.

[0007] On the other hand, a method has been proposed in which an analysis method using a disk is proposed in which antigen-carrying beads, a fluorescent IgG antibody-labeled reagent, and a sample are mixed in a reaction field provided on the disk, and a complex is formed by binding with a target substance, and then the beads are introduced from the reaction field into a separation chamber filled with a density gradient medium by centrifugal force generated by rotating the disk, and the complex is detected by irradiation with an electromagnetic irradiation beam. The separation chamber is filled with a density gradient medium, and the introduced beads are moved to an isopycnic point in the density gradient medium by centrifugal force and stop there. Then, the target immune complex can be detected by detecting the fluorescence from the isopycnic point. According to this analysis method, surplus fluorescent labels and the like that are not bound to immune complexes whose isopycnic points are different from the beads can be separated from the target immune complex, and the immune complex can be detected without a washing operation (Patent Document 5). [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Special Publication No. 6-505802 [Patent Document 2] JP 2008-185423 A [Patent Document 3] Special Publication No. 2002-503331 [Patent Document 4] JP 2012-255772 A [Patent Document 5] Special Publication No. 2006-505766 Summary of the Invention [Problem to be solved by the invention]

[0009] However, the technology described in Patent Document 1 only discloses liberating a reporter molecule, which is a labeling substance, in a free liquid medium and reacting it with a substrate in the liquid, and is insufficient to realize improved detection sensitivity and improved reactivity that would enable detection of low concentrations of a target substance.

[0010] Patent Document 2 does not disclose anything about performing immunoassays with high sensitivity. Patent Document 3 merely discloses a conventional ELISA process implemented in a microreactor. Furthermore, while a microreactor can improve reaction speed and reactivity by reducing the reaction field, there is a problem in that the signal intensity is reduced due to the reduced reaction volume. For this reason, if a high-sensitivity measurement of immune complexes is to be performed using the techniques described in Patent Documents 2 and 3, a high-sensitivity detector and camera are required, resulting in a problem of high cost.

[0011] Patent Document 4 discloses a technique for performing immunoassays using a general-purpose optical disk device by using labeled beads as detection targets. Generally, the transportation of particulate matter in a microreactor can cause clogging. In the technique of Patent Document 4, antibodies are immobilized in the detection region, and labeled beads carrying antigens that flow into the detection region are captured. This can lead to partial accumulation of the labeled beads, which can make actual handling difficult in some cases. In addition, there is a problem that if the labeled beads captured in the detection region come close to each other, it can cause a detection error.

[0012] In addition, in the technology described in Patent Document 4, an antigen-antibody reaction is carried out between the immobilized antibody and the antigen supported on the labeled beads while the labeled beads are flowing down, so the entire detection area becomes the reaction field of the antigen-antibody reaction. Since reactivity is affected by the frequency of contact between the antigen and the antibody, the technology in which the entire detection area becomes the reaction field tends to have low antigen-antibody reaction efficiency. In addition, since the capture of the labeled beads and its detection (i.e., detection of the antigen) are carried out in the same reaction field, the detection sensitivity decreases when the reaction efficiency is low. Furthermore, depending on the flow speed of the antibody, it becomes difficult for the immobilized antibody to capture the labeled beads, which causes problems such as further deterioration of the detection sensitivity and detection speed.

[0013] In addition, the technology described in Patent Document 5 detects the complex by concentrating (distributing) beads carrying the complex at isopycnic points in a density gradient medium. Therefore, the signal detection becomes more difficult as the amount of accumulation decreases, making it difficult to detect the target substance in the sample with higher sensitivity.

[0014] The present invention has been made in consideration of the above problems, and one of its objects is to provide a technique that can detect a target substance in a sample with higher sensitivity and at lower cost than conventional techniques. [Means for solving the problem]

[0015] According to one aspect of the present invention, a complex is formed by sandwiching a target substance between a first capture substance bound to a labeling substance and a second capture substance immobilized on a solid phase, and then a portion containing the labeling substance is separated from the formed complex, The separated portion containing the labeled substance is moved through a liquid filled in a tubular passage by centrifugal force; The present invention provides a method for detecting a target substance, comprising detecting the portion containing the labeling substance by scattered light produced by irradiating light into the tubular passage and scattering the portion containing the labeling substance.

[0016] According to a preferred embodiment of the present invention, Before separating the portion containing the labeling substance from the complex, a washing process is performed to wash the complex in a state in which the inflow of the complex into the tubular passage is inhibited.

[0017] According to a preferred embodiment of the present invention, when the cleaning process is performed, the tubular passage is filled with the cleaning liquid used for cleaning.

[0018] According to a preferred embodiment of the present invention, the cleaning liquid used in the cleaning process is stored downstream of the tubular path.

[0019] According to a preferred embodiment of the present invention, the cleaning process is carried out in a state in which the composite is held in a predetermined chamber to prevent the composite from flowing into the tubular passage.

[0020] According to another preferred embodiment of the present invention, the method of the present invention comprises the steps of: A first storage chamber for storing the cleaning liquid, the chamber, and a second storage chamber for storing a liquid flowing out from the chamber are arranged in this order from a rotation center side of the device toward an outward side, the first storage chamber and the chamber are communicated with each other through a flow path that can be opened and closed, and the chamber and the second storage chamber are communicated with each other through the tubular path; The cleaning process is carried out by rotating the device to supply the cleaning solution from the first reservoir to the chamber and then discharging the solution through the tubular path into the second reservoir.

[0021] According to another preferred embodiment of the present invention, the first storage chamber and the chamber are communicated with each other through an openable flow path.

[0022] According to another preferred embodiment of the invention, the washing process is performed until the second reservoir and the tubular passage are filled with the washing liquid.

[0023] According to another preferred embodiment of the present invention, the method of the present invention comprises the steps of: a third storage chamber for storing a liquid containing a reagent for separating a portion containing the labeling substance from the complex is disposed closer to a rotation center than the chamber of the device, and the third storage chamber and the chamber are communicated with each other through a flow path that can be opened and closed; The method includes rotating the device to supply a liquid containing the reagent to the chamber after the cleaning process to separate a portion containing the labeled substance, and then moving the separated portion containing the labeled substance into the tubular path.

[0024] According to another preferred embodiment of the present invention, the third storage chamber and the chamber are communicated with each other through an openable flow path.

[0025] According to another preferred embodiment of the present invention, scattered light intermittently generated within a predetermined time from the tubular path is detected.

[0026] According to another preferred embodiment of the present invention, the marker substance is a substance having a higher specific gravity than the liquid filled in the tubular passage. According to another preferred embodiment of the present invention, the labeling substance is a fine particle whose main component is one selected from the group consisting of metal, ceramic, glass, and resin. According to another preferred embodiment of the present invention, the labeling substance is a metal colloid particle.

[0027] According to another preferred embodiment of the present invention, the solid phase is at least one particulate material selected from the group consisting of glass particles, ceramic particles, magnetic particles, and resin particles. According to another preferred embodiment of the present invention, the solid phase is a structure inside a container used in forming the complex.

[0028] According to another preferred embodiment of the present invention, separation of the portion containing the labeling substance from the complex is carried out by at least one treatment selected from the group consisting of a heat treatment, a pH adjustment treatment, a denaturation treatment, an oxidation treatment, a reduction treatment, an enzyme treatment, and a competitive reaction treatment.

[0029] According to another preferred embodiment of the present invention, the first capture substance is at least one selected from the group consisting of an antibody, an antibody fragment, a modified antibody, an antigen, an aptamer, and a nucleic acid that specifically binds to a target substance.

[0030] According to another preferred embodiment of the present invention, the second capture substance is at least one selected from the group consisting of an antibody, an antibody fragment, a modified antibody, an antigen, an aptamer, and a nucleic acid that specifically binds to a target substance.

[0031] According to another aspect of the present invention, there is provided a reagent for use in the above-mentioned method for detecting a target substance of the present invention, the reagent having the effect of separating a portion containing the labeling substance from the complex. According to another preferred embodiment of the present invention, the reagent of the present invention includes at least one selected from the group consisting of a pH adjuster, a denaturant, a reducing agent, an oxidizing agent, an enzyme, and a competing agent. According to another preferred embodiment of the present invention, the reagent of the present invention contains a pH adjuster. Effect of the Invention

[0032] According to the method for detecting a target substance of the present invention, the portion containing the labeling substance separated from the complex (also referred to as "labeling substance-containing fragment" in this specification) is moved by centrifugal force through a liquid filled in a tubular path, and the labeling substance-containing fragment is detected by the scattered light that is generated by the labeling substance-containing fragment reflecting and scattering the light irradiated into the tubular path. In general, the smaller the signal generation source (substance that generates an optical signal, labeling substance) in the detection target, the more advanced technology is required to detect the detection target using the change in absorbance as a signal. However, the method of the present invention obtains scattered light as an optical signal, so that minute detection targets such as the labeling substance-containing fragment can be sufficiently detected.

[0033] Here, when detecting a target substance using scattered light, scattered light is also generated from substances other than the target substance, so that a problem often occurs in that the target substance cannot be specifically detected. However, in the method of the present invention, the labeled substance-containing fragments in the liquid move at their own moving speed due to the action of centrifugal force, so that the labeled substance-containing fragments that are the target substance can be specifically detected. Furthermore, in the method of the present invention, the labeled substance-containing fragments move in the liquid filled in the tubular passage due to the action of centrifugal force, so that they move at a slower speed than when they are transported with the flow of the liquid. Therefore, according to the method of the present invention, scattered light from the labeled substance-containing fragments can be easily obtained even while the labeled substance-containing fragments are moving in the tubular passage due to centrifugal force.

[0034] Furthermore, according to the method of the present invention, for example, when beads are used as the solid phase, even if multiple target substances are carried on one bead, the number of label-containing fragments separated from the beads can be sequentially detected (counted) rather than counting one bead at a time, thereby achieving the effect of enabling the target substance to be detected with high sensitivity.

[0035] According to the reagent of the present invention, the portion of the complex that contains the labeling substance, i.e., the labeling substance-containing fragment, can be separated satisfactorily, and therefore a reagent suitable for detecting a target substance can be provided. [Brief description of the drawings]

[0036] [Figure 1] FIG. 1 is a diagram for explaining an overview of an embodiment of a detection method of the present invention. [Figure 2a] 1 is a schematic plan view showing an example of a disk suitable for carrying out a detection method according to a first embodiment. FIG. [Figure 2b] FIG. 2b is a partially enlarged view of FIG. 2a. [Diagram 3] FIG. 4 is a schematic plan view showing another example of a disk suitable for carrying out the detection method of the first embodiment. [Figure 4] FIG. 2 is a schematic diagram illustrating an example of a detection device. [Diagram 5] FIG. 2 is a graph showing the relationship between the pH value and cleavage characteristics of each reagent in Example 1. [Figure 6] 13 is a graph showing the relationship between the concentration (pH value) of NaOH and the cutting characteristics in Example 2. FIG. [Figure 7] FIG. 13 is a diagram showing the cleavage characteristics of various denaturing agents in Example 3. [Figure 8] FIG. 1 shows the change in gold colloid particle diameter due to sensitization treatment in Example 4. [Figure 9] FIG. 13 is a graph showing the relationship between the particle size of metal colloid particles and the scattered light intensity in Example 5. [Figure 10] FIG. 13 is a graph showing the relationship between the concentration (corresponding to the antigen concentration) of eluted colloidal gold particles (portion containing colloidal gold particles) and scattered light intensity in Example 6. [Figure 11] FIG. 13 is a diagram illustrating selective outflow of gold colloid particles (portions containing gold colloid particles) in Example 7. [Figure 12] FIG. 13 shows the results of measuring gold colloid particles (portion containing gold colloid particles) in Example 7. [Figure 13a] FIG. 13 shows the results of detecting gold colloid particles (parts containing gold colloid particles) in Example 8, with scattered light being converted into a voltage value. [Figure 13b] FIG. 13B is a partially enlarged view of FIG. 13A. [Figure 14a] FIG. 13 shows the results of detecting scattered light from colloidal gold particles (parts containing colloidal gold particles) as voltage values ​​when no antigen was used for immune complex formation (blank) in Example 8. [Figure 14b] FIG. 14B is an enlarged view of a portion of FIG. 14A. [Figure 15] FIG. 13 is a graph showing the relationship between the spot count number and the antigen concentration measured in Example 8. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] <Detection method of the present invention> In the detection method of the present invention, the portion containing the labeled substance separated from the complex is moved in the liquid filled in the tubular passage by centrifugal force, so that the portion containing the labeled substance is intermittently supplied to the liquid filled in the tubular passage. The portion containing the labeled substance is detected by the scattered light generated by scattering the irradiated light irradiated to the tubular passage by the portion containing the labeled substance. The complex is formed by sandwiching the target substance between a first capture substance bound to the labeled substance and a second capture substance fixed to a solid phase. Therefore, the target substance can be detected by detecting the portion containing the labeled substance separated from the complex.

[0038] In this specification, when each element is connected with a hyphen, such as "solid phase-second capture substance-target substance-first capture substance-labeled substance", it means that they are physically or chemically bonded to each other. Furthermore, "first capture substance-labeled substance" may be written as "first capture substance bound to a labeled substance" or simply as "first capture substance". "Outer side", "outer direction" and "outer" refer to the centrifugal direction. "Main component" refers to the component with the highest content, for example, a component with a content of more than 50% by mass.

[0039] Hereinafter, one embodiment of the detection method of the present invention will be described with reference to Fig. 1. Fig. 1 is a schematic diagram showing the concept of one embodiment of the detection method of the present invention. For ease of explanation, Fig. 1 shows a case in which the target substance is an antigen and the first and second capture substances are primary antibodies.

[0040] In the initial state, a solid phase on which a second capture substance for capturing a target substance is fixed is prepared in a reaction field (first reaction field, a space in which a reaction is carried out) (step 1, FIG. 1(a)). When a sample solution containing a target substance is introduced therein (FIG. 1(b)), the target substance in the sample solution is specifically captured by the second capture substance (step 2). After removing anything other than the captured target substance from the reaction field by a washing process as necessary, a first capture substance bound to a labeling substance is introduced into the reaction field, and this first capture substance binds to the target substance, forming an immune complex (hereinafter also simply referred to as an "immune complex") in which the target substance is sandwiched between the second capture substance and the first capture substance on the solid phase (step 3, FIG. 1(c)). It is preferable to carry out a washing process next. It is preferable to remove substances that affect the detection results when mixed into the tubular path by this washing process beforehand from the system, and such substances include, for example, an excess of the first capture substance that has not formed a complex and a free labeling substance. This washing treatment may also be used to remove other contaminants, resulting in the labeling substance being supported on the solid phase in an amount corresponding to the target substance captured on the solid phase.

[0041] Then, the formed complex is treated with a reagent such as an enzyme, an oxidizing agent, a denaturant, an acid, or a base, or an external field such as heat, light (electromagnetic waves), or vibration (FIG. 1(d)), to separate the portion containing the labeling substance from the solid phase (step 4, FIG. 1(e)). The portion to be separated (the portion containing the labeling substance) may be the labeling substance alone, or may be in a state in which some molecular group is bound to the labeling substance. The molecular group is not limited to a second capture substance-target substance-first capture substance complex, a target substance-first capture substance complex, or a first capture substance, in which the original molecular group remains intact (i.e., a molecular group formed by cleavage of intermolecular bonds), but may also be a molecular group formed by cleavage of intramolecular bonds within the original molecular group and fragmentation.

[0042] The reaction field is a space partitioned into which a reaction is carried out in which a target substance is sandwiched between a first capture substance bound to a labeling substance and a second capture substance fixed to a solid phase to form a complex, and a reaction in which a portion containing the labeling substance is separated from the formed complex. Examples of the reaction field include a reaction vessel and a reaction chamber. In this method, steps 1 to 4 shown in Fig. 1(a) to (e) may be carried out in separate reaction fields, or consecutive steps may be carried out in the same reaction field, as long as the portion containing the labeling substance can be moved in a tubular path by centrifugal force. For example, steps 1 to 3 may be completed using a single vessel, and step 4 may be completed in another single vessel.

[0043] In this embodiment, a reaction chamber provided in a rotatably configured device may be used as the reaction field. It is possible to appropriately select from which stage of steps 1 to 4 or the washing treatment that is appropriately performed, the reaction chamber is used. For example, a solid phase to which a second capture substance is immobilized may be loaded into the reaction chamber of the device, and all of steps 1 to 4 may be performed using the reaction chamber, or steps 1 to 3 may be performed in a container outside the device, and steps 4 and after may be performed in the reaction chamber of the device. In the latter case, regardless of whether the product of step 4 is subjected to solid-liquid separation or not, a solution containing a portion containing a labeling substance is introduced into the reaction chamber, and in such a case, the reaction chamber is simply used as a detection field, i.e., a front chamber for supplying the solution into a tubular channel with a small diameter.

[0044] After step 4, the portion containing the labeled substance contained in the solution is moved by centrifugal force into the detection field, i.e., into the liquid filled in the tubular path with a small diameter (FIG. 1(f)). In the example shown in FIG. 1(f), a tubular path with a small diameter is connected from the reaction chamber toward the outside direction (right side of the figure) from the center of rotation. Therefore, if the portion containing the labeled substance is stored in the reaction chamber of the device in a state of being contained in the solution, the portion containing the labeled substance can enter the liquid filled in the tubular path with a small diameter by the centrifugal force generated when the device is rotated, and move further outward in the liquid. Also, in the example shown, the hole diameter of the tubular path with a small diameter is set to a dimension that allows the portion containing the labeled substance to pass but not the solid phase to pass, so that the portion containing the labeled substance can enter the tubular path while preventing the inflow of the solid phase. Here, the portion containing the labeled substance moves by centrifugal force in the liquid filled in the tubular path. The movement is due to so-called centrifugal sedimentation. In other words, the movement speed of the portion containing the labeled substance is relatively large compared to the movement speed of the liquid filled in the tubular path. In the present invention, the liquid filled in the tubular passage hardly moves due to centrifugal force, and the movement speed is usually almost zero, but in principle, slight movement is acceptable as long as it does not substantially affect the movement of the part containing the target substance.

[0045] The other end of the tubular path is connected to a second storage chamber (not shown in FIG. 1) provided in the device. The tubular path and the second storage chamber may be always in communication with each other, or may be freely opened and closed by a valve. This second storage chamber may be provided downstream of the chamber in the device, and is preferably provided at a position outside the chamber. Before the portion containing the labeled substance is moved to the tubular path, the tubular path is filled with liquid. When the second storage chamber is provided at a position outside the chamber, the tubular path and the second storage chamber are always in communication with each other, and both the tubular path and the second storage chamber are filled with liquid. The timing of filling the tubular path with liquid varies depending on the timing of using the reaction chamber. For example, when the reaction chamber is used before the above-mentioned step 3, the tubular path is filled with liquid after the cleaning process after step 3 and before step 4. Furthermore, when the reaction chamber is used after step 4, the tubular path is preferably filled with liquid before the solution containing the portion containing the labeling substance is introduced into the reaction chamber. A valve may be provided in the reaction chamber, and after the filling of the tubular path with the liquid is completed, the valve may be opened to introduce the portion containing the labeling substance into the liquid filled in the tubular path. In such a case, the timing for introducing the solution containing the portion containing the labeling substance into the reaction chamber may be before or after the filling of the tubular path with the liquid is completed.

[0046] The detection field is a field for detecting scattered light from the tubular passage, and is equipped with a light source and a light receiving element, and is a field for causing an optical change in which scattered light is generated based on the irradiated light (hereinafter, it may be referred to as the "second reaction field" as appropriate). By forming at least a part of the tubular passage onto which the irradiated light is irradiated from a light-transmitting material, the irradiated light irradiated from the light source toward the tubular passage passes through the wall of the tubular passage and is scattered by the labeling substance moving through the tubular passage, generating scattered light. By detecting this scattered light with a light receiving element, the presence or absence of the labeling substance can be detected (Figure 1(g)).

[0047] The speed at which a particle in a liquid moves when placed in a centrifugal field is explained by Stokes' law. Specifically, when a particle with mass m rotates with a rotation radius r1 and an angular velocity ω, it experiences a centrifugal force f as shown in the following formula (1).

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[0048] It is possible to know the particle size, density, and even the viscosity of the solution of the labeled substance in advance. In addition, if the labeled substance is sufficiently large or heavy in the part containing the labeled substance compared to the other parts, the part containing the labeled substance can be approximated by the labeled substance. This makes it possible to derive the moving speed of the part containing the labeled substance and the arrival time to a predetermined position (detection area) of the tubular path theoretically or experimentally based on formula (5). Note that, since formula (5) is a theoretical formula, if the preconditions of formula (5) are not met, the moving speed, etc. may be calculated after correcting formula (5).

[0049] As described above, the time it takes for the portion containing the labeled substance to pass through the detection area can be estimated based on the derived migration speed. Furthermore, the portion containing the labeled substance passes through the detection area within a predetermined time from the start of centrifugation. Therefore, it can be determined that the scattered light detected in the detection area during the predetermined time is scattered light caused by the target detection object. Furthermore, since the detection target moves through a tubular passage, the scattered light from the target becomes an intermittent signal, and the continuously occurring scattered light can be identified as background noise due to some factor, and the scattered light from the detection target can be specifically detected.

[0050] In this way, the detection method of the present invention can appropriately detect minute detection targets. The labeled substance (the portion containing the labeled substance) supplied to the tubular passage is derived from the compound formed with the target substance in the reaction field, so that the presence of the target substance can be detected by detecting the labeled substance. In addition, the number of occurrences of scattered light that occurs intermittently within a given period of time can be counted. Based on this count, the number of portions containing the labeled substance that have been supplied to the tubular passage, i.e., the number of target substances, can be quantitatively evaluated. This is particularly effective in the case of trace analysis in which the number of target substances in a sample is small.

[0051] Furthermore, in the detection method of the present invention, a portion containing a labeled substance is separated from the solid phase, and the separated portion containing a labeled substance is introduced into the detection field. Therefore, compared with the conventionally proposed method of performing immunoassays by counting beads that have formed immune complexes using a disk-like microreactor, the size of the substance passing through the flow path can be significantly reduced, and the substance to be detected can be introduced into the detection field smoothly and easily.

[0052] <Target substance> The target substance to be analyzed in the measurement method of the present invention is one that binds to a specific biomolecule, and may be one that binds to a specific biomolecule by a biochemical reaction. For example, it may be one that can be detected immunologically or genetically, and is not particularly limited. Specific examples of target substances include pathogens such as bacteria and viruses, proteins, peptides, DNA, RNA, nucleic acids such as aptamers, exosomes, sugar chains, and various other compounds. Further examples include physiologically active substances such as hormones, and their agonists, antagonists, and alkaloids.

[0053] <First capture substance> The first capture substance of the present invention binds to the target substance captured by the second capture substance, sandwiching the target substance between the second capture substance immobilized on the solid phase and the second capture substance immobilized on the solid phase to form a complex immobilized on the solid phase, i.e., a complex bound in the order of solid phase-second capture substance-target substance-first capture substance (first capture substance bound to a labeling substance). When the target substance is an antigen, an antibody that specifically reacts with the target substance is used as the first capture substance. On the other hand, when the target substance is an antibody, the first capture substance may be an antigen to which the antibody specifically binds, or an antibody that binds to the antibody and is different from the second capture substance, or an antibody produced in a host other than the host that produces the antibody may be used. Therefore, when the target substance is an antibody, a complex is formed which is solid phase-antigen (second capture substance)-target substance-antigen (first capture substance) or antibody (first capture substance), or a complex is formed which is solid phase-antibody (second capture substance)-target substance-antigen (first capture substance) or antibody (first capture substance).

[0054] Furthermore, as the first capture substance of the present invention, in addition to the above-mentioned antigens and antibodies, nucleic acids, aptamers, either biotin-avidin, or either ligand-receptor may be used depending on the target substance. For example, when the target substance is DNA derived from bacteria, a nucleic acid molecule having a sequence complementary to a partial region of the DNA can be used as the first capture substance. Furthermore, when the target substance is a sequence-specific DNA-binding protein such as a transcription factor, DNA having a specific sequence can be used as the first capture substance.

[0055] The antibody used as the first capture substance may be a polyclonal antibody or a monoclonal antibody, but from the viewpoint of reaction specificity, a monoclonal antibody is preferable.

[0056] The antibody used as the first capture substance includes an antibody, as well as an antibody fragment and a modified antibody having substantially the same reactivity as the antibody. The antibody fragment includes a Fab fragment, a F(ab')2 fragment, a Fab' fragment, and a scFv fragment.

[0057] <Second capture substance> The second capture substance of the present invention has the function of capturing a target substance onto a solid phase and is used by immobilizing it on a solid phase; for example, when the target substance is an antigen, an antibody that specifically reacts with the target substance is used, and when the target substance is an antibody, an antigen or antibody to which the antibody specifically reacts is used.

[0058] Furthermore, as the second capture substance of the present invention, in addition to the above-mentioned antigens and antibodies, nucleic acids, aptamers, ligands (substrates, inhibitors, and antagonists of the target substance), and receptors may be used depending on the target substance. For example, when the target substance is DNA derived from bacteria, a nucleic acid molecule having a sequence complementary to a partial region of the DNA can be used as the second capture substance. In addition, when the target substance is a sequence-specific DNA-binding protein such as a transcription factor, DNA having a specific sequence can be used as the second capture substance.

[0059] The antibody used as the second capture substance may be a polyclonal antibody or a monoclonal antibody, but from the viewpoint of reaction specificity, a monoclonal antibody is preferable.

[0060] The antibody used as the second capture substance includes an antibody, as well as an antibody fragment and a modified antibody having substantially the same reactivity as the antibody. The antibody fragment includes a Fab fragment, a F(ab')2 fragment, a Fab' fragment, and a scFv fragment.

[0061] Here, the size of the solid phase is much larger than that of the second capture substance. Therefore, multiple second capture substances are easily immobilized on the solid phase. In a conventional method in which a target substance is captured on a solid phase such as beads and the number of beads that have captured the target substance is counted to detect the target substance, the detection result may be significantly affected if multiple target substances are captured on one bead. However, in this embodiment, the detection target is a portion containing a labeling substance separated from a complex that is not bound to beads, so that highly sensitive detection can be achieved. In addition, if steric hindrance of the labeling substance significantly affects the access of the first capture substance to the target substance captured on the solid phase, the concentration, etc. of the second capture substance may be appropriately adjusted when binding to the solid phase so that the distribution of the second capture substance on the solid phase is sparse, i.e., sparsely and densely distributed.

[0062] <Labeling substance> The labeling substance is not particularly limited as long as it labels the first capture substance and generates scattered light. For example, inorganic substances such as glass, ceramics, and metals, and fine particles mainly composed of fluorescent substances or resins are included. Examples of resin particles include synthetic latex such as polystyrene latex, latex such as natural rubber latex, and synthetic resin particles such as polystyrene. The labeling substance may be composed of a single material or multiple materials. Furthermore, it may be a combination of different inorganic-organic materials. Examples of combinations of different materials include composite particles in which metal particles are supported on resin particles or ceramic particles, and composite particles in which metal is coated on resin particles. In addition, it may be a resin particle supported by a fluorescent substance, but is not limited thereto.

[0063] The particle size of these labeling substances is preferably 80 nm to 3 μm in terms of scattered light intensity and mobility within the device. Note that, since scattered light intensity, migration speed in a centrifugal field, reactivity to the first capture substance, and the like vary depending on the type and size of the labeling substance, an appropriate particle size is selected taking these factors into consideration.

[0064] Considering the contribution of centrifugal force to the movement speed, a substance with a higher specific gravity than the liquid filling the tubular passage is preferable as the labeling substance. Inorganic particles with a higher density than organic matter and resin particles with a relatively large particle size are suitable as labeling substances. This is because the movement speed of the part containing the labeling substance can be made to differ greatly from the movement speed of the organic matter, which is the main impurity in the sample, in the detection field. Among these, metal particles and metal colloid particles, which are expected to obtain good scattered light intensity, are particularly suitable as labeling substances.

[0065] As the metal colloid particles that can be used as the labeling substance, various metal colloid particles can be appropriately selected and used, for example, metal colloids such as gold colloid, silver colloid, platinum colloid, etc., and composite metal colloid particles thereof can be mentioned. Examples of the composite metal colloid include platinum-supported gold colloid, platinum-supported palladium colloid, etc.

[0066] In the present invention, preferred metal colloid particles include gold colloid particles, composite metal colloid particles thereof, and silver colloid particles, and more preferred are gold colloid particles.

[0067] The particle diameter of the metal colloid particles is usually 1 nm to 500 nm. The particle diameter of the metal colloid particles suitable for detection varies depending on the sensitivity, accuracy, measurement conditions, etc. of the optical detection system selected, but for example, when using a simple detection system that detects scattered light by combining a laser light source and a photodiode, the particle diameter of the metal colloid particles is 80 nm or more, more preferably 150 nm or more, and more preferably 200 nm or more for easier detection. The particle diameter is preferably 80 nm to 500 nm, more preferably 100 nm to 500 nm, even more preferably 120 nm to 300 nm, and particularly preferably 180 nm to 250 nm.

[0068] When the first capture substance is an antibody, the case where the antibody serving as the first capture substance is labeled and the case where an antibody that recognizes the antibody (for example, a secondary antibody) is labeled are included.

[0069] The method of binding the labeling substance and the first capture substance is not limited, and can be, for example, binding by physical adsorption, chemical binding, binding using affinity, and combinations of these. The labeling substance and the first capture substance can be bound by a known method.

[0070] The size of the labeling substance is relatively larger than that of the first capture substance. Therefore, there may be cases where a plurality of first capture substances are bound to the labeling substance. When a large number of first capture substances are bound to the labeling substance, the reactivity between the first capture substance and the target substance is improved, while the higher the concentration of the target substance, the more likely it is that a plurality of target substances correspond to one labeling substance. In that case, the concentration of the target substance is evaluated at a lower concentration than the actual concentration. Therefore, in order to broaden the concentration range in which quantitative detection is possible to a higher concentration side, a process for optimizing the ratio of the labeling substance to the first capture substance may be performed. As such a process, for example, a process for adjusting the charge ratio of the first capture substance bound to the labeling substance and the target substance in a reaction in which the first capture substance bound to the labeling substance and the target substance are bound to each other may be mentioned.

[0071] Furthermore, a tubular path, which is a hollow conduit, is used to introduce the sample (the portion containing the labeling substance) into the detection field, and preferably a tubular path with a small diameter is used. The small diameter tubular path may be, for example, a microchannel. When the labeling substance is an insoluble substance such as a metal colloid particle, the smaller the particle size of the labeling substance bound to the first capture substance is, the more preferable it is to avoid clogging of the channel. On the other hand, in consideration of the fact that the scattered light intensity decreases as the particle size becomes smaller, the labeling substance is suitably selected from those preferably in the particle size range of 80 nm to 3 μm, as described above.

[0072] Here, when the labeling substance is a metal colloid particle, for example, a sensitization reaction may be performed to grow the colloid particle and increase the particle size. According to this, when the first capture substance and the metal colloid particle of the labeling substance are bound to each other, smaller metal colloid particles are used, so that the number of the first capture substance bound to one metal colloid particle can be suppressed. And, if the particle size of the metal colloid particle is increased by a sensitization reaction before the labeling substance is introduced to the detection field, the scattered light intensity can be increased. In the present invention, it is sufficient to introduce the labeling substance to the detection field, and it is not necessary to introduce the target substance or the first capture substance bound to the target substance to the detection field. Therefore, there is no problem even if the target substance bound to the labeling substance or the first capture substance is decomposed or denatured by the sensitization reaction.

[0073] Specifically, when the labeling substance is a metal colloid particle, for example, by utilizing the catalytic activity of the metal colloid or by using a reducing agent in combination, metal ions in a metal salt solution containing gold ions or silver ions that has been added separately can be reduced and accumulated on the surface of the metal colloid of the labeling substance, thereby increasing the particle size of the metal colloid.

[0074] The metal colloid particles to be subjected to this sensitization reaction preferably have a core metal species that is highly stable against oxidation and reduction. The core metal colloid particles may be formed of a single metal or a composite metal. Examples of such metal colloid particles include gold colloid particles, platinum colloid particles, and palladium colloid particles. Examples of composite metal colloid particles include platinum-gold colloid particles in which platinum colloid particles are supported on gold colloid particles.

[0075] In such a sensitization reaction of metal colloid particles, examples of metal salts serving as a source of metal ions include salts of metals belonging to the 4th, 5th, and 6th periods of the periodic table, and it is preferable to use salts of gold, platinum, silver, palladium, nickel, cobalt, copper, etc. The metal to be precipitated may be the same metal as the metal colloid particles or a different metal, and is not limited as long as the reduction reaction can be carried out under easy conditions.

[0076] For example, palladium-platinum colloidal particles can be grown by using a solution containing any one of the metal ions, nickel ions, cobalt ions, and copper ions, as a sensitizer solution.

[0077] For example, gold colloid particles can be grown by using a sensitizer solution containing chloroauric acid as a gold ion source to accumulate gold colloids on the surface of the gold colloid particles as a labeling substance. Gold colloid particles can be grown as composite metal colloid particles by using a sensitizer solution containing silver nitrate to accumulate silver colloids on the surface of the gold colloid particles. Platinum-gold colloid particles can be grown by utilizing the catalytic activity of platinum and using a sensitizer solution containing silver nitrate, copper salts such as copper sulfate, and the like.

[0078] The sensitizer solution contains various metal salts as essential components, which are the supply sources of the above-mentioned metal ions, and may further contain, as necessary, a complexing agent, a pH adjusting agent, a buffering agent, a stabilizer, etc. As such a sensitizer solution, a conventionally known electroless plating solution may also be used.

[0079] Examples of the complexing agent include ammonia, citrate, tartrate, lactate, etc. Examples of the pH adjusting agent include acetate, propionate, ammonium salt, etc. Examples of the stabilizer include various surfactants, etc.

[0080] In order to promote the reduction reaction of the metal ions, a solution containing a reducing agent may be used. Examples of reducing agents include, but are not limited to, citric acid, ascorbic acid, sodium hypophosphite, hydrazine hydrate, hydrazine sulfate, sodium borohydride, dimethylamine borane, formaldehyde, Rochelle salt, glucose, ethylene glycol, etc. The reducing agent used is appropriately selected from those suitable for the metal ions to be reduced.

[0081] The sensitization reaction may be carried out using a sensitizer solution containing both a metal ion component and a reducing agent. In this case, a metal salt solution containing a metal ion component and a reducing agent solution containing a reducing agent may be prepared separately and mixed immediately before the sensitization reaction. Preferably, the metal salt solution and the reducing agent solution are prepared separately and each is added to the reaction system. Depending on the catalytic activity of the metal colloid particles, the sensitization reaction may be carried out without adding a reducing agent solution.

[0082] This sensitization reaction is preferably carried out after the complex is formed and before the portion containing the labeling substance is separated.

[0083] <How to cut out the part containing the labeling substance> In the detection method of the present invention, after a complex of the second capture substance-target substance-first capture substance (the first capture substance bound to the labeling substance) is formed on the solid phase in the reaction field, a portion containing the labeling substance is excised (separated) from the complex and released from the solid phase into the liquid. This excision and separation are performed in a state where the complex is impregnated with the liquid. In addition to the tubular path being filled with liquid, if the reaction field is also filled with liquid and the solid phase is immersed in the liquid, the tubular path and the reaction field are communicated with each other through the liquid. The portion containing the labeling substance in the liquid of the reaction field separated from the solid phase by the excision is intermittently supplied to the liquid filled in the tubular path by centrifugal force. As described above, the separated portion only needs to contain the labeling substance, and does not need to be in the state of the first capture substance containing the labeling substance. In other words, any bond in the complex may be cut as long as the labeling substance or the portion containing the labeling substance is separated.

[0084] It is preferable that the portion containing the labeling substance separated from the complex does not contain the solid phase (the portion of the solid phase) from the viewpoint of transportability, etc. Therefore, it is preferable to separate the complex between the solid phase and the second capture substance, in the middle of the second capture substance, between the second capture substance and the target substance, in the middle of the target substance, between the target substance and the first capture substance, in the middle of the first capture substance, or between the first capture substance and the labeling substance, to liberate the portion containing the labeling substance.

[0085] As described above, the method of binding the labeling substance to the first capture substance is not limited, and for example, binding methods such as binding by physical adsorption, chemical binding, binding using affinity, and combinations of these can be used. The labeling substance and the first capture substance can be bound by a known method.

[0086] Examples of means for cleaving (separating) the portion containing the labeling substance include a method of cleaving by applying physical energy such as heat, light, or vibration from an external field, and a method of adding a cleaving reagent consisting of an oxidizing agent, a reducing agent, or the like. The cleaving reagent may cleave the bond between the solid phase and the second capture substance, or may cleave other bonds within the complex. There are no particular limitations on the cleaving reagent as long as it can separate the portion containing the label from the complex formed on the solid phase and does not significantly impair the properties of the labeling substance, but it is preferable for it to cleave the bond between the target substance and the second capture substance or the first capture substance, or between the first capture substance and the labeling substance.

[0087] Examples of cleavage reagents that cleave bonds include pH adjusters, denaturants, reducing agents, enzymes, competing agents, and oxidizing agents, and preferred examples include pH adjusters, denaturants, reducing agents, and enzymes, and more preferred examples include pH adjusters and denaturants.

[0088] The pH adjuster is a compound or composition that contains an acid, a base, or a salt thereof and changes the pH. The acid may be either an inorganic acid or an organic acid, and the base may be either an inorganic base or an organic base. Examples of such acids and bases include Bronsted acids, bases, and salts thereof, and examples thereof include hydrochloric acid, boric acid, carbonic acid, nitric acid, acetic acid, boronic acid, formic acid, citric acid, phosphoric acid, oxalic acid, lactic acid, malic acid, salicylic acid, glycine hydrochloric acid, glycine sodium hydroxide, sodium hydroxide, amines such as triethylamine, dimethylamine, and methylamine, ammonia, and sodium bicarbonate, but are not limited thereto. Among these, strong acids and strong bases are preferred, and specific examples thereof include hydrochloric acid, nitric acid, formic acid, phosphoric acid, oxalic acid, glycine hydrochloric acid, glycine sodium hydroxide, sodium hydroxide, amines such as triethylamine, dimethylamine, and methylamine.

[0089] Denaturants are reagents that change the higher-order structure of proteins and nucleic acids, and examples of such denaturants include protein denaturants such as urea and guanidine salts, and surfactants such as sodium dodecyl sulfate. Other denaturants include, but are not limited to, Lewis acids not included in the above pH adjusters, alcohols or esters, and derivatives thereof.

[0090] Examples of reducing agents that cleave disulfide bonds include 2-mercaptoethanol and dithiothreitol.

[0091] The enzyme hydrolyzes a part of the components of the complex, and examples thereof include pepsin, papain, ficin, nuclease, and enzymes that act specifically on specific bonds of the components.

[0092] Examples of the competitor include an agonist, antagonist, or competitive antibody that competes with the binding between the target substance and the first or second capture substance.

[0093] Furthermore, peroxides including hydrogen peroxide and other oxidizing agents may be used as cleavage reagents as long as they do not affect the target substance.

[0094] The type and concentration of the cleavage reagent are appropriately selected depending on the structure and binding strength of the target complex, and are adjusted within a range that does not significantly impair the function or structure of the labeling substance.

[0095] Here, a linker may be introduced in advance into a predetermined portion of the complex so that an arbitrary portion of the complex is specifically cleaved. This linker can be used, for example, for the purpose of linking a solid phase to a second capture substance, or a labeling substance to a first capture substance when these two substances are bound together. If the linker has a predetermined functional group or reactive group capable of forming a chemical bond at both ends, the solid phase and the second capture substance, or the labeling substance and the first capture substance can be linked through the functional groups at both ends of the linker. This linker can be used for the purpose of linking a solid phase to a second capture substance, or a labeling substance to a first capture substance, but is not limited thereto. For example, a linker may be introduced into the inside of the first capture substance or the second capture substance. Examples of functional groups or reactive groups at both ends of the linker include those that form covalent bonds such as amide bonds, disulfide bonds, and ester bonds. In this case, the bond at the terminal portion of the linker can be decomposed by hydrolysis or treatment with a reducing agent, and the complex can be cleaved.

[0096] Furthermore, the binding mechanism between the linker and the target substance is not limited to the above-mentioned covalent bond, but may be, for example, physical adsorption, ionic bonding, bonding utilizing intermolecular affinity, or a combination of these bonds.

[0097] Furthermore, the molecular structure constituting the linker is not limited, and synthetic polymers such as proteins, peptides, sugar chains, nucleic acids, and polyethylene glycols can be used. Furthermore, biotin-avidin, which forms an affinity bond, may also be used.

[0098] The linker portion can be cleaved by physical energy from an external field or by a reagent, such as an enzyme (e.g., endopeptidase, glycolytic enzyme, endonuclease, etc.), a reducing agent, an acid, a base, a surfactant, or a competing agent, depending on the type of linker.

[0099] Specifically, for example, disulfide bonds can be cleaved by the action of a reducing agent having a thiol group in the molecule, such as 2-mercaptoethanol or dithiothreitol. In the case of biotin-avidin bonds, the bonds can be cleaved by changing the pH of the reaction system by adding a pH adjuster or by using an elution buffer. Furthermore, when the linker molecule is composed of a sugar chain, a specific bond can be cleaved by the action of an enzyme specific to the bond between the constituent monosaccharides.

[0100] Furthermore, when a linker having a histidine tag is used, for example, a linker molecule having a histidine tag at its end is bound to the second capture substance, and nickel is immobilized on the surface of the solid phase with a chelating agent, thereby immobilizing the second capture substance on the solid phase via the histidine tag. If histidine or imidazole, which is a competitor for the histidine tag, is added as a cleavage reagent, the histidine tag can be dissociated from nickel, and the complex containing the labeled substance can be separated from the solid phase.

[0101] The cleavage reagent for separating the portion containing the label from the complex may be removed or inactivated after the cleavage reaction. For example, when the reaction field is shifted from neutral to acidic or alkaline by the addition of a pH adjuster, a further pH adjuster is added to return the reaction field to neutral.

[0102] The reagent of the present invention is configured to include the above-mentioned cleavage reagent. The reagent of the present invention may be configured in the form of a reagent kit. In addition to the reagent of the present invention (cleavage reagent), the reagent kit may include, for example, one or more of a washing solution, a dispersion in which a first capture substance bound to a labeling substance is dispersed, and a dispersion of a solid phase on which a second capture substance is supported.

[0103] <Solid phase> The solid phase is an insoluble solid to which the second capture substance binds, and is processed to a form, shape, and size that can be placed in the reaction field.

[0104] The form or shape of the solid phase is not particularly limited, and may be any of a structure such as a protrusion or structure formed on the inner wall of a reaction vessel serving as a reaction field or on the inside of the reaction vessel and integrated with the reaction vessel, or a structure formed separately from the reaction vessel.

[0105] Examples of the solid phase formed separately from the reaction vessel include a plate-like body, a granular body, a fibrous body, a woven fabric, a nonwoven fabric, a film, a sheet, and the like that can be accommodated in the reaction vessel. The plate-like body, the granular body, and the fibrous body may be any of hollow, solid, and porous. The shape of the granular body may be any of spherical, annular, flat, cylindrical, and amorphous. The granular body may be at least one selected from, for example, magnetic particles, glass particles, ceramic particles, and resin particles made of polymers such as latex and polystyrene. As the solid phase, glass particles, ceramic particles, magnetic particles, or resin particles are preferable. As such a solid phase, a particulate material, a fibrous material, a woven fabric, or a nonwoven fabric is preferably used, more preferably a particulate material or a fibrous material is used, and even more preferably a particulate material is used.

[0106] The binding between the solid phase and the second capture substance can be achieved by a binding mode such as physical adsorption, covalent bond, ionic bond, affinity binding, or a combination of these. Preferably, the second capture substance is immobilized on the solid phase by a covalent bond or ionic bond that is stronger than the binding force due to affinity or adsorption. In addition, the second capture substance is immobilized on the solid phase in an amount necessary to capture the maximum detectable amount of the target substance within a set detection range (a concentration range of the target substance set as detectable).

[0107] Considering the capture of the target substance, such granular material is preferably one with a larger specific surface area, but when it is introduced into a reaction chamber communicating with a tubular passage, a size is selected that does not flow into the tubular passage from the inlet of the tubular passage. For example, if the smallest dimension of the length, width, and thickness of the granular material is about 1.2 to 5 times the inlet diameter, the inlet diameter is sufficiently prevented from flowing into the tubular passage. In addition, when the inlet shape of the tubular passage is anisotropic, the inlet diameter is the dimension in the minor axis direction. As the granular material, various beads with diameters of about 1 μm to 100 μm are commercially available. Therefore, an appropriate one can be appropriately selected depending on the size of the inlet. The granular material used is preferably resin beads, more preferably resin beads with a particle size of 10 μm to 100 μm, and more preferably resin beads with a particle size of 10 μm to 50 μm, particularly 15 μm to 30 μm.

[0108] <Reaction field> In this embodiment, the method for detecting a target substance includes introducing a sample into a reaction field having a solid phase on which a second capture substance is immobilized in advance, reacting the second capture substance on the solid phase with the target substance in the sample, and then introducing a first capture substance to form a complex sandwiching the target substance. Thereafter, the excess first capture substance is discharged outside the reaction system. Then, as described above, the bond of the complex is cut to separate the portion containing the labeling substance. The process up to separating the portion containing the labeling substance is performed in the reaction field. The surface density of the immobilized second capture substance on the solid phase can be adjusted in advance. Therefore, by reacting the target substance with the second capture substance in this way and then introducing the first capture substance, the quantitativeness of detection can be improved in a wide range of concentrations of the target substance contained in the sample, from low to high.

[0109] In addition, in the reaction field, the reaction to form a complex, i.e., the process of mixing the first capture substance, the solid phase carrying the second capture substance, and the sample containing the target substance, may be introduced into the reaction field in any order, and some or all of them may be introduced simultaneously, as long as a complex is formed. That is, the step of forming a complex is not limited to the step of reacting a target substance with a second capture substance immobilized on a solid phase as described above, and then reacting the first capture substance bound to a labeling substance to form a complex. For example, the target substance may be reacted with a first capture substance bound to a labeling substance, and then reacted with a second capture substance immobilized on a solid phase to form a complex, or the first capture substance bound to a labeling substance, the target substance, and the second capture substance immobilized on a solid phase may be simultaneously reacted to form a complex.

[0110] In addition, the reaction field is not particularly limited as long as it is a field that can form a complex of solid phase-second capture substance-target substance-first capture substance (first capture substance bound to a labeling substance) and separate the part containing the labeling substance from this complex. In order to carry out this reaction step, only one reaction field may be used, or multiple reaction fields may be used. In other words, the formation of the complex and the separation of the part containing the labeling substance may be carried out in the same reaction field (space), or in different reaction fields (spaces).

[0111] The reaction field may be formed, for example, using a partitioned container or chamber. For example, a chamber or channel formed in a test tube, a microtube, a microplate, a microreactor, a biodisk, or the like may be used as the reaction field. Furthermore, the operation in the reaction field may be performed manually or automatically. When the operation in the reaction field is automated, for example, a device in which the reaction field is implemented in the form of a chamber may be used, and further, the device may be in the form of a disk that can rotate around a central axis.

[0112] Preferably, the device has at least one reaction field in the form of a reaction chamber. In the case of a device in the form of a disk, the reaction chamber is disposed closer to the central axis of the disk than the tubular passage, and communicates with the tubular passage on the outer side. The reaction chamber is usually formed with a size larger in both the depth direction and the width direction than the inner diameter of the tube of the tubular passage. Preferably, the reaction chamber may be formed in a shape (cone shape) narrowed toward the tubular passage. When the disk rotates, the portion containing the labeling substance in the liquid of the reaction chamber separated from the solid phase by the cleavage reagent is intermittently supplied from the large-capacity reaction chamber to the liquid filled in the narrow tubular passage.

[0113] The communication port (the entrance of the tubular path) that connects the reaction chamber and the tubular path must be formed to a size that allows the portion containing the labeled substance to pass through, and prevents the solid phase from flowing out when a solid phase formed separately from the reaction chamber is introduced into the reaction chamber (for example, when the solid phase is a solid such as a bead and has fluidity within the reaction chamber). Considering general processing accuracy, the communication port is, for example, 5 μm or more, preferably 10 μm or more, and more preferably 15 μm or more, but is designed to a size that is easy to process, assuming a shape and size that prevents the solid phase from flowing into the tubular path.

[0114] It is desirable that the portion containing the labeled substance to be detected is introduced into the detection field in a state where impurities are reduced. In the method of the present invention, the labeled substance that has been moved to the tubular path by centrifugal force is detected, so if excess labeled substance is present in the sample introduced into the tubular path, it will have a significant effect on the detection accuracy. For this reason, it is important to remove excess first capture substances (first capture substances that bind to the labeled substance and include those that have not captured the target substance and free labeled substances) from the sample provided to the tubular path.

[0115] A washing process may be selected as a method for removing the excess first capture substance from the sample. Specifically, after forming a complex sandwiching the target substance, a washing process can be performed to remove the excess first capture substance that has not been involved in the reaction. Meanwhile, the first capture substance that has captured the target substance is fixed to the solid phase via the target substance and the second capture substance, and is therefore retained in the reaction field. If a cleavage reagent is added in this state, the portion containing the separated labeling substance can be introduced into the detection field under conditions in which the excess first capture substance has been removed to a level that does not affect the detection result.

[0116] In addition, removal by centrifugation may be selected as a method for removing the excess first capture substance from the sample. Removal of the excess first capture substance by centrifugation can be easily performed by using a disk. Specifically, for example, when a complex is formed (or introduced) in the reaction chamber, the first capture substance that has captured the target substance is immobilized on the solid phase and placed in the reaction chamber. On the other hand, since the excess first capture substance is not immobilized, if centrifugal force is applied in a state in which the above-mentioned tubular path is filled with liquid, the solid phase of the reaction chamber is impregnated with the liquid, and the reaction chamber and the tubular path are connected by the liquid, the excess first capture substance can be moved to the tubular path by centrifugal force and discharged outside the reaction chamber. If a cleavage reagent is then added to the reaction chamber to separate the portion containing the labeling substance from the complex, the portion containing the labeling substance can be introduced into the detection field without the coexistence of the excess first capture substance. In this way, when the excess first capture substance is removed using centrifugal force, a washing process using a washing solution such as a detergent is not necessary. From the viewpoint of improving the cleaning power, the cleaning treatment may be carried out by combining removal of the excess first capture substance by centrifugal force and cleaning with a cleaning liquid.

[0117] In addition, in order to fix the excess first capture substance discharged from the reaction chamber with a binder that specifically binds to the first capture substance, a binder may be held in the migration path of the excess first capture substance. This binder is supported in a location where the portion containing the labeling substance does not get in the way of passing through the tubular path. In this way, the excess first capture substance discharged from the reaction chamber is fixed to a predetermined location in the device.

[0118] The binder may be, for example, an antigen or an antibody that specifically binds to the first capture substance, and may be the same as the target substance of the first capture substance.

[0119] In addition to the above-mentioned antigens and antibodies, a nucleic acid, an aptamer, a ligand (substrate, inhibitor, or antagonist) or a receptor to which the first capture substance binds may be used as a binding agent.

[0120] The antibody used as the binding agent may be a polyclonal antibody or a monoclonal antibody, but from the viewpoint of reaction specificity, it is preferable to use a monoclonal antibody.

[0121] The antibody used as the binding agent also includes an antibody, as well as an antibody fragment and a modified antibody having substantially the same reactivity as the antibody. Antibody fragments include Fab fragments, F(ab')2 fragments, Fab' fragments, scFv fragments, etc.

[0122] <Detection field> The detection field (second reaction field) is a field where the part containing the labeled substance separated in the reaction field (first reaction field) is introduced and scattered light caused by the part containing the labeled substance is detected. In this detection field, a tubular path along which the part containing the labeled substance moves is arranged, and a light source that irradiates irradiation light toward the tubular path and a light receiving element that detects scattered light emitted from the labeled substance in the tubular path are provided.

[0123] After the complex is formed in the above-mentioned reaction field (first reaction field), the above-mentioned washing process and centrifugation process are performed to discharge elements other than the complex from the system. After that, by cutting the part containing the labeling substance, the sample is introduced into the detection field in a state in which the excess first capture substance and other impurities that affect detection have been reduced.

[0124] The tubular passage is provided in a rotatably formed device, and is formed of a light-transmitting material so that the irradiated light emitted from the light source can reach the inside of the tubular passage, and the scattered light generated due to the detection target can be emitted therefrom.

[0125] This tubular path is preferably formed in a hollow cylindrical shape with both ends open, and is extended from the rotation center side toward the outside along the rotation radius. The open end (hereinafter sometimes referred to as "one open end") on the rotation center side (centripetal direction side) of the tubular path communicates with a reaction field (first reaction field) that holds a solution containing a portion containing a labeling substance. The cross-sectional shape of the tubular path is not limited as long as it is a hollow tube. It may be a circular tube or a square tube, or may have a flat shape. It may also be composed of one seamless part, or may be composed of a combination of multiple parts.

[0126] The inner diameter of the tubular path is designed to be in a range of about 3 to 150 times the size of the detection target so as not to cause clogging, and to have a shape that does not allow the solid phase in the reaction field (reaction chamber) to flow in. As described above, when the solid phase is introduced into the reaction chamber, that is, when the solid phase is formed separately from the reaction chamber, the inner diameter of the tubular path is smaller than the size of the solid phase, for example, about 1 / 1.2 to 1 / 5 times the size of the solid phase. In detail, for example, the smallest value among the maximum values ​​of the length, width, and height of the solid phase is set as a reference value, and the inlet diameter of the tubular path may be made smaller than the reference value. When the solid phase is isotropic, the length, width, and height are equal, so any of them may be used as the reference value. The inner diameter of the tubular path may be formed uniformly with the same width as the inlet diameter, or may be formed with a width different from the inlet diameter, and may be formed to be wider or narrower in a tapered or stepped shape, for example.

[0127] Specifically, for example, the inner diameter of the tubular passage is 1 μm to 100 μm, preferably 5 μm to 50 μm, and more preferably 10 μm to 20 μm. The smaller the inner diameter, the smaller the amount (absolute amount) of impurities present in the tubular passage, and therefore the more the scattered light noise caused by impurities can be reduced. In addition, when the cross section of the tubular passage is an anisotropic shape such as an ellipse or a rectangle, the short side direction of the cross section is the flow path height of the tubular passage, and the long side direction is the flow path width. The tubular passage is designed so that the height direction is the above-mentioned inner diameter.

[0128] If the height of the tubular channel is set so that the solid phase does not flow in, the channel width can be designed regardless of the size of the solid phase. The minimum value of the channel width of the tubular channel is the size of the part containing the labeling substance, and the upper limit value is the width of the contact surface of the connected reaction chamber. The wider the tubular channel, the smoother the flow of the liquid in the tubular channel, but as a result of the enlargement of the contact area with the reaction chamber, the inclination of the mortar shape from the reaction chamber to the tubular channel becomes shallower, which may affect the accumulation range of the solid phase. From this point of view, it is preferable that the channel width of the tubular channel is narrow. Therefore, the channel width of the tubular channel is set to an appropriate width in consideration of the flow of the liquid and the accumulation state of the solid phase in the reaction chamber, and is, for example, 100 μm to 1 cm, preferably 100 μm to 5 mm, more preferably 500 μm to 3 mm, and even more preferably 500 μm to 1 mm.

[0129] The open end on the outer side of the tubular path (hereinafter, sometimes referred to as "the other open end") communicates with a waste liquid storage chamber as a second storage chamber. This waste liquid storage chamber is a partitioned chamber that stores the liquid that has passed through the tubular path. When a cleaning process is performed in a reaction field (first reaction field) formed in the device, the waste liquid from the cleaning process is discharged via the tubular path and stored in this waste liquid storage chamber. The waste liquid storage chamber may be disposed downstream of the tubular path, and may be disposed, for example, partially on the rotation center side along the rotation radius of the tubular path, or entirely on the outer side.

[0130] Here, in the method of the present invention, in order to detect the labeling substance, it is necessary to fill the tubular path with liquid. In other words, even when centrifugal force is applied, it is necessary to prevent the liquid in the tubular path from flowing out from the open end. For this reason, a limiting mechanism is provided to limit the flow of liquid in the tubular path. This limiting mechanism can be realized, for example, by extending a narrow flow path from the end of the drainage storage chamber toward the rotation center. According to this, the liquid supplied to the tubular path under the action of centrifugal force is stored in the drainage storage chamber, while the overflowing liquid advances through the narrow flow path toward the rotation center. Here, since the liquid level is formed at the same distance from the rotation center, if the liquid is supplied until it reaches the open end (the one open end) of the tubular path on the rotation center side, the liquid level is formed at the same position as the open end (the one open end) of the tubular path on the rotation center side in the flow path toward the rotation center. At that time, if centrifugal force is applied, the liquid can be prevented from returning to the upstream side even if the supply of the liquid is interrupted. This causes the tubular path to be filled with liquid.

[0131] A drainage reservoir may also be used as the limiting mechanism. For example, when at least a portion of the drainage reservoir is disposed on the rotation center side of the tubular path, the end of the drainage reservoir that is not connected to the tubular path may be disposed so as to reach the open end of the tubular path on the rotation center side (the one open end) or to be closer to the rotation center than the open end of the tubular path on the rotation center side. When the liquid is supplied until it reaches the open end of the tubular path on the rotation center side (the one open end), the liquid level of the drainage reservoir and the liquid level of the tubular path are formed at the same distance from the rotation center. This causes the tubular path to be filled with liquid. In this case, the drainage reservoir acts to prevent the liquid in the tubular path from flowing out of the open end, so there is no need to provide a separate limiting mechanism to limit the flow of the liquid in the tubular path.

[0132] When no cleaning process is performed within the device, the waste liquid storage chamber may be omitted, and a thin flow path may be provided extending from the end of the tubular path toward the center of rotation.

[0133] Another example of the restriction mechanism is to provide an on-off valve at the other open end of the tubular passage or in the space connected to the open ends. By doing so, the other end of the tubular passage is blocked by closing the on-off valve, so that the tubular passage can be filled with liquid. On-off valves include not only those that operate electrically, electromagnetically, or mechanically, but also capillary burst valves (a mechanism that functions as a valve because liquid does not flow out of the open end until a centrifugal force exceeding a predetermined value is applied), which will be described later, and an appropriate one can be selected and used from these.

[0134] During detection, centrifugal force is applied to the liquid supplied to the tubular path while the tubular path is filled with the liquid, causing the portion containing the labeled substance to move through the tubular path from the reaction field on the rotation center side (first reaction field) to the outer side of the device.

[0135] When a portion containing a labeling substance to be detected is introduced into the tubular passage, the irradiated light is scattered to generate scattered light, which is detected by a light-receiving element such as a photodiode, thereby detecting the target substance of interest.

[0136] The scattered light intensity is correlated with the particle size when the particle size of the labeling substance is within a predetermined range, and the scattered light intensity in a specific direction changes depending on the particle size. Since the particle size of the particles used in the labeling substance is known in advance, the light receiving element can be placed at a position where the scattered light can be detected, and the scattered light can be detected appropriately. If scattered light occurs in all directions, the light receiving element can be placed at any position. The scattered light to be detected may be forward scattered light or backward scattered light. The light receiving element is placed appropriately depending on the direction in which the scattered light to be detected is scattered. In addition, reflected light that becomes noise is generated at the interface where the irradiated light is incident, but by adjusting the positions and angles of the light source and the light receiving element, the noise in the reflected light can be reduced and the scattered light from the labeling substance can be properly detected.

[0137] Here, for example, a small and inexpensive detection device can be realized by using an optical system using a general-purpose laser light source such as that used in an optical pickup, or a rotation mechanism. However, when a part containing a labeling substance to be detected passes through a detection field at high speed, it becomes difficult for a detection system using inexpensive general-purpose materials to accurately detect scattered light due to a minute labeling substance.

[0138] In this embodiment, when the laser spot of the laser light source has a diameter of D μm, in a device rotating at N rpm (1 rotation: 60 / N seconds), the part containing the labeling substance moves at a moving speed of v μm / s so that the labeling substance to be detected enters the laser spot at least once. In this case, the following formula is established.

number

number

[0139] According to this, for example, when the laser spot diameter is 10 μm and the disk is rotated at 2000 rpm, the moving speed of the labeling substance needs to be 333 μm / sec or less.

[0140] Unlike the method of the present invention, it is also possible to pass the portion containing the labeling substance through a tubular passage by passing a liquid, and detect scattered light from the labeling substance moving through the tubular passage. For this reason, the moving speed of the liquid flowing through the device by centrifugal force was calculated using the following formula.

number

[0141] When the medium is water at 20°C and rotated at 2000 rpm, the moving speed of the medium flowing due to centrifugal force at a given position in the radial direction on the disk is calculated using formula (8), and the moving speed of 200 nm gold colloid particles moving in the medium due to the generated centrifugal force is calculated based on Stokes' law to be 60 mm / sec for the former and 84 μm / sec for the latter. It was also confirmed that the liquid in the disk actually moves at high speed due to rotation. Therefore, when the movement of the gold colloid particles (labeling substance) is governed by the flow of the liquid, it becomes difficult to detect scattered light.

[0142] Therefore, in the present invention, the tubular passage is filled with liquid to function like a centrifuge tube, and the part containing the labeled substance is caused to move through the liquid by centrifugal force, allowing the scattered light to move through the tubular passage at a speed that allows it to be detected.

[0143] In the present invention, where the movement of the portion containing the marker substance in the tubular passage is not governed by the liquid flow, the speed at which the portion containing the marker substance moves through the tubular passage varies depending on the density and particle size of the portion containing the marker substance (the marker substance) and the centrifugal force, provided that the liquid filled in the tubular passage is the same. Therefore, by appropriately selecting the rotation speed of the device and the marker substance, the movement speed can be set within a desired range.

[0144] <Device> The device is also called a biodisc or a microreactor, and is equipped with at least a reaction chamber (corresponding to the above-mentioned reaction field) for holding a complex and performing a reaction as necessary, and a tubular path (corresponding to the above-mentioned detection field) for moving a detection target, and is used for analyzing biological samples by forming a micro-diameter flow path for liquid circulation and a storage chamber for storing a reagent or liquid as necessary. In addition, the device is formed to be rotatable in order to apply centrifugal force.

[0145] The device can be produced by a known method using known materials such as various plastics such as acrylic resin, polycarbonate resin, and silicone resin, and inorganic materials such as glass and silicone. The material is preferably translucent so that the detection target can be optically detected, and is more preferably transparent.

[0146] This device can be fabricated using known techniques. For example, it can be manufactured by laminating a sheet or plate having a pattern of flow channels or chambers with a flat sheet or plate. The internal structure of the reaction chambers and the like may be formed by machining, etching using photolithography, or other general molding techniques. Furthermore, a reflective layer may be disposed on the back surface of the tubular path in order to increase the amount of light of the signal incident on the light receiving element.

[0147] The storage chamber provided in the device for storing a reagent or liquid is disposed on the rotation center side with respect to the reaction chamber in order to supply the liquid to the reaction chamber by centrifugal force. In the method for detecting a target substance of the present invention, the detection target is moved through the liquid in the tubular path by centrifugal force while the tubular path is filled with the liquid. For this reason, when the liquid to be filled in the tubular path is stored in the storage chamber in advance, the liquid stored in the storage chamber is filled into the tubular path at a predetermined timing. In addition, in this embodiment, when a washing process is performed on the complex held in the reaction chamber, a washing liquid storage chamber as a first storage chamber and a waste liquid storage chamber as a second storage chamber are provided, and the washing liquid is stored in the washing liquid storage chamber. The waste liquid storage chamber may be disposed downstream of the tubular path, and at least a part of the waste liquid storage chamber may be disposed on the rotation center side with respect to the tubular path, or the whole of the waste liquid storage chamber may be disposed on the outer side with respect to the tubular path. The cleaning liquid may also serve as the liquid to be filled in the tubular path, and in such a case, the storage chamber for storing the liquid to be filled in the tubular path and the cleaning liquid storage chamber may be integrated into one chamber. In the case where the tubular path is filled with the washing liquid stored in the washing liquid storage chamber, for example, when at least a part of the drainage liquid storage chamber is disposed on the rotation center side of the tubular path, the end of the drainage liquid storage chamber that is not connected to the tubular path may be disposed at a distance equal to or shorter than the rotation center from the open end (the one open end) of the tubular path on the rotation center side. Then, the washing storage chamber may be stored with a volume of washing liquid that reaches at least the open end of the tubular path on the rotation center side in the drainage liquid storage tank and the tubular path. When the entire drainage liquid storage chamber is disposed on the outer side of the tubular path, the washing liquid may be stored in an amount that exceeds the total capacity of the drainage liquid storage chamber and the tubular path, but does not reach the total capacity of the drainage liquid storage chamber, the tubular path, and the reaction chamber. By storing such an amount of washing liquid, after washing, the tubular path is filled with the washing liquid, liquid is stored in the reaction chamber, and the remaining space of the reaction chamber can receive the cleavage reagent of the portion containing the labeling substance.

[0148] Furthermore, when the tubular path is filled with the washing liquid stored in the washing liquid storage chamber, the washing process may be performed until the drainage storage chamber and the tubular path are filled with the washing liquid, and may be performed until the washing liquid exceeds the capacity of the tubular path and flows into the reaction chamber, provided that the remaining space in the reaction chamber can accommodate the cleavage reagent of the portion containing the labeling substance. When the washing liquid flows into the reaction chamber in addition to the tubular path and is stored in the reaction chamber, the tubular path and the reaction chamber are liquid-communicated.

[0149] Furthermore, in this embodiment, a cleavage process is carried out to separate the portion containing the labeling substance from the complex, and when this is carried out in the device, a cleavage reagent storage chamber is provided as a third storage chamber to store the necessary cleavage reagent. Also, as described above, when a sensitization reaction of metal colloid particles is carried out, a sensitizer storage chamber is provided to store a sensitizer solution required for the sensitization reaction.

[0150] The reagents stored in the storage chambers may be stored in a liquid state or in a solid state. In the case of storing the reagents in a solid state, a dissolving liquid for dissolving the reagents flows into the storage chambers in which the reagents are stored, and then the reagents are delivered from the storage chambers to the reaction chambers.

[0151] When a cleavage reaction is carried out in which a portion containing a labeling substance is separated by applying energy from an external field such as heat or electromagnetic waves, the cleavage reagent storage chamber may not be provided.

[0152] <Control system> Each device arranged in the detection field for detecting scattered light is electrically controlled by an information processing device. Signal processing is performed based on the received scattered light, and the labeling substance (the portion containing the labeling substance) is detected using the scattered light as a signal. In the detection method of the present invention, the labeled substance to be detected is moved in the tubular path by the centrifugal force generated by rotating the device. The moving speed can be calculated if the labeled substance, the liquid filled in the tubular path, and the centrifugal force applied are known. In addition, the allowable moving speed of the labeled substance can also be calculated from the rotation speed and the spot diameter of the laser light source (the above formula (7)). Based on these, the settable range of the rotation speed can be derived. The light receiving element detects scattered light from a position of a predetermined rotation radius. In other words, the position of the predetermined rotation radius is irradiated with light from a laser light source, and the spot diameter becomes the detection area. The light receiving element is disposed at a position where it detects scattered light from the detection area. Examples of the light receiving element include, but are not limited to, a photodiode, an avalanche photodiode, a photomultiplier tube, a CMOS, and a CCD.

[0153] When the labeling substance enters the spot diameter of the laser light source, the scattered light is incident on, for example, a photodiode as a light receiving element. Then, the incident light is photoelectrically converted and output from the photodiode, and a voltage value corresponding to the incident scattered light intensity is input to an A / D converter. Then, the A / D converter samples the light at a predetermined sampling time to convert it into a digital signal, which is input from the A / D converter to an information processing device. The A / D converter is not particularly limited as long as it can convert an analog signal into a digital signal, but may be, for example, an integrated circuit or an oscilloscope. The sampling time is designed based on the resolution for the flow path width. For example, it is set so that data can be acquired at intervals of 1 / 10 or more and less than 1 / 1 of the spot diameter of the laser light source. For example, when the spot diameter of the laser light source is 10 μm at a position of a rotation radius of 50 mm for a tubular path rotating at 2000 rpm, in order to acquire data at intervals of approximately 1 μm, the sampling time is set to approximately 100 ns.

[0154] In the information processing device, blank data is stored in association with a predetermined position in the width direction of the flow path of the tubular path, for example, immediately after the tubular path is filled with liquid and before the marker substance is supplied to the tubular path. Thereafter, at each predetermined sampling time, the input voltage value is stored in association with the same position, and the difference from the blank data is calculated. If the difference between the two is equal to or less than a predetermined threshold, it is determined that the marker substance is not detected at that position or that the scattered light is noise, and the scattered light generation is not counted (the count is canceled). On the other hand, if the calculated difference exceeds a predetermined threshold, it is determined that the scattered light is based on the marker substance. Note that since the marker substance is smaller than the spot diameter of the laser light source, the result is that the scattered light is detected multiple times before it passes through the spot diameter (detection area). For this reason, the number of occurrences of scattered light 1 may be counted when the voltage value drops after multiple measurements (the difference between the blank and the input voltage value becomes equal to or less than a predetermined threshold).

[0155] Furthermore, according to the detection method of the present invention, the labeled substance (part containing the labeled substance) and impurities can be sufficiently separated and detected, but for some reason, it is possible that foreign matter other than the labeled substance may be mixed into the tubular path at the timing of detecting the labeled substance (a predetermined time range based on the moving speed of the labeled substance from the start of detection) or scattered light may be generated due to the condition of the tubular path such as a scratch. In such a case, the signal pattern of the scattered light caused by the foreign matter or the tubular path will be a different signal pattern from that of the labeled substance, so that the two can be distinguished and the target detection target can be accurately detected. When the target detection target can be distinguished and detected, it is not necessarily necessary to obtain blank data before detecting the labeled substance as described above.

[0156] Furthermore, in this detection method, the labeling substance, the liquid to be filled in the tubular passage, and even the laser light source (spot diameter) can be determined in advance, and the rotation speed (number of rotations) of the device optimal for detection can also be derived in advance. Therefore, these values ​​can be stored in the information processing device as defaults. Note that the configuration may be such that the user inputs the necessary values ​​as appropriate upon detection. Furthermore, if it is known in advance that a unique scattered light will be generated due to a scratch on the tubular passage, the signal pattern may be stored as blank data by default.

[0157] <Target substance detection method 1 (antibody test)> An exemplary detection method 1 of a target substance (antibody) according to this embodiment will be described. This detection method is an embodiment of the outline of the detection method described with reference to FIG. 1 in an antibody test. In this method, first, an antigen (second capture substance) for the target substance is immobilized on the surface of polystyrene beads (solid phase) according to a conventional method. Next, blocking is performed, and polystyrene beads (second capture substance immobilized on the solid phase) are introduced into a container of a predetermined volume. The polystyrene beads are introduced in a state of being dispersed in a buffer, and then filtration, centrifugation, or the like is appropriately performed to remove the solvent. Thereafter, a sample such as serum containing the target substance (antibody) is added, and the beads are contacted with each other for a certain period of time to react, and then washed with a buffer if necessary, and then a secondary antibody solution (first capture substance bound to a labeling substance) is added and reacted for a certain period of time. Here, the labeling substance is metal colloid particles. As a result, an immune complex of an antigen-antibody-secondary antibody sandwich type is formed on the beads. After washing with a buffer, a cleavage reagent (pH adjuster) is introduced to adjust the pH to 0 to 4 or 10 to 14, and the portion containing the metal colloid particles is released from the immune complex formed on the solid phase.

[0158] In addition, in such antibody tests, the user can freely select at what stage after immobilizing the antigen on the polystyrene beads (solid phase) to use the reaction chamber of the device as the reaction field, as described above. When using the reaction chamber of the device, the washing process may be performed only once after the antigen-antibody-secondary antibody immune complex is formed on the polystyrene beads.

[0159] After the cutting process, the part containing the separated metal colloid particles is detected. In this method, the part containing the metal colloid particles separated by the cutting process is introduced into a tubular path of the device and detected. A communication port (inlet) formed with a height smaller than the diameter of polystyrene beads is provided in a reaction chamber connected to the tubular path, and the part containing the metal colloid particles is introduced or stored in this reaction chamber, which is the front chamber of the tubular path. Then, the metal colloid particles are introduced into the tubular path through the communication port (inlet) and move outward through the tubular path by centrifugal force. Here, the tubular path is filled with liquid, and the movement of the liquid does not substantially occur, so the part containing the metal colloid particles moves within this liquid phase by centrifugal force. Irradiation light is irradiated onto this tubular path, and scattered light is generated when the metal colloid particles pass through the tubular path. Scattered light is generated intermittently every time a metal colloid particle passes through. The signal corresponding to each metal colloid particle is not continuous and is obtained as a unique signal, so by measuring this, the metal colloid particle can be detected, and as a result, the target substance, the antibody, can be detected.

[0160] As described above, the method for detecting a target substance can be carried out by the above method.

[0161] <Target substance detection method 2 (antigen test)> An exemplary detection method 2 of a target substance (antigen) according to this embodiment will be described. This detection method is a further concrete example of the immunoassay method called the sandwich method, the outline of which is explained in FIG. 1. In this method, first, an antibody (second capture substance) against the target substance is diluted to an appropriate concentration using phosphate buffered saline (hereinafter referred to as PBS) or a buffer solution, and the antibody is immobilized on the surface of polystyrene beads (solid phase) according to a conventional method. Next, after blocking, a container of a predetermined volume is filled with a predetermined volume of polystyrene beads (second capture substance immobilized on the solid phase). A sample such as serum containing the target substance (antigen) is added, and the beads are contacted and reacted for a certain period of time, and then washed with a buffer, and a primary antibody (first capture substance bound to the labeling substance) labeled with metal colloid particles is added to bind to the antigen. This forms an antibody-antigen-antibody sandwich-type immune complex on the styrene beads.

[0162] In this method, as in the above detection method 1, the user can freely select at what stage to use the reaction chamber of the device. When using the reaction chamber of the device, the washing process may be performed only once after the antibody-antigen-antibody immune complex is formed on the polystyrene beads. The detection of the part containing the metal colloid particles is the same as in the above detection method 1, and this allows an immunoassay to be performed to detect the target substance.

[0163] An example of the device of this embodiment will be described in more detail below with reference to FIG. 2. FIG. 2 is a diagram showing an outline of a disk 1 for detecting a target substance, with FIG. 2(a) showing an overall view of the disk and FIG. 2(b) showing an enlarged view of a portion thereof. This disk 1 is a disk-shaped plastic disk such as a compact disk (CD), DVD, or Blu-ray disk (BD) in which a micro-diameter flow passage or chamber through which a liquid can flow is formed. In order to apply centrifugal force, a disk shape suitable for rotation is adopted, and the center of the disk is the center of rotation. The disk 1 is provided with six units 2, and each unit 2 is configured to be able to detect a target substance independently.

[0164] Each unit 2 includes a reaction chamber 10 in which the complex is held, a tubular path 11 connected to the reaction chamber 10, a waste liquid storage chamber 12 connected to the tubular path 11, a washing liquid storage chamber 13 for storing a washing liquid, and a cleavage reagent storage chamber 14 for storing a cleavage reagent.

[0165] The reaction chamber 10 has a supply port 10a on its upper surface that penetrates the inside and outside of the chamber, and is formed so that a solution containing a solid phase, a reagent, etc. can be introduced from the supply port 10a. The reaction chamber 10 is the reaction field in this disk 1. When centrifugal force is applied to the solid phase introduced into the reaction chamber 10, it accumulates on the inner wall (including the upper surface, bottom surface, and side surface) on the tubular path 11 side. Here, the outer side of the reaction chamber 10 is formed in a mortar shape when viewed from above so that the solid phase accumulates while suppressing the spread in the width direction of the reaction chamber 10 (the length in the direction intersecting the rotation radius). In addition, the inside of the reaction chamber 10 on the tubular path 11 side is formed so that the bottom surface is inclined obliquely so that the bottom gradually becomes shallower toward the tubular path 11 side.

[0166] In this disk 1, the necessary steps can be carried out in order according to the steps shown in Fig. 1. In this disk 1, a process for separating a portion containing a labeling substance from a complex can be carried out in a reaction chamber 10, and the formed complex (e.g., a complex consisting of a solid phase-a second capture substance-a target substance-a first capture substance bound to a labeling substance) is held in the reaction chamber 10.

[0167] The tubular path 11 is a hollow tube with both ends open, and is extended along the rotation radius of the disk 1. The open end on the rotation center side forms a communication port (inlet) 11a communicating with the reaction chamber 10, and the other open end forms a communication port (outlet) 11b communicating with the drainage storage chamber 12. This tubular path 11 is made of a light-transmitting material. The flow path width of the tubular path 11 is formed narrower than the width of the reaction chamber 10. In addition, the depth is formed smaller than the minimum diameter of the outer shape of the solid phase to be introduced so that the solid phase introduced into the reaction chamber 10 can be blocked. In this disk 1, the tubular path 11 abuts against the upper side of the reaction chamber 10 and communicates with the reaction chamber 10. The tubular path 11 is structured to block the solid phase introduced into the reaction chamber 10 by defining its depth direction, so that the flow path width is designed regardless of the solid phase size.

[0168] The waste liquid storage chamber 12 is connected to the tubular path 11 on the outer side in the rotational radial direction of the disk 1, and stores waste liquid generated in the reaction chamber 10. The tubular path 11 and the waste liquid storage chamber 12 are connected to each other through a communication port (outlet) 11b, and waste liquid from the reaction chamber 10 is discharged to the waste liquid storage chamber 12 via the tubular path 11. The waste liquid storage chamber 12 also develops on both sides with the communication port 11b as the center, and is connected to a thin flow path 17 that extends from both ends toward the rotation center. The flow path 17 communicates with the cleaning liquid storage chamber 13 at its tip. Liquid supplied in excess of the capacity of the waste liquid storage chamber 12 enters the flow path 17 and also enters the tubular path 11.

[0169] The cleaning liquid storage chamber 13 is a partitioned chamber for storing the cleaning liquid to be added to the reaction chamber 10, and has an inlet 13a formed therethrough on the upper surface side of the disk for introducing the cleaning liquid. The cleaning liquid storage chamber 13 is disposed on the rotation center side of the reaction chamber 10, and is connected to the reaction chamber 10 by a flow path 15. Only one cleaning liquid storage chamber 13 is provided in the unit 2, and a single amount of cleaning liquid is stored therein. In this disk 1, the cleaning liquid storage chamber 13 stores an amount of cleaning liquid that exceeds the total capacity of the storage chamber 12 and the tubular path 11, but does not reach the total capacity of the storage chamber 12, the tubular path 11, and the reaction chamber 10.

[0170] The cleavage reagent storage chamber 14 is a defined chamber for storing the cleavage reagent to be added to the reaction chamber 10, and has an inlet 14a formed therethrough on the upper surface side of the disk for introducing the reagent. The cleavage reagent storage chamber 14 is also disposed on the rotation center side with respect to the reaction chamber 10, and is connected to the reaction chamber 10 by a flow channel 16.

[0171] The cleavage reagent stored in the cleavage reagent storage chamber 14 is adjusted to a high concentration so that the desired final concentration will be achieved when the entire amount stored in the cleavage reagent storage chamber 14 is added to the reaction chamber 10.

[0172] The flow channels 15 and 16 are hollow tubes with a small diameter and openable at both ends. Here, "openable" refers to a state in which the movement of fluid into and out of the flow channel can be controlled. That is, the open state and the closed state can be switched depending on the rotation speed. In this embodiment in which the rotation speed is increased stepwise, when the flow channels 15 and 16 are opened, they remain open until a series of processes are completed, and have a function of being openable and closable, but are actually formed to be openable. That is, in the detection method of the present invention, the cleavage reagent storage chamber 14 and the reaction chamber 10 may be communicated with each other by an openable flow channel instead of an openable flow channel. The flow channels 15 and 16 have a function called a capillary burst valve, and when a centrifugal force of a predetermined value or more is applied, they are in an open mode, and liquid does not flow at a centrifugal force lower than that. The inner diameter of the flow channel 15 is designed to be larger than the inner diameter of the flow channel 16, and opens at a lower centrifugal force (lower rotation speed) than the flow channel 16 opens. This causes the washing solution to be supplied from the washing solution storage chamber 13 to the reaction chamber 10. Thereafter, by increasing the rotation speed, the flow path 16 opens, the cleavage reagent in the cleavage reagent storage chamber 14 flows, and the cleavage reagent is added to the reaction chamber 10.

[0173] The rotation speed at which the cleaning liquid is supplied to the reaction chamber 10 is determined based on the ease of bursting. The ease of bursting varies depending on the shape of the flow path and the characteristics of the liquid flowing through the flow path. For example, it is affected by the cross-sectional area of ​​the flow path, the length of the flow path, the inlet and outlet positions (radius of rotation) and shapes of the flow path, the surface tension, contact angle, and viscosity of the liquid. Therefore, the rotation speed is selected theoretically or experimentally taking these factors into consideration. In view of the mechanical load, the rotation speed is preferably set to 10,000 rpm or less. This disk 1 is designed to perform cleaning once, but if cleaning is performed multiple times, the cleaning liquid storage chamber 13 is divided into multiple sections, and a flow path is provided for each section. Each flow path is connected to the reaction chamber 10. Each flow path is designed to open at a rotation speed that is lower than the rotation speed at which the flow path 16 opens, and at different rotation speeds.

[0174] When a target substance is detected using this disk 1, a solution in which beads (solid phase) on which a second capture substance is immobilized are dispersed is first introduced into the reaction chamber 10 from the supply port 10a. The reaction chamber 10 is formed with a communication port 11a with the tubular path 11, and the solid phase is selected to have a size that does not pass through this communication port 11a. Therefore, the solid phase in the introduced solution is retained in the reaction chamber 10.

[0175] Next, a sample diluted to an appropriate concentration is supplied to the reaction chamber 10 through the supply port 10a, and the target substance in the sample is captured by the second capture substance on the beads. After a predetermined time has passed, a solution containing the first capture substance is supplied to the reaction chamber 10 through the supply port 10a, whereby the first capture substance binds to the target substance and a complex is formed. This causes the complex to be retained in the reaction chamber 10. The solvent in the reaction chamber 10 is discharged from the communication port 11a as the reaction chamber 10 rotates, passes through the tubular path 11, and is stored in the waste liquid storage chamber 12.

[0176] When the disk 1 is rotated at a predetermined rotation speed and the flow path 15 is opened, the washing liquid in the washing liquid storage chamber 13 is supplied to the reaction chamber 10 to perform washing. As a result, the excess first capture substance and other impurities in the reaction chamber 10 are transferred to the waste liquid storage chamber 12 by the washing liquid.

[0177] As the washing liquid is discharged from the washing liquid storage chamber 13, the washing liquid is gradually stored in the waste liquid storage chamber 12. Since the washing liquid stored in the washing liquid storage chamber 13 exceeds the capacity of the waste liquid storage chamber 12, the washing liquid overflows from the waste liquid storage chamber 12 and enters the flow path 17, and the liquid level also rises in the tubular path 11. Here, since an outward centrifugal force is acting on the disk 1, the washing liquid does not flow disorderly toward the reaction chamber 10. In addition, the flow path 17 is an air vent, and the air in the reaction chamber 10, tubular path 11, and waste liquid storage chamber 12 is expelled into the flow path 17 and pushed out into the washing liquid storage chamber 13. When the washing liquid is completely discharged from the washing liquid storage chamber 13, the waste liquid storage chamber 12 and the tubular path 11 are filled with liquid, and further, the liquid also accumulates in the reaction chamber 10, forming a state in which the solid phase is immersed while leaving a gap on the rotation center side. Most of the first capture substance transferred to the waste liquid storage chamber 12 by the flow of the cleaning liquid settles to the bottom surface (outer side) of the waste liquid storage chamber 12 due to the acting centrifugal force.

[0178] Next, the rotation speed is increased to a predetermined rotation speed at which the flow path 16 is opened, and the cleavage reagent stored in the cleavage reagent storage chamber 14 is caused to flow out into the reaction chamber 10. This gradually increases the concentration of the cleavage reagent in the reaction chamber 10, and the portion containing the labeled substance is cleaved and separated from the complex. The separated portion containing the labeled substance moves through the tubular path 11 to the drainage storage chamber 12.

[0179] A predetermined position of the tubular path is irradiated with light from a light source. Therefore, scattered light is generated when a portion containing a labeled substance passes through the tubular path 11. By detecting this with a light receiving element, it is possible to appropriately detect the portion containing the labeled substance, and as a result, it is possible to detect the target substance.

[0180] Fig. 3 is a diagram showing another example of a disk, which is a modification of the disk 1 of the first embodiment. Specifically, this modification is a modification of the unit 2 of the disk 1, and Fig. 3 shows the unit 2 of this modification. The same parts as those of the disk 1 described above are given the same reference numerals and their explanation is omitted.

[0181] In this unit 2, from the rotation center toward the outside, there are provided a first storage chamber 101 for holding a sample, a flow path 102 communicating with the first storage chamber 101, a second storage chamber 103 for storing liquid to be filled into the tubular path 11, and the tubular path 11, and further there is provided a thin flow path 17 extending from the end of the branch flow path branching off from the tip of the tubular path 11 toward the rotation center.

[0182] This unit 2 is for introducing a sample, which has been subjected to separation of the portion containing the labeled substance in a reaction field outside the disk 1, into the disk 1 and detecting the portion containing the labeled substance. For this reason, a cleaning process is not performed on the disk 1.

[0183] In this unit 2, first, a predetermined amount of sample solution is introduced into the first storage chamber 101 through the opening 101a on the upper surface of the first storage chamber 101. Here, since the flow path 102 is configured with a function of a capillary burst valve, the sample solution introduced in a stationary state does not flow out to the second storage chamber 103 through the flow path 102. In addition, a predetermined amount of liquid to be filled into the tubular path 11 is introduced into the second storage chamber 103 through the opening 103a on the upper surface of the second storage chamber 103. The second storage chamber 103 is provided with an introduction part at the end on the rotation center side, at a position off the extension of the flow path 102 and protruding toward the rotation center side. The opening 103a is formed through the upper surface of this introduction part. This makes it possible to prevent the liquid from leaking out from the opening 103a when the sample solution held in the first storage chamber 101 is introduced into the second storage chamber 103.

[0184] The second storage chamber 103 is structured so that the tubular passage 11 abuts against the upper portion in the depth direction of the second storage chamber 103 and communicates with it. The second storage chamber 103 stores the amount of liquid necessary to fill the tubular passage 11, but in order to fill the tubular passage 11 with liquid, the tubular passage 11, its branch passage ahead, and the passage 17 communicating therewith are filled with liquid up to a position corresponding to the communication port (inlet) 11a of the tubular passage 11, so the second storage chamber 103 is designed based on the total internal volume of the tubular passage 11, the branch passage ahead, and the corresponding portion of the passage 17. In addition, the structure of the second storage chamber 103 is designed so that the liquid level of the liquid stored in the liquid is lower than the bottom surface of the communication port 11a of the tubular passage 11 when the liquid is stored. Furthermore, when device 1 is formed from a general-purpose resin material, the insides of first and second storage chambers 101, 103 have hydrophobic surfaces, which act to cause the introduced aqueous sample solution and liquid for filling the tubular path to remain within the chambers.

[0185] Then, the device 1 is rotated at a predetermined rotation speed that does not open the flow path 102, and the liquid in the second storage chamber 103 is introduced into the tubular path 11. Here, the flow path 17 functions as an air vent, so that the liquid in the second storage chamber 103 is smoothly introduced into the tubular path 11 when the disk 1 is rotated at a predetermined rotation speed.

[0186] Next, when the rotation speed is increased to a rotation speed at which the flow channel 102 opens, the sample solution flows out from the first storage chamber 101 to the second storage chamber 103. Then, the portion containing the labeled substance moves outward in the tubular path 11 by centrifugal force. In this way, when the cutting process is performed in a reaction field outside the disk 1, the target substance can be detected with a simple device configuration.

[0187] For example, the device shown in FIG. 4 is used to rotate the disk 1. The device includes a motor and a rotating shaft connected to the motor, and a mounting table for rotating the disk 1 in a horizontal direction is supported on the rotating shaft. When the motor is driven, the disk 1 mounted on the upper surface of the mounting table rotates, and centrifugal force is applied. The motor is connected to a microcontroller (not shown), and is connected to an information processing device (personal computer (PC)) via the microcontroller. The information processing device transmits and receives signals to and from each device connected thereto, and controls each device. The information processing device (PC) is not particularly limited as long as it is a device capable of implementing signal control. When a control signal from the information processing device is input to the microcontroller, the microcontroller controls the motor to rotate at a predetermined number of rotations for a predetermined time.

[0188] The mounting table is provided with a window penetrating in the thickness direction at a position facing the tubular path 11 when the disk 1 is mounted. A laser light source (LD) is installed above the mounting table, and is adjusted so that irradiated light is incident at an oblique angle to the upper surface of the mounting table. A focus lens is disposed in front of the laser light source, and is adjusted so that the irradiated light from the laser light source is focused inside the tubular path 11. A PC is connected to this laser light source via the microcontroller, and the output is controlled by the microcontroller that receives instructions from the PC.

[0189] A collecting lens (Lens) and photodiode (PD) are placed below the window of the mounting table and are configured to receive the scattered light. The scattered light intensity is converted into a voltage by photoelectric conversion by the photodiode and input to a signal detector (oscilloscope). The signal detector samples at a specified sampling time, and the input analog signal (voltage value) is converted into a digital signal and input to a PC. The PC then outputs the scattered light measurement results. This makes it possible to obtain the scattered light of the labeling substance and detect the target substance.

[0190] The present invention has been described above based on an embodiment, but the present invention is not limited to the above embodiment, and it can be easily inferred that various improvements and modifications are possible within the scope of the present invention without departing from the spirit of the present invention.

[0191] For example, the disk 1 may be configured by appropriately providing a mechanism for controlling the movement of the fluid. Examples of such mechanisms include valves. Such valves switch the communication ports of the flow paths or chambers between a closed state and an open state, and in addition to the capillary burst valves provided in the flow paths 15 and 16, various valves that operate by electricity, magnetic force, electromagnetic force, mechanical mechanism, inertial force, centrifugal force, or heat can be used.

[0192] Furthermore, for example, the disk 1 has been described as a circular disk, but in the present invention, it is not limited to a disk and may have other shapes. In other words, in the method of the present invention, the device is not significantly limited in shape as long as it can apply centrifugal force. For this reason, for example, a chip-type device having only one unit 2 and molded into a fan-shaped or rectangular outer shape can be exemplified. In such a case, a mechanism or tool for holding the chip can be provided on the mounting table of the device that rotates the device, and the chip can be fitted into the mechanism or tool and rotated to apply centrifugal force.

[0193] The following inventions can be exemplified as modified examples of the present invention. The present invention provides a method for detecting a target substance, which comprises contacting a binding agent that specifically binds to the first capture substance with a sample containing a mixture of a complex formed by the capture of a target substance by a first capture substance bound to a labeling substance and the first capture substance that has not captured the target substance, thereby binding the first capture substance that has not captured the target substance to the binding agent, placing the binding agent in a chamber in which a sample is stored, applying centrifugal force to extract the complex from the chamber and move it through a tubular path filled with liquid, and detecting the complex by the scattered light generated when the complex moving through the tubular path scatters irradiated light.

[0194] In this modification, the detection target is a complex formed by binding of a target substance and a first capture substance. Here, the excess first capture substance that is not bound to the target substance is also bound to a labeling substance, so if this is introduced into the detection field, scattered light is also detected from the unreacted first capture substance, causing an error in the detection result. In the above-mentioned first embodiment, the target substance is immobilized on a solid phase, the first capture substance is temporarily supported on the solid phase, and the excess first capture substance is removed by an appropriate washing process. However, in this modification, the excess first capture substance is bound to a binder, and the binder is retained in a chamber, so that the excess first capture substance can be retained in the chamber, and the introduction of the excess first capture substance into the detection field is suppressed, making it possible to improve the accuracy of complex detection while eliminating the need for washing.

[0195] The formation of the complex and the binding of the excess first capture substance to the binder are carried out in a state where the liquid is stored. For example, if a tubular path and a front chamber (chamber) for supplying a sample to the tubular path are provided in one device, and a binder is placed in the front chamber, the excess first capture substance contained in the sample introduced into the front chamber can be bound to the binder and retained in the front chamber. The chamber in which the binder is placed does not necessarily have to be formed integrally with the device, and another chamber provided outside the device may be used to place the excess first capture substance. When placing the excess first capture substance in the chamber, a method such as fixing the binder to the inner wall of the chamber or fixing the binder to a solid and introducing it into the chamber can be appropriately selected.

[0196] In addition, when the binder is introduced into the anterior chamber, if the solid to which the binder is fixed is made larger than the tubular passage, the excess first capture substance can be retained. Furthermore, a valve may be provided in the chamber, and after the filling of the tubular passage with the liquid is completed, the valve may be opened to introduce the complex formed by the binding of the target substance and the first capture substance into the tubular passage.

[0197] <Binding agent> The binder specifically binds to the first capture substance, and a suitable example is an antigen. The antigen may be directly supported on a solid phase such as an inner wall or a solid, or may be indirectly immobilized on the solid phase via an antibody or the like. For example, when an antigen is used as the binder, a tag such as biotin may be added to the antigen and avidin may be bound to the solid phase, whereby the binder can be immobilized on the solid phase by biotin-avidin binding.

[0198] The binding between the solid phase and the binder can be achieved by any of a variety of binding modes, including physical adsorption, covalent bonding, ionic bonding, affinity bonding, and combinations thereof. Preferably, the binder is immobilized on the solid phase by a covalent bond or ionic bond that is stronger than the binding force due to affinity or adsorption.

[0199] In addition, when the labeling substance is larger than the first capture substance, it is expected that multiple first capture substances will bind to the labeling substance. However, in this modified example, the smaller the number of first capture substances that bind to the labeling substance, the better the realization, and ideally, it is desirable for only one first capture substance to bind to the labeling substance. EXAMPLES

[0200] The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to the description of the examples.

[0201] (Reference Example 1) Preparation of platinum-gold colloidal suspension All glassware used was washed with aqua regia. 390 mL of ultrapure water was placed in a flask and boiled, and 30 mL of chloroauric acid solution (1 g of gold per 1 liter of solution, Katayama Chemical Industry Co., Ltd.) was added to the boiling water, followed by 60 mL of 1 wt% sodium citrate solution. After the addition of the sodium citrate solution, several tens of mL of chloroplatinic acid solution (1 g of platinum per 1 liter of solution, Wako Pure Chemical Industries Co., Ltd.) was added. Five minutes later, 60 mL of 1 wt% sodium citrate solution was added and refluxed for 4 hours to obtain a platinum-gold colloid suspension. In order to accurately detect the portion containing the labeling substance, reagents and reducing agents were added as appropriate to adjust the particle size of the colloid to 100 nm or more.

[0202] (Reference Example 2) Preparation of monoclonal antibodies An anti-SARS-CoV-2NP monoclonal antibody (hereinafter simply referred to as "anti-NP antibody") was obtained according to conventional methods using a commercially available SARS-CoV-2 nucleocapsid protein (hereinafter simply referred to as "SARS-CoV-2NP") (CUSABIO) as an immunization antigen.

[0203] Specifically, mice (BALB / c, 5 weeks old, Japan SLC) were immunized three times with 100 μg of SARS-CoV-2NP, and their spleen cells were used for cell fusion. Mouse myeloma cells, Sp2 / 0-Ag14 cells (Shulman et al., 1978), were used for cell fusion. The cells were cultured in Dulbecco's Modified Eagle Medium (Gibco) supplemented with 0.3 mg / ml L-glutamine, 100 units / ml penicillin G potassium, 100 μg / ml streptomycin sulfate, and 40 μg / ml gentacin (DMEM), to which fetal bovine serum (JRH) was added at 10%. Cell fusion was performed by mixing spleen cells from immunized mice with Sp2 / 0-Ag14 cells and adding polyethylene glycol solution (Sigma) to the mixture. The fused cells were cultured in HAT-RPMI [serum-supplemented RPMI containing 0.1 mM sodium hypoxantine, 0.4 μM aminepterin, and 0.1 mM thymidine (Gibco)], and antibody production in the culture supernatant was confirmed by enzyme-linked immunosorbent assay (ELISA). Cells that produced positive antibodies were cultured and expanded in HT-RPMI [serum-supplemented RPMI containing 0.1 mM sodium hypoxantine and 0.1 mM thymidine].

[0204] The cloned cells were intraperitoneally inoculated into mice (BALB / c, Retire, Japan SLC) that had been inoculated with 2,6,10,14-Tetramethylpentadecane (Kishida Chemical), and the ascites was collected. The ascites was subjected to a protein G column to purify the monoclonal antibodies. Finally, 83 clones of monoclonal antibody-producing cells against SARS-CoV-2NP were obtained.

[0205] (Reference Example 3) Preparation of colloidal gold suspension Gold colloid particles were prepared according to a conventional method using an aqueous solution of chloroauric acid (1 g of gold per 1 liter of aqueous solution, manufactured by Katayama Chemical Industry Co., Ltd.) as a raw material. Specifically, 1 ml of 1% (v / w) aqueous solution of chloroauric acid was added to 99 ml of pure water that had been heated to a boil, and one minute later, 1.5 ml of 1% (v / w) aqueous solution of sodium citrate was added, heated, boiled for 5 minutes, and then allowed to cool at room temperature. Next, a 200 mM aqueous solution of potassium carbonate was added to this solution to adjust the pH to 9.0, and ultrapure water was added to this to make the total volume 100 ml, thereby obtaining a gold colloid suspension in which gold colloid particles were suspended. If the desired particle size was not obtained, the amount of reducing agent added to chloroauric acid was appropriately adjusted to produce gold colloid particles of the required particle size.

[0206] (Example 1) Separation of fractions containing labeled substances (platinum-gold colloidal particles) using a pH adjuster (1) Preparation of platinum-gold colloid-labeled antibody solution One anti-NP antibody selected from the anti-NP antibodies obtained in Reference Example 2 was labeled with platinum-gold colloid by the following procedure. 1 μg of protein equivalent weight of the anti-NP antibody (hereinafter, when the protein equivalent weight is indicated, it is indicated by the weight value obtained by gravimetric analysis of the purified protein) was mixed with 1 mL of the platinum-gold colloid suspension with a particle size of 120 nm prepared in Reference Example 1, and the mixture was allowed to stand at room temperature for 2 minutes to bind the anti-NP antibody to the surface of the platinum-gold colloid particles. Then, a 10% bovine serum albumin (hereinafter, referred to as "BSA") aqueous solution was added so that the final concentration was 1.0% relative to the total liquid volume, and the remaining surface of the platinum-gold colloid particles was blocked with BSA to prepare a platinum-gold colloid-labeled anti-NP antibody solution. Then, this solution was centrifuged (600×g, 25 minutes) to precipitate the platinum-gold colloid-labeled anti-NP antibody, and the supernatant was removed to obtain the platinum-gold colloid-labeled anti-NP antibody. The obtained platinum-gold colloid-labeled anti-NP antibody was suspended in 50 mM Tris-HCl buffer (pH 7.4) containing 10% sucrose, 1% BSA, and 0.5% Triton-X100 (trade name) to prepare a platinum-gold colloid-labeled antibody solution.

[0207] (2) Cutting treatment with pH adjuster Among the anti-NP antibodies obtained in Reference Example 2, an antibody other than the anti-NP antibody used in the platinum-gold colloid-labeled anti-NP antibody described above was adjusted to 20 μg / mL in Tris-HCl buffer to prepare an antibody dilution solution, and 100 μL of the antibody dilution solution was added to each well of a 96-well microplate and left to stand for 15-18 hours to immobilize the antibody. After that, a blocking treatment was performed, and then 50 μL of SARS-CoV-2NP solution adjusted to 10 ng / mL and 50 μL of platinum-gold colloid-labeled antibody solution were added and left to stand at 37 ° C. for 10 minutes. Next, the plate was washed with TBS-T (Tris Buffered Saline with Tween 20), and after the washing solution was discharged, 100 μL of various reagents with different pH values ​​were added, and the plate was left to stand at room temperature for 2 minutes, and the solution in the well was collected. The types of reagents used and their pH values ​​are shown in Table 1. The pH value was measured using a pH meter (LAQUA, Horiba).

[0208] The pH of the citric acid solution was adjusted to the desired pH (pH 2.94 to pH 6.96) by mixing citric acid and trisodium citrate. The phosphoric acid solution was adjusted to the desired pH (pH 6.99 to 8.56) near neutral by mixing sodium dihydrogen phosphate and disodium hydrogen phosphate, and was adjusted to the desired alkaline pH (pH 11.33 to 12.85) by mixing disodium hydrogen phosphate and trisodium phosphate. The glycine hydrochloric acid buffer, glycine NaOH buffer, and triethylamine solution were adjusted to the desired pH by adding hydrochloric acid or sodium hydroxide. The pH of the hydrochloric acid solution and sodium hydroxide solution was adjusted by changing the dilution ratio.

[0209] [Table 1]

[0210] (3) Measurement of platinum-gold colloid particles The amount of platinum-gold colloid particles contained in the solution recovered by the above procedure was measured. Specifically, the recovered solution was introduced into a sample cell, and the sample cell was irradiated with a 650 nm laser beam, and the scattered light obtained was measured with a photodiode set at a predetermined position.

[0211] Since the scattered light intensity increases in proportion to the concentration of platinum-gold colloidal particles in the solution, the concentration of platinum-gold colloidal particles in the solution can be quantitatively analyzed by measuring the scattered light intensity. In other words, by treating the immune complex with a pH adjuster, the "part containing platinum-gold colloidal particles" separated from the solid phase or immune complex can be detected.

[0212] In addition, if the platinum-gold colloid-labeled anti-NP antibody is nonspecifically adsorbed (to a place other than the antigen) and is not removed by washing, it will be separated by the pH adjuster and mixed into the measurement sample. Therefore, only 100 μL of the platinum-gold colloid-labeled anti-NP antibody solution was added to the well in which the antibody was immobilized in the same manner as above, and the well was left to stand at 37°C for 10 minutes. Next, the well was washed with TBS-T, and 100 μL of the pH adjuster solution was added. After standing at room temperature for 2 minutes, the solution was recovered from the well and scattered light measurement was performed. The same operation was performed for each pH adjuster, and the obtained scattered light intensity was used as the blank value for each pH adjuster. In addition, if the pH adjuster has a weak processing ability to cleave the complex, the difference between the scattered light intensity of the sample and the scattered light intensity of the blank becomes small, so that the scattered light intensity of the sample measurement result may be shown as a negative value in the result of the difference between the two.

[0213] The blank value can be estimated as the scattered light intensity of the platinum-gold colloid particles derived from the platinum-gold colloid-labeled anti-NP antibody nonspecifically adsorbed in the well, so this blank value was subtracted from the measured value to obtain the scattered light intensity of the "part containing platinum-gold colloid particles" of the formed immune complex. The results are shown in Figure 5.

[0214] Figure 5 shows the relationship between the pH value and the cleavage characteristics of each reagent. The lower part of Figure 5 shows the pH value of each reagent and the corresponding scattered light intensity value, and the upper part of Figure 5 shows each value in a graph. The horizontal axis of the graph is the pH value, and the vertical axis is the scattered light intensity. As can be seen from Figure 5, when the pH value is near neutral, the scattered light intensity is low. In other words, it is shown that the added neutral reagent has low cleavage performance of the "part containing platinum-gold colloid particles" of the complex. On the other hand, when the added reagent is acidic or basic, the scattered light intensity increases, indicating that the "part containing platinum-gold colloid particles" of the complex (solid phase-anti-NP antibody-SARS-CoV-2NP-platinum-gold colloid-labeled anti-NP antibody) bound to the solid phase can be cleaved by a pH adjuster such as an acid or base.

[0215] In particular, it was shown that pH adjusters (acids) with a pH of less than 4 and pH adjusters (bases) with a pH of 10.6 or higher have good cleavage performance, and furthermore, pH adjusters (bases) with a pH of 11.6 or higher have excellent cleavage performance.

[0216] (Example 2) Separation of fractions containing labeled substances (platinum-gold colloidal particles) using sodium hydroxide As a result of Example 1, the cleavage ability of the sodium hydroxide solution was particularly good, so the cleavage performance of the portion containing the labeled substance from the complex bound to the solid phase (solid phase-anti-NP antibody-SARS-CoV-2NP-platinum-gold colloid-labeled anti-NP antibody) was further evaluated by changing the concentration of sodium hydroxide.

[0217] The sodium hydroxide concentrations were 0.01 mM, 0.1 mM, 1 mM, 10 mM, 100 mM, and 1 M. The treatment was carried out in the same manner as in Example 1, except that 100 μL of an aqueous sodium hydroxide solution adjusted to each concentration was used. The results are shown in FIG.

[0218] Figure 6 shows the cutting performance of the composite by sodium hydroxide. The pH value and scattered light intensity at each concentration of sodium hydroxide are shown in the lower part of Figure 6, and each value is shown in a graph in the upper part of Figure 6. The horizontal axis of the graph is the molar concentration of sodium hydroxide, and the vertical axis is the scattered light intensity. As can be seen from Figure 6, it was revealed that sodium hydroxide at a concentration of 10 mM or more separates (liberates) the portion of the immune complex that contains the platinum-gold colloid particles. The pH of the 10 mM sodium hydroxide solution was 11.87.

[0219] (Example 3) Separation of the portion containing the labeling substance (platinum-gold colloid particles) using a denaturing agent A microplate having wells in which anti-NP antibodies were immobilized and blocked was prepared in the same manner as in Example 1. The well solution was collected in the same manner as in Example 1, except that a denaturant was used instead of a pH adjuster on the complex (solid phase-anti-NP antibody-SARS-CoV-2NP-platinum-gold colloid-labeled anti-NP antibody) formed using the wells of this microplate as the solid phase, and the amount of platinum-gold colloid particles was measured in the same manner as in Example 1(3). The denaturant used was 8M urea, 6M guanidine hydrochloride, or 1% SDS. The results are shown in FIG. 7. For comparison, the results of a similar treatment using 1M-NaOH are also shown in FIG. 7.

[0220] Figure 7 shows the effect of denaturants on immune complexes bound to a solid phase, as measured by the scattered light intensity of separated (released) platinum-gold colloids (portions containing platinum-gold colloidal particles). For comparison, the data shown is the case where 1M-NaOH was used. As can be seen from Figure 7, it was clear that all denaturants liberated the portions containing platinum-gold colloidal particles.

[0221] (Example 4) Sensitization treatment of gold colloid particles The gold colloid particle suspension (particle size about 12 nm) prepared in Reference Example 3 was adjusted to a concentration of 4.36 nM with pure water, and 2 μL of the suspension was added to 100 μL of 1M NaOH and mixed. This 1M NaOH treatment was a schematic example of a cleavage treatment of the complex with a pH adjuster. 100 μL of 1M HCl was added thereto for neutralization. 6.35 μL of this neutralization solution was added to a mixture of 972 μL of 10 mM Walpole buffer (pH 2.0) and 1.65 μL of 12.1 M chloroauric acid solution, and 20 μL of 375 mM hydroxylamine hydrochloride was added and mixed, and the mixture was left to stand at room temperature for 10 minutes to carry out a sensitization reaction. After this, the gold colloid particles in the solution were precipitated by centrifugation, the supernatant was removed, and the solution was washed with ultrapure water. 1μL of the resulting gold colloid particle dispersion was dropped onto a sample holder with carbon tape attached, the air was removed, and the sample was photographed using an electron microscope TM4000Plus (Hitachi, Ltd.). The applied voltage was 15kV and the magnification was 1000. Five or more fields of view were photographed, and the images were analyzed to calculate the average particle size and standard deviation. For comparison, a sample was subjected to the same sensitization treatment using ultrapure water that did not contain gold colloid particles instead of the 4.36nM gold colloid particle dispersion. The results are shown in Figure 8.

[0222] FIG. 8 shows the change in colloidal gold particle diameter due to the sensitization reaction. The vertical axis in FIG. 8 shows particle diameter. As can be seen from FIG. 8, the sensitization reaction allowed the gold colloid particles in the 4.36 nM colloidal gold particle dispersion to grow to particles with a particle diameter of about 3 to 5 μm. On the other hand, precipitation of colloidal gold particles was observed even when no colloidal gold particles were included, but the particles were significantly smaller than those in the case where the sensitization treatment was performed, so the two were clearly distinguishable in detection. Note that the particle diameter can naturally be adjusted by adjusting the reaction conditions, so it is clear that colloidal gold particles with a particle diameter suitable for the detection system can be produced. This demonstrated that minute gold colloid particles could be enlarged by a sensitization reaction, and that the sensitized gold (colloid) particles could be adequately detected optically.

[0223] (Example 5) Change in scattered light intensity due to metal particle size (1) Preparation of colloidal gold particles and colloidal platinum particles of various sizes Platinum-gold colloidal particles with a particle size of 120 nm were prepared according to Reference Example 1. Furthermore, gold colloidal particles with particle sizes of 12 nm and 200 nm were prepared according to Reference Example 3, and gold (colloidal) particles with particle sizes of 500 nm and 1000 nm were commercially available (NANOPARTz, A11-500-CIT-DIH-1-50, A11-1000-NPC-DIH-1-100). Each particle was prepared in a state of being dispersed in a solution.

[0224] (2) Measurement of scattered light intensity The gold colloid particle dispersions of each particle size prepared above were diluted to a concentration within the range in which scattered light could be measured, and the scattered light was measured using the same method as in Example 1. The obtained scattered light intensity value was divided by the concentration to calculate the scattered light intensity at 1 nM. As a result, it was found that the scattered light intensity increased as the particle size of the gold colloid particles increased. The results are shown in Figure 9.

[0225] Figure 9 shows the relationship between the particle size of metal colloid particles and the scattered light intensity. The horizontal axis shows the particle size (nm) and the vertical axis shows the scattered light intensity in logarithmic scale. As can be seen from Figure 9, the scattered light intensity increases significantly from 120 nm, demonstrating that metal colloid particles can be detected from around 100 nm using a simple scattered light detection system consisting of a laser light source and a photodiode.

[0226] Example 6: Antigen detection based on moieties containing labeling substances (colloidal platinum-gold particles) The anti-NP antibody used for immobilization in Example 1 was adjusted to 20 μg / mL with Tris-HCl buffer to prepare an antibody dilution solution. This was mixed with magnetic particles (manufactured by JSR Life Sciences Corporation), and the anti-NP antibody was immobilized on the magnetic particles by chemical bonding. Then, the anti-NP antibody immobilized on the magnetic particles, SARS-CoV-2NP solutions adjusted to concentrations of 10, 100, 1000, and 10000 pg / mL, and the platinum-gold colloid-labeled anti-NP antibody prepared in Example 1 were reacted at 37°C for 30 minutes. After the reaction, each solution was centrifuged with a 0.45 μm centrifugal filter (Merck) to recover the magnetic particles, which were then washed five times with TBS-T. Then, 1M-NaOH was added to the magnetic particles, a cleavage treatment (elution) was performed on the complex (magnetic particle-anti-NP antibody-SARS-CoV-2NP-platinum-gold colloid-labeled anti-NP antibody) bound to the magnetic particles, and the scattered light intensity of the recovered eluate was measured. The measurement results are shown in FIG. 10. Note that in the examples, elution means that a portion containing a labeling substance, which is a part of the complex, is cleaved from the complex, and as a result, that portion is separated from the solid phase and released into the liquid, and is not limited to being interpreted as dissolving the separated portion in the liquid.

[0227] FIG. 10 is a diagram showing the concentration of eluted platinum-gold colloid particles (part containing platinum-gold colloid particles) in terms of scattered light intensity. The vertical axis shows the scattered light intensity in logarithmic scale, and the horizontal axis shows the antigen concentration. As can be seen from FIG. 10, the scattered light intensity based on the platinum-gold colloid particles was proportional to the antigen concentration. In other words, as the antigen concentration increased, the amount of eluted platinum-gold colloid particles also increased, demonstrating that the complex formed on the solid phase by the antigen-antibody reaction was effectively cleaved, and further that the part containing the cleaved labeling substance was separated, eluted, and appropriately detected.

[0228] (Example 7) Detection of scattered light from a rotating disk (1) Experiment to evaluate the outflow of colloidal gold particles This example is an experimental evaluation of the outflow properties of gold colloid particles. For this experiment, a disk was prepared that had a chamber defined at the center of the disk, and a flow path that was connected to the chamber and extended radially outward from the disk. This disk had a 50 μm thick polypropylene sheet (hereinafter abbreviated as "PP sheet") attached to the top surface of the disk, in which the chamber and flow path were recessed, via an adhesive layer, and the top surface of the chamber and flow path were formed by the PP sheet.

[0229] FIG. 11 is a schematic diagram showing a cross section of a part of the flow path of the disk used in this embodiment. The inside of the chamber is formed so that the depth in the thickness direction is 1000 μm, and the flow path is connected to the upper part of the wall opposite the center of rotation of the chamber. The flow path is formed with a width of 230 μm and a depth of 15 μm, has a rectangular flow path cross section, and is a capillary burst valve that opens and allows liquid to flow when the disk rotates beyond a predetermined rotation speed. Also, as shown in FIG. 11, the wall of the chamber that contacts the flow path is inclined so as to rise obliquely from the bottom surface. Therefore, the depth of the chamber gradually becomes shallower toward the flow path. In the prepared disk, the PP sheet on the upper surface of the chamber was punctured to make a through hole penetrating the inside and outside of the chamber.

[0230] A dispersion of polystyrene beads (PS beads) (Micromod, particle size 20 μm, 25 mg / ml aqueous dispersion) was injected into this chamber through the drilled through-hole. Since the injected dispersion was small compared to the volume of the chamber, a liquid pool was formed near the through-hole due to surface tension. Next, the chamber was rotated at 2000 rpm for 1 minute to drain the solvent in the chamber into the flow path. The solvent that had passed through the flow path was collected by opening a hole in the PP sheet on the top surface at the chamber beyond the flow path and sucking up the entire amount with a pipette.

[0231] Then, 10 μL of the gold colloid particle dispersion prepared in Reference Example 3 was injected into the chamber through the through hole. As a result, a pool of gold colloid particle dispersion was formed near the through hole due to surface tension. Then, the chamber was rotated at 2000 rpm for 1 minute to allow the solution in the chamber to flow into the flow path, and the liquid that passed through the flow path was similarly collected from the chamber at the end of the flow path. The collected solutions of the gold colloid particle dispersion and the PS bead dispersion prepared in Reference Example 3 were measured for absorbance or scattered light intensity using a microspectrophotometer or a scattered light intensity measuring device, and the two were compared. As a result, neither of the collected solutions contained PS beads, while the collected solution derived from the gold colloid particle dispersion contained gold colloid particles at more than 60% of the initial concentration. This confirmed that the gold colloid particles flowed out of the chamber into the flow path by weaving between the blocked PS beads.

[0232] (2) Specific detection of labeled substances using scattered light Next, scattered light detection of the labeling substance in the presence of a biomaterial was performed. BSA was used as a typical impurity. A dispersion of 200 nm-diameter colloidal gold particles prepared in the same manner as in Reference Example 3 was used as the colloidal gold particles. A 10% BSA solution (manufactured by Oriental Yeast Co., Ltd., Cat 47408903) or water was prepared, and a 2 pM colloidal gold particle dispersion was diluted to 2 fM using each of these as a solvent. The diluted colloidal gold particle dispersion and the 10% BSA solution were each added to a 300 μL cuvette, and the scattered light intensity was measured using the scattered light detection device used in Example 1.

[0233] For this experiment, a disk was prepared that had a chamber partitioned on the rotation center side and a flow path that was connected to the chamber and extended toward the radial outside of the disk. In this disk, the chamber and the flow path were recessed into a resin disk, and a 50 μm thick PP sheet was attached to the top surface of the disk via an adhesive layer. The PP sheet formed the top surface of the chamber and the flow path. Both the chamber and the flow path were formed to a depth of 15 μm, and their bottom surfaces were continuous with each other. The width of the flow path was 1 mm, and the tip of the extended flow path was connected to a branch path that was perpendicular to the flow path, and both ends of the branch path were connected to a thin flow path that headed toward the rotation center. The thin flow path was connected to the end of the chamber on the rotation center side and communicated with the end of the chamber on the rotation center side. The chamber was formed to be wider than the flow path. In addition, a through hole was drilled in the top surface of the chamber.

[0234] A BSA solution or a gold colloid particle dispersion diluted to 2 fM with a BSA solution was injected into the chamber of this disk. The disk was set on the mounting table of the device shown in Figure 4 and rotated at 2000 rpm for 2 minutes, and the number of scattered lights generated was counted. The results are shown in Table 2. [Table 2]

[0235] Table 2 shows the measurement results for gold colloid particle dispersion (GNP), BSA solution (BSA), and gold colloid particle dispersion diluted with BSA solution (GNP+BSA) from the top to the bottom. The middle column shows the results of measuring the scattered light intensity using a scattered light detector, and the rightmost column shows the count results of the number of scattered lights generated from the rotating disk. As can be seen from these figures, in the measurement of scattered light intensity using a cuvette, scattered light was measured for both the gold colloid particle dispersion and the BSA solution, and stronger scattered light was measured for the sample in which the two were mixed. This result shows that in a measurement method in which a relatively large capacity cell such as a 300 μL cuvette is used and scattered light is detected by irradiating the sample with irradiation light while it is left stationary, it is difficult to specifically detect the target substance such as gold colloid particles by scattered light when impurities are present. On the other hand, when a disk was used, the results of the measurement using water as a blank and the measurement of the BSA solution were similar, and the generation of scattered light caused by BSA was not counted (detected). Furthermore, when a gold colloid particle dispersion was measured, the generation of scattered light originating from the gold colloid particles was counted, indicating the detection of gold colloid particles.

[0236] FIG. 12 shows the results of gold colloid particle detection using a disk. The horizontal axis of FIG. 12 shows the position in the flow path width direction in the tubular path, and the vertical axis shows the intensity of scattered light in voltage values. The peaks of the counted gold colloid particles are indicated by ▼. FIG. 12 shows the change in the detection peak over time from top to bottom. According to this, it can be seen that there are peaks that are observed continuously without changing over time, and intermittent peaks that appear only for a short time. The peaks that do not change over time can be determined as noise. Therefore, noise can be canceled by obtaining the intensity (voltage value) of the blank scattered light at the same position of the tubular path and subtracting it from the intensity (voltage value) of the scattered light obtained at any rotation during measurement. On the other hand, the intermittent peaks that occur intermittently can be determined to be due to the dynamic gold colloid particles passing through the flow path. Therefore, it was shown that even if impurities are present, the target detection target can be separated and specifically detected.

[0237] (Example 8) Separation of a portion containing a labeling substance (colloidal gold particles) and detection of scattered light using a rotating chip-type device (1) Preparation of colloidal gold-labeled antibody solution Except for using, as the labeling substance, colloidal gold particles having a particle size of 200 nm prepared in Reference Example 3 instead of platinum-gold colloidal particles, colloidal gold-labeled anti-NP antibody was prepared in the same manner as in the preparation of platinum-gold colloidal labeled antibody in Example 1. The colloidal gold-labeled anti-NP antibody prepared was suspended in a specimen extract to prepare a colloidal gold-labeled antibody solution.

[0238] (2) Preparation of polystyrene bead-immobilized antibodies Among the anti-NP antibodies prepared in Reference Example 2, an antibody other than the anti-NP antibody used in the above-mentioned gold colloid-labeled anti-NP antibody was selected and bound to polystyrene beads (Micromod, particle size 20 μm, 25 mg / ml aqueous dispersion) by the maleimide method to prepare polystyrene bead-immobilized anti-NP antibody.

[0239] (3) Formation of an antibody-antigen-antibody sandwich-type immune complex SARS-CoV-2NP was used as the antigen, and the antigen, the gold colloid-labeled antibody solution, and the polystyrene bead-immobilized anti-NP antibody were each adjusted to a predetermined concentration with the sample extract. These were then mixed and reacted at room temperature for 10 minutes to generate an antibody-antigen-antibody sandwich-type immune complex.

[0240] (4) Cleaning process A chip-type device was prepared in which a single unit was formed independently, comprising a reaction chamber, a flow channel, a tubular path, a drainage reservoir, a washing liquid reservoir, and a reagent reservoir (cleavage reagent reservoir) similar to that shown in FIG. 2b.

[0241] Then, the solution containing the immune complex prepared above was introduced into the chip-type device. Specifically, the solution containing the immune complex prepared above was injected into the reaction chamber, and the chip-type device was set on the mounting table of the apparatus shown in FIG. 4 and rotated at 2000 rpm for 10 seconds to discharge the liquid from the reaction chamber, after which the rotation was stopped and a cleaning liquid was injected into the reaction chamber. Then, the chip-type device was rotated at 2000 rpm for 9 minutes to perform a cleaning process. After the cleaning process, the tubular path was filled with liquid. In addition, the cleaning liquid was filled and stored beyond the inlet of the tubular path to a position where the polystyrene beads in the reaction chamber were almost immersed.

[0242] (5) Separation of the portion containing the labeled substance (gold colloid particles) and specific detection of the labeled substance by scattered light After the washing process, 1M NaOH was injected into the reaction chamber as a cleavage reagent. The chip-type device was then rotated at 2000 rpm for 3 minutes to separate the gold colloid particles (the portion containing the gold colloid particles) from the complex, i.e., to dissolve them into the liquid stored in the reaction chamber. The gold colloid particles dissolved in the liquid in the reaction chamber pass through a tubular passage by centrifugal force, and the scattered light generated by the irradiation of the irradiated light is detected. The detected scattered light is converted into an electrical signal and obtained as a voltage value. The scattered light detection results when the antibody concentration was adjusted to 0.5 pg / mL and an immune complex was formed are shown in Figures 13a and 13b.

[0243] 13a and 13b are diagrams showing the results of detecting gold colloid particles (parts containing gold colloid particles) by scattered light in this embodiment. In FIG. 13a, the horizontal axis indicates the rotation time (time (round)) of the chip-type device, with 1000 time corresponding to 30 seconds. The vertical axis indicates the widthwise position (position (smp: sampling)) in the tubular path, with 100 smp corresponding to 492 μm. The voltage value corresponding to the detected scattered light is output and displayed as grayscale image data. The grayscale shading corresponds to the high and low voltage values, with the brightness decreasing as the voltage value increases. FIG. 13b is a partially enlarged view of FIG. 13a, with the horizontal axis indicating a range of 1000 to 2000 rounds and the vertical axis indicating a range of 200 to 300 smp.

[0244] Here, the sample was a mixture of polystyrene bead-immobilized anti-NP antibody and gold colloid-labeled antibody solution without adding antigen, and the results of the above steps (4) washing treatment and (5) separation (elution) of the part containing the labeling substance (gold colloid particles) and specific detection of the labeling substance by scattered light in this example are shown in Figures 14a and 14b. The vertical and horizontal axes and displayed data in Figures 14a and 14b are the same as those in Figures 13a and 13b, respectively, and Figure 14b is a partial enlargement of Figure 14a.

[0245] The gold colloid-labeled anti-NP antibody introduced into the reaction chamber may also adsorb (non-specifically adsorb) to things other than antigens. When NaOH (a cleavage reagent) is added to this non-specifically adsorbed gold colloid-labeled anti-NP antibody, the part containing the gold colloid particles is separated from the adherend. This phenomenon occurs due to the addition of the cleavage reagent, so that non-detection target gold colloid particles are introduced into the tubular passage along with the detection target gold colloid particles (gold colloid particles that have formed a complex), and are unintentionally detected. However, by determining in advance the amount of non-specifically adsorbed gold colloid-labeled anti-NP antibody using the method described above, the obtained results can be corrected.

[0246] In addition, in Figures 13a and 14a, strong continuous signals are observed near both ends of the tubular passage in the width direction without changing over time. Since this signal is also observed in Figure 14a, it is difficult to assume that such signals are due to gold colloid particles, and it can be determined that such signals are noise caused by light refraction, scattering, distortion, etc. that occurs near the ends of the tubular passage. When such signals are measured, the areas near both ends of the tubular passage are excluded from the effective detection range. Furthermore, signals that are continuously detected at the same position (in Figures 13a and 14a, they are displayed as linear images because they are output with the same intensity along the time axis) are scattered light originating from something that is not moving through the tubular pathway, and can therefore be judged to be noise due to scratches or the like within the tubular pathway. Furthermore, when only colloidal gold particles were flowed through the tubular passage of the chip-type device to detect scattered light, and the shape of the signal indicating the scattered light of colloidal gold particles was confirmed from the obtained image data, it was found that the roughly circular minute spots in Figures 13a to 14b corresponded to the signal of colloidal gold particles. As a result, the anisotropic and slightly larger dot-like spots observed in Figure 14a have shapes different from the signal derived from colloidal gold particles, and therefore can be determined to be scattered light (noise) due to foreign matter. Therefore, these noise signals can be excluded from the detection results.

[0247] On the other hand, the intermittent occurrence of minute circular spots can be determined to be due to scattered light originating from dynamic gold colloid particles passing through the tubular passage. The spots corresponding to these gold colloid particles were counted in each of Figures 13a and 14a within the effective detection range excluding the vicinity of both ends of the tubular passage in the width direction.

[0248] The count number of the colloidal gold particles corresponding to the antigen was determined by subtracting the count number in Fig. 14a from the count number in Fig. 13a. The relationship between the count number (count(-blank)) and the antigen concentration when the immune complex was formed is shown in Fig. 15. The horizontal axis shows the antigen concentration in common logarithm, and the vertical axis shows the count number of scattered light corresponding to the antigen.

[0249] As can be seen from a comparison between FIG. 13a and FIG. 14a, the number of spots showing gold colloid particles displayed in FIG. 14a is much more than that in FIG. 13a. Furthermore, as shown in FIG. 15, the detected count number (count(-blank)) is a "positive value" in the thousands, which is clearly significantly different from the case where the antigen is not present. Therefore, it was shown that the immune complex fixed to the solid phase can be washed and cleaved with a cleavage reagent using the device, and a portion containing a labeled substance can be separated from the complex, and the separated portion containing the labeled substance can be moved inside the tubular passage by centrifugal force, and the labeled substance can be detected by the scattered light derived from the moving labeled substance. Furthermore, as shown in Figures 13b and 14b, the spots corresponding to the colloidal gold particles were clearly visible, indicating that the signals based on the scattered light of individual colloidal gold particles could be counted and quantitative evaluation of the colloidal gold particles could be achieved.

[0250] In particular, Figure 15 shows that there is a tendency for the number of counts of scattered light due to colloidal gold particles to correlate well with the antigen concentration when the antigen concentration is low. This suggests the possibility of more quantitative detection of colloidal gold particles corresponding to the antigen concentration, particularly when the antigen concentration in a sample is low.

[0251] To examine whether this method can detect antigens at lower concentrations than the conventional immunochromatography method, the above results were compared with those of the conventional immunochromatography method. For the conventional immunochromatography method, ImmunoAce SARS-CoV-2 II (Towns Co., Ltd.) was used, and the antigen detection results were evaluated visually. SARS-CoV-2NP adjusted to a specified concentration was used as the antigen. The results are shown in Table 3.

[0252] [Table 3]

[0253] In Table 3, the leftmost column shows the final concentration of the antigen used when forming the immune complex. The middle column shows the results of the conventional immunochromatography method. The rightmost column shows the results of the method of Example 8. "+" indicates that the antigen was detected by the immunochromatography method, and that the scattered light was counted by the method of this example. "ND" indicates that the antigen was not detected by the immunochromatography method, and that the scattered light was not counted by the method of this example. The lowest antigen concentration at which detection was confirmed was 0.5 pg / mL by the method of this example, while it was 50 pg / mL by the conventional immunochromatography method. From this, it can be said that the method of this example can detect lower concentrations of antibodies compared to the conventional immunochromatography method, and has superior sensitivity.

[0254] In the method of the present embodiment described above, the cleaning liquid was directly injected from the outside into the reaction chamber during the cleaning process. Alternatively, the cleaning liquid may be stored in the cleaning liquid storage chamber and the chip-type device may be rotated to supply the cleaning liquid from the cleaning liquid storage chamber to the reaction chamber. In a separate experiment, after the cleaning liquid was injected into the cleaning liquid storage chamber and the chip-type device was rotated at 2000 rpm for 1 minute, it was visually confirmed that the amount of cleaning liquid in the cleaning liquid storage chamber decreased and the amount of cleaning liquid in the reaction chamber increased. This shows that the cleaning liquid stored in the cleaning liquid storage chamber can be introduced into the reaction chamber by rotation and the cleaning process can be performed.

[0255] In addition, the chip-type device has a reagent storage chamber (cleavage reagent storage chamber) disposed on the rotation center side of the reaction chamber, and connected to the reaction chamber by a flow path. This reagent storage chamber is connected to the reaction chamber and the tubular path by a flow path similar to that of the washing liquid storage chamber, so from the above experiment of transferring the washing liquid by rotation, if the cleavage reagent is also stored in the reagent storage chamber, it can be said that by rotating the chip-type device, the cleavage reagent can be supplied to the reaction chamber by the centrifugal force generated, just like the washing liquid, and the cleavage process can be performed in the reaction chamber. [Industrial Applicability]

[0256] According to the present invention, a target substance can be detected with high sensitivity and at low cost, and therefore the present invention can be used as a substance detection method and device in various industrial fields as well as the medical field. [Explanation of symbols]

[0257] 1 Disc 2 units 10 Reaction chamber 11 Tubular tract 12 Drainage Reservoir 13 Cleaning fluid storage chamber 14 Cleavage Reagent Storage Room

Claims

1. A device rotatably formed around a rotation center for detecting a portion containing a labeled substance separated from a composite formed by sandwiching a target substance between a first capture substance bound to a labeled substance and a second capture substance fixed to a solid phase, The separated portion containing the labeling substance is provided with a tubular passage for moving it through the filled liquid by centrifugal force. The labeling substance is a substance with a higher specific gravity than the liquid. The device wherein at least a portion of the tubular passage transmits scattered light that scatters the light irradiated onto the portion containing the labeling material.

2. A first storage chamber for storing cleaning fluid, a chamber for holding the composite so as to prevent the composite from flowing into the tubular passage, and a second storage chamber for storing liquid flowing out from the chamber are arranged in this order from the rotation center side of the device outward, the first storage chamber and the chamber are connected by a flow path, and the chamber and the second storage chamber are connected by the tubular passage, By rotating the device, the cleaning solution is supplied from the first storage chamber to the chamber, and then discharged to the second storage chamber via the tubular passage to perform the cleaning process. The device according to claim 1.

3. The device according to claim 2, wherein the flow path connecting the first storage chamber and the chamber is openable and closable.

4. The device according to claim 2, wherein the flow path connecting the first storage chamber and the chamber is openable.

5. The device according to any one of claims 2 to 4, wherein the amount of cleaning fluid stored in the first storage chamber is sufficient to fill the tubular passage.

6. A third storage chamber is located on the rotational side of the chamber of the device, storing a liquid containing a reagent for separating the portion containing the labeled substance from the composite, and the third storage chamber and the chamber are connected by a flow path. The device according to claim 2, wherein, after the cleaning process, a liquid containing the reagent is supplied to the chamber by rotating the device, the portion containing the labeling substance is separated, and the separated portion containing the labeling substance is moved into the tubular passage.

7. The device according to claim 6, wherein the flow path connecting the third storage chamber and the chamber is openable.

8. The device according to claim 1, wherein the labeling substance is a fine particle whose main component is one selected from the group consisting of metal, ceramics, glass, and resin.

9. The device according to claim 8, wherein the labeling substance is a metal colloid particle.

10. The device according to claim 1, wherein the solid phase is at least one granular body selected from the group consisting of glass particles, ceramic particles, magnetic particles, and resin particles.

11. The device according to claim 1, wherein the solid phase is an internal structure of a container used when forming the composite.

12. The device according to claim 1, wherein the separation of the portion containing the labeling substance from the composite is performed by at least one treatment selected from the group consisting of heat treatment, pH adjustment treatment, denaturation treatment, oxidation treatment, reduction treatment, enzymatic treatment and competitive reaction treatment.

13. The device according to claim 1, wherein the first capture substance is at least one selected from the group consisting of antibodies, antibody fragments, modified antibodies, antigens, aptamers, and nucleic acids that specifically bind to a target substance.

14. The device according to claim 1, wherein the second capture substance is at least one selected from the group consisting of antibodies, antibody fragments, modified antibodies, antigens, aptamers, and nucleic acids that specifically bind to a target substance.

15. A target substance detection device comprising the device described in Claim 1, a rotating means for rotating the device, an irradiating means for irradiating light into the tubular passage, and a detection means for detecting the scattered light.

16. The detection device according to claim 15, wherein the detection means detects scattered light that is intermittently generated from the tubular passage within a predetermined time.

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