Method and reagent for detecting target substance

Abstract

[Problem] To provide a technique with which it is possible to detect a target substance in a sample with high sensitivity and at low cost compared with conventional techniques. [Solution] A method for detecting a target substance, the method including forming a complex by sandwiching the target substance between a first capture substance immobilized in a solid phase and a second capture substance labeled with a marker in a first reaction field, separating a portion including the marker from the complex, and detecting the target substance in a second reaction field. The liquid is filled in the tubular channel by a centrifugal force, and the marker-containing portion is detected by scattered light scattered by the marker-containing portion.

Description

Technical Field

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

[0002] It has always been known that immunoassay methods are used to detect specific antigens or antibodies related to diseases as biomarkers to discover diseases or qualitatively or quantitatively analyze treatment effects. In recent years, in order to detect biomarkers more effectively, the demand for high-sensitivity detection of trace biomarkers has increased. Enzyme-linked immunosorbent assay (ELISA) (also referred to as "ELISA method" in this manual), which is one of the immunoassay methods, is a method that uses specific antibodies, antigens bound to target antibodies, and complementary nucleic acids to capture target antigens, antibodies or nucleic acids contained in a sample solution and detects them using enzyme reactions. It has been widely popularized due to advantages such as cost.

[0003] ELISA is a method for quantitatively detecting target substances. Currently, the most popular ELISA is performed on a 96-well microplate, which has many steps and requires complicated operations. In addition, since it undergoes multiple reaction steps, it requires a lot of labor and time to obtain the measurement results. Therefore, shortening the operation time and reaction time is being sought. Further high-sensitivity detection is also sought.

[0004] In order to achieve high-sensitivity detection, it is thought to improve the reactivity of enzymes and substrates. Therefore, a technology (patent document 1) is proposed, for example, to free an immune complex (labeled compound) to which a reporter substance is added from a solid phase and react it in a free liquid medium. In this patent document 1, the constituent elements of the immune complex are designed in a manner that biotin or functionalized azo dyes and avidins are introduced into the immune complex. For example, as a labeled antibody, an antibody that is bound to a reporter group by the combination of streptavidin-biotin is prepared. When the immune complex is formed on the solid phase, the reporter group is introduced into the immune complex by the combination of streptavidin-biotin. If an excess of streptavidin is added afterwards, the streptavidin in the complex is replaced by the added streptavidin, and the reporter molecule in the immune complex is freed. Therefore, in this technology, the reporter molecule can be detected in a free liquid medium and is associated with the concentration of the subject in the subject to be tested.

[0005] In addition, in order to detect immune complexes with high sensitivity, microreactors that react in microcontainers at the μm level have been developed using microfabrication technology. Using a microreactor with a reaction field as a microspace is expected to increase the reaction rate. The method of using tiny flow paths and reaction chambers to implement experimental operations such as extraction and separation on a microreactor is also called Lab on a chip. In addition, a technology has been proposed to form a microreactor on a disc-shaped disk and use the centrifugal force generated by rotation to distribute sample liquid and separate biological materials (Patent Document 2). In addition, the following technology has been proposed: a plurality of chambers, flow paths, storage tanks, etc. are formed on a disc-shaped disk, and the reagent is delivered and discharged by rotating the disk, and the immunoassay step performed manually in the hole is implemented in the microreactor (Patent Document 3).

[0006] In the immunoassay in the microreactor, a method using labeled beads is proposed to detect low concentrations of target substances (Patent Document 4). For example, Patent Document 4 discloses a disc-shaped disk, the surface of which is provided with a bead filling portion filled with antibody-modified labeled beads and a detection area. It is also disclosed that: the antibodies on the labeled beads are used to capture the antigens in the sample liquid injected into the disk, and the labeled bead complex bound to the antigen on the labeled beads is sent into the detection area by the rotation of the disk and captured by the antibodies fixed in the detection area; the number of labeled bead complexes in a state of being fixed in the detection area is counted by the optical reading means of the optical disk device, thereby detecting the target substance in the sample.

[0007] On the other hand, in the analysis method using a disk, the following method is proposed: antigen-loaded beads, fluorescent IgG antibody labeling reagent and sample are mixed in a reaction field set on the disk, and the disk is rotated after binding with the target substance to form a complex, and the beads are introduced from the reaction field into a separation chamber filled with a density gradient medium by the centrifugal force generated, and the complex is detected by irradiating an electromagnetic irradiation beam. The separation chamber is filled with a density gradient medium, and the introduced beads move to an isopycnic point in the density gradient medium due to the centrifugal force and remain stationary there. In addition, the target immune complex can be detected by detecting the fluorescence from the isopycnic point. According to this analysis method, it is possible to separate the excess fluorescent markers that are not bound to the immune complex different from the isopycnic point of the beads from the target immune complex, and the immune complex can be detected without performing a washing operation (Patent Document 5).

[0008] Prior art literature

[0009] Patent Literature

[0010] Patent Document 1: Japanese Patent Publication No. 6-505802

[0011] Patent Document 2: Japanese Patent Application Publication No. 2008-185423

[0012] Patent Document 3: Japanese Patent Application No. 2002-503331

[0013] Patent Document 4: Japanese Patent Application Publication No. 2012-255772

[0014] Patent Document 5: Japanese Patent Application No. 2006-505766 Summary of the invention

[0015] Problem that the invention aims to solve

[0016] However, the technology described in Patent Document 1 only discloses that a reporter molecule as a label is released into a free liquid medium and reacts with a substrate in the liquid, which is insufficient to achieve improved detection sensitivity and improved reactivity such as enabling detection of a target substance at a low concentration.

[0017] In patent document 2, there is no disclosure on the point of performing immunoassay with high sensitivity. Patent document 3 only discloses that the process of the previous ELISA method is installed in a microreactor. In addition, the microreactor can improve the reaction speed and reactivity by reducing the reaction field on the one hand, but there is a problem that the signal intensity decreases due to the decrease in the reaction volume on the other hand. Therefore, if you want to implement the high-sensitivity measurement of immune complexes by the technology recorded in patent documents 2 and 3, you need a highly sensitive detector and camera, which has the problem of increased cost.

[0018] Patent document 4 discloses a technique of implementing immunoassay by setting the detection object as a marker bead and using a general optical disk device. Usually, the transportation of granular matter in a microreactor is also a cause of blockage, but in the technique of patent document 4, antibodies are fixed in the detection region, and the marker bead loaded with antigens that is captured and flows into the detection region is captured, so local accumulation of the marker bead is likely to occur, and sometimes there is a problem that practical operation becomes difficult. In addition, there is the following problem: when the marker beads captured in the detection region are close to each other, detection errors may be caused.

[0019] Moreover, the technology described in patent document 4 is to make the label beads flow down while the fixed antibody and the antigen loaded by the label beads react with each other, so the detection area as a whole becomes the reaction field of the antigen-antibody reaction. Reactivity is affected by the contact frequency of antigen and antibody, so the antigen-antibody reaction efficiency in the technology where the entire detection area becomes the reaction field is easy to decrease. In addition, since the capture of the label beads and its detection (that is, the detection of the antigen) are carried out in the same reaction field, the detection sensitivity will decrease when the reaction efficiency is low. And then, the speed of the antibody flowing down may cause the fixed antibody to be difficult to capture the label beads, so there is a problem of further deterioration of the detection sensitivity and the detection speed.

[0020] In addition, the technique described in Patent Document 5 is to aggregate (localize) the beads loaded with the complex at isopycnic points in the density gradient medium for detection. Therefore, the smaller the amount of aggregation, the more complicated the signal detection is, and it is difficult to detect the target substance in the sample with higher sensitivity.

[0021] The present invention has been made in view of the above-mentioned problems, and one object of the present invention is to provide a technology capable of detecting a target substance in a sample with high sensitivity and at low cost compared with conventional technologies.

[0022] Solutions for solving problems

[0023] According to one aspect thereof, the present invention provides a method for detecting a target substance, comprising:

[0024] The target substance is sandwiched between a first capture substance bound to a label and a second capture substance immobilized on a solid phase to form a complex, and a portion containing the label is separated from the formed complex.

[0025] The separated portion containing the marker moves in the liquid filled in the tubular channel by centrifugal force.

[0026] The portion containing the labeled substance is detected by the scattered light generated by scattering the light irradiated into the tubular channel by the portion containing the labeled substance.

[0027] According to a preferred embodiment of the present invention,

[0028] Before separating the portion containing the labeling substance from the complex, a washing treatment is performed to wash the complex in a state where the inflow of the complex into the tubular channel is suppressed.

[0029] According to a preferred embodiment of the present invention, when the cleaning process is performed, the tubular channel is filled with a cleaning liquid for cleaning.

[0030] According to a preferred embodiment of the present invention, the cleaning liquid used in the cleaning process is stored downstream of the tubular passage.

[0031] According to a preferred embodiment of the present invention, the cleaning process is performed in a state where the inflow of the complex into the tubular channel is suppressed by holding the complex in a predetermined chamber.

[0032] According to other preferred embodiments of the present invention, in the method of the present invention,

[0033] In the device formed in a rotatable manner, a first storage chamber storing the cleaning liquid, the chamber, and a second storage chamber storing the liquid flowing out of the chamber are sequentially arranged from the rotation center side of the device toward the outside, the first storage chamber and the chamber are connected by a flow path that can be opened and closed freely, and the chamber and the second storage chamber are connected by the tubular channel.

[0034] The method of the present invention includes: rotating the device to deliver the cleaning liquid from the first storage chamber into the chamber and then discharging the cleaning liquid into the second storage chamber through the tubular passage to perform the cleaning process.

[0035] According to another preferred embodiment of the present invention, the first storage chamber and the cavity are communicated with each other via an openable flow path.

[0036] According to another preferred embodiment of the present invention, the cleaning process is performed until the second storage chamber and the tubular channel are filled with the cleaning liquid.

[0037] According to other preferred embodiments of the present invention, in the method of the present invention,

[0038] A third storage chamber is arranged on the rotation center side of the chamber in the device, the third storage chamber stores a liquid containing a reagent for separating the portion containing the label from the complex, and the third storage chamber is connected to the chamber via a flow path that can be opened and closed freely.

[0039] The method of the present invention comprises: rotating the device, introducing the liquid containing the reagent into the chamber after the cleaning process to separate the portion containing the marker, and moving the separated portion containing the marker in the tubular channel.

[0040] According to another preferred embodiment of the present invention, the third storage chamber and the cavity are communicated with each other via an openable flow path.

[0041] According to another preferred embodiment of the present invention, scattered light intermittently generated from the tubular passage within a predetermined time is detected.

[0042] According to another preferred embodiment of the present invention, the marker is a substance having a specific gravity greater than that of the liquid filling the tubular channel.

[0043] According to another preferred embodiment of the present invention, the labeling substance is a microparticle having one selected from the group consisting of metal, ceramic, glass and resin as a main constituent component.

[0044] According to another preferred embodiment of the present invention, the marker is metal colloidal particles.

[0045] According to another preferred embodiment of the present invention, the solid phase is at least one kind of granular body selected from the group consisting of glass particles, ceramic particles, magnetic particles, and resin particles.

[0046] According to another preferred embodiment of the present invention, the solid phase is a structure inside a container used to form the composite.

[0047] According to other preferred embodiments of the present invention, the separation of the above-mentioned portion containing the marker from the above-mentioned complex is carried out by at least one treatment selected from the group consisting of heating treatment, pH adjustment treatment, denaturation treatment, oxidation treatment, reduction treatment, enzyme treatment and competitive reaction treatment.

[0048] According to another preferred embodiment of the present invention, 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 the target substance.

[0049] According to another preferred embodiment of the present invention, 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 the target substance.

[0050] According to another aspect of the present invention, there is provided a reagent used in the method for detecting a target substance of the present invention, the reagent having a function of separating the portion containing the label from the complex.

[0051] According to another preferred embodiment of the present invention, the reagent of the present invention comprises at least one selected from the group consisting of a pH adjuster, a denaturant, a reducing agent, an oxidizing agent, an enzyme, and a competitive agent.

[0052] According to another preferred embodiment of the present invention, the reagent of the present invention comprises a pH adjuster.

[0053] Effects of the Invention

[0054] According to the method for detecting the target substance of the present invention, the above-mentioned portion containing the marker (also referred to as "fragments containing the marker" in this specification) separated from the above-mentioned complex is moved in the liquid filled in the tubular channel by centrifugal force, and the fragments containing the marker are detected by the scattered light generated by the reflection and scattering of the light irradiated into the tubular channel by the fragments containing the marker. Generally, the smaller the signal generating source (substance generating optical signal, marker) in the detection object, the more complicated technology is required when detecting the detection object by absorbance change as signal. However, the method of the present invention obtains scattered light in the form of optical signal, and therefore can fully detect tiny detection objects such as the above-mentioned fragments containing the marker.

[0055] Here, when using scattered light to detect the detection object, substances other than the detection object will also generate scattered light, so the problem of not being able to specifically detect the detection object often arises. However, in the method of the present invention, the fragments containing the marker in the liquid are moved at an inherent moving speed by the action of centrifugal force, so the detection object, i.e., the fragments containing the marker, can be specifically detected. Furthermore, in the method of the present invention, the fragments containing the marker are moved in the liquid filled in the tubular channel by the action of centrifugal force, so they are moved at a slower speed than when they are transferred with the flow of the liquid. Therefore, it can be said that according to the method of the present invention, in the process of the fragments containing the marker moving in the tubular channel by the centrifugal force, the scattered light from the fragments containing the marker can be easily obtained.

[0056] Furthermore, according to the method of the present invention, for example, when beads are used as a solid phase, even if multiple target substances are loaded on one bead, the number of fragments containing the marker separated from the beads can be detected (counted) one by one because the fragments are not counted as one per bead unit, thereby achieving the effect of being able to detect the target substance with high sensitivity.

[0057] According to the reagent of the present invention, the portion containing the label in the complex, that is, the fragment containing the label can be separated well, and thus a reagent suitable for detecting a target substance can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 This is a diagram for explaining the outline of one embodiment of the detection method of the present invention.

[0059] Figure 2a It is a schematic plan view showing an example of a disk suitable for implementing the detection method of the first embodiment.

[0060] Figure 2b for Figure 2a A partial enlarged view of .

[0061] Figure 3 It is a schematic plan view showing another example of a disk suitable for implementing the detection method of the first embodiment.

[0062] Figure 4 This is a schematic diagram showing an example of a detection device.

[0063] Figure 5 This is a graph showing the relationship between the pH value of each reagent in Example 1 and the shutdown characteristics.

[0064] Figure 6 This is a graph showing the relationship between the NaOH concentration (pH value) and the shutdown characteristics in Example 2.

[0065] Figure 7This is a graph showing the cutting characteristics of various denaturants in Example 3.

[0066] Figure 8 This is a graph showing the change in the gold colloid particle size caused by the sensitization treatment in Example 4.

[0067] Fig. 9 This is a graph showing the relationship between the particle size of metal colloid particles and the scattered light intensity in Example 5.

[0068] Fig.10 This is a graph showing the relationship between the concentration of gold colloid particles (the portion including gold colloid particles) dissolved in Example 6 (corresponding to the antigen concentration) and the scattered light intensity.

[0069] Fig.11 This is a diagram illustrating the selective outflow of gold colloid particles (the portion including gold colloid particles) in Example 7.

[0070] Fig.12 This is a diagram showing the measurement results of gold colloid particles (part including gold colloid particles) in Example 7.

[0071] Fig.13a This is a diagram showing the results of detecting scattered light of gold colloid particles (a portion including gold colloid particles) in Example 8 as voltage values.

[0072] Fig.13b for Fig.13a A partial enlarged view of .

[0073] Fig.14a This is a graph showing the results of detecting scattered light of gold colloid particles (a portion including gold colloid particles) in the form of voltage values ​​when no antigen was used in immune complex formation in Example 8 (blank).

[0074] Fig.14b for Fig.14a A partial enlarged view of .

[0075] Fig.15 This is a graph showing the relationship between the count value of spots measured in Example 8 and the antigen concentration. DETAILED DESCRIPTION

[0076] <Detection method of the present invention>

[0077] In the detection method of the present invention, the portion containing the marker separated from the complex is moved in the liquid filled in the tubular channel by centrifugal force, so that the portion containing the marker is intermittently supplied to the liquid filled in the tubular channel. And the portion containing the marker is detected by the scattered light generated by the irradiation light irradiated to the tubular channel being scattered by the portion containing the marker. The complex is formed by sandwiching the target substance between the first capture substance bound to the marker and the second capture substance fixed to the solid phase. Therefore, by detecting the portion containing the marker separated from the complex, the target substance can be detected.

[0078] It should be noted that, in this specification, when the elements are connected by hyphens such as "solid phase-second capture substance-target substance-first capture substance-label", it means that they are physically or chemically bound to each other. In addition, "first capture substance-label" is sometimes written as "first capture substance bound to a label" or simply as "first capture substance". "Outside", "outside direction" and "outward" refer to the centrifugal direction. "Main constituent" refers to the constituent with the largest content, for example, it refers to a component with a content exceeding 50% by mass.

[0079] The following uses Figure 1 One embodiment of the detection method of the present invention is described. Figure 1 This is a schematic diagram showing the concept of an embodiment of the detection method of the present invention. It should be noted that, in order to simplify the description, Figure 1 , shows the case where the target substance is an antigen and the first and second capture substances are primary antibodies.

[0080] In the initial state, a solid phase (step 1, step 2) on which a second capture substance for capturing a target substance is immobilized is prepared in a reaction field (first reaction field, space for performing a reaction). Figure 1 (a)). When a sample solution containing a target substance is introduced therein ( Figure 1 (b)), the target substance in the sample solution can be specifically captured by the second capture substance (step 2). After removing substances other than the captured target substance from the reaction field by washing treatment as needed, the first capture substance bound to the marker is introduced into the reaction field, and the first capture substance binds to the target substance to form an immune complex (hereinafter also referred to as "immune complex") on the solid phase in which the target substance is sandwiched between the second capture substance and the first capture substance (step 3, Figure 1(c)). Next, a cleaning treatment is preferably performed. It is preferred that the cleaning treatment removes substances that may affect the detection results once mixed into the tubular channel out of the system in advance. Such substances include, for example, the redundant first capture substance and free marker that do not form a complex. In addition, other inclusions may also be removed by the cleaning treatment. Thus, a state is formed in which the solid phase is loaded with a marker corresponding to the amount of the target substance captured by the solid phase.

[0081] Then, the formed complex is subjected to reagents such as enzymes, redox agents, denaturants, acids, bases, or external fields such as heat, light (electromagnetic waves), and vibrations ( Figure 1 (d)), thereby separating the portion containing the marker from the solid phase (step 4, Figure 1 (e)). It should be noted that the separated part (the part containing the marker) may be only the marker, or may be a state where certain molecular clusters are bound to the marker. The molecular clusters are not limited to the complex of the second capture substance-target substance-first capture substance and the complex of the target substance-first capture substance, the original molecular clusters such as the first capture substance remaining as they are (i.e., generated by the severance of intermolecular bonds), and may also be fragmented by the severance of intramolecular bonds within the original molecular clusters.

[0082] The reaction field is a place where a reaction is carried out by sandwiching the target substance between the first capture substance bound to the marker and the second capture substance fixed to the solid phase to form a complex, and a reaction is carried out by separating the part containing the marker from the formed complex, and refers to a separated space. Examples of the reaction field include a reaction container, a reaction chamber (chamber), etc. It should be noted that in this method, the above Figure 1 As long as the portion containing the marker can be moved in the tubular channel by centrifugal force, steps 1 to 4 shown in (a) to (e) can be performed in different reaction fields, or multiple consecutive steps can be performed in the same reaction field. For example, steps 1 to 3 can be performed in one container and step 4 can be performed in another container.

[0083] It should be noted that, in the present embodiment, a reaction chamber provided in a device configured in a rotatable manner can be used as a reaction field. The reaction chamber can be appropriately selected and used starting from any stage in steps 1 to 4 or in a properly implemented cleaning process. For example, a solid phase having a second capture substance fixed thereto can be loaded into the reaction chamber of the above-mentioned device and all of steps 1 to 4 can be implemented using the reaction chamber, or steps 1 to 3 can be implemented in a container outside the above-mentioned device and step 4 and subsequent steps can be implemented in the reaction chamber of the above-mentioned device. In the latter case, regardless of whether the product of step 4 is subjected to solid-liquid separation, the solution containing the portion containing the marker is introduced into the reaction chamber, in which case the reaction chamber is used only as an antechamber for supplying the solution to the detection field, i.e., the small-diameter tubular channel.

[0084] After step 4, the portion containing the marker contained in the solution is moved to the detection field, that is, the liquid filled in the small-diameter tubular channel, by centrifugal force ( Figure 1 (f)). Figure 1 In the example shown in (f), a tubular channel with a small diameter is continuously arranged from the reaction chamber to the outside direction (right side of the figure) of the rotation center. Therefore, if the part containing the marker is stored in the reaction chamber of the device in a state contained in the solution, the part containing the marker can enter the liquid filled in the tubular channel with a small diameter by the centrifugal force generated when the device is rotated, and move further outward in the liquid. In addition, in the example shown in the figure, the aperture of the tubular channel with a small diameter is set to a size that the part containing the marker can pass through but the solid phase cannot pass through, so the inflow of the solid phase can be prevented and the part containing the marker can enter the tubular channel. Here, the part containing the marker moves in the liquid filled in the tubular channel by centrifugal force. This movement is based on so-called centrifugal sedimentation. That is, the moving speed of the part containing the marker is relatively greater than the moving speed of the liquid filled in the tubular channel. In the present invention, the liquid filled in the tubular channel hardly moves due to the centrifugal force, and the moving speed is usually almost zero, and in principle, as long as it does not substantially affect the movement of the part containing the target substance, a slight movement is also acceptable.

[0085] The other end of the tubular passage is connected to the second storage chamber ( Figure 1(not shown in the figure). The tubular channel and the second storage chamber can always be connected, or can be freely opened and closed by a valve. As the second storage chamber, it can be arranged at a downstream side of the above-mentioned chamber in the above-mentioned device, for example, it is preferably arranged at a position closer to the outside than the above-mentioned chamber. Before the part containing the marker is moved in the tubular channel, the tubular channel is in a state filled with liquid. When the second storage chamber is arranged at a position closer to the outside than the above-mentioned chamber, the tubular channel and the second storage chamber are both filled with liquid when the tubular channel and the second storage chamber are always connected. The timing of filling the tubular channel with liquid varies depending on the timing of using the reaction chamber. For example, when the reaction chamber is used from before the above-mentioned step 3, the tubular channel is filled with liquid during the cleaning treatment after step 3 and thereafter and before step 4. In addition, when the reaction chamber is used in step 4 and thereafter, it is suitable to fill the tubular channel with liquid before introducing the solution containing the part containing the marker into the reaction chamber. It should be noted that the structure may also be such that: the reaction chamber is provided with a valve, and after the tubular channel is filled with liquid, the valve is opened to introduce the portion containing the marker into the liquid filled in the tubular channel. In this case, the timing of introducing the solution containing the portion containing the marker into the reaction chamber may be before or after the tubular channel is filled with liquid.

[0086] The detection field is a place where scattered light from the tubular channel is detected, and is equipped with a light source and a light receiving element. It is a place where optical changes such as scattered light based on the irradiated light occur (hereinafter sometimes appropriately referred to as the "second reaction field"). By forming at least a portion of the tubular channel irradiated with irradiated light with a light-transmitting material, the irradiated light irradiated from the light source to the tubular channel passes through the wall of the tubular channel and is scattered by the marker moving in the tubular channel to generate scattered light. By detecting the scattered light with the light receiving element, it is possible to detect whether the marker exists ( Figure 1 (g)).

[0087] The moving speed of particles in a liquid in a centrifugal field can be described by Stokes' law. Specifically, when a particle with a mass of m rotates with a rotation radius r1 and an angular velocity ω, it is subjected to a centrifugal force f as shown in the following equation (1).

[0088] f=mr1ω 2 …………(1)

[0089] m: mass of the particle (g)

[0090] r1: Radius of rotation (cm)

[0091] ω: angular velocity of rotation (rad / s)

[0092] In addition, if the radius of the particle is r2 and the particle density is ρ p, the liquid density is ρ1, then the centrifugal force Fc acting on the particle is as shown in the following formula (2).

[0093]

[0094] According to Stokes' drag law, the drag Fr experienced by a sphere with a radius of r2 moving at a speed vs in a liquid with a viscosity of η is expressed as follows (3).

[0095] F r =6πηr2v s ……(3)

[0096] Fc and Fr reach equilibrium at the final velocity of the particle, so as shown in the following equation (4), the particle radius r2 in equation (4) is replaced by the diameter D p The moving speed of the particle v s As shown in the following formula (5).

[0097]

[0098]

[0099] That is, when centrifugal force acts, the particle diameter (Dp: cm), particle density (ρp: g·cm -3 ), liquid density (ρ1: g·cm -3 ), solution viscosity (η: poise; g·cm -1 ·s -1 ) are related to each other and determine the particle movement speed (v s : cm / sec). In other words, in the same liquid, particles with higher density and larger particle size will move faster in the liquid. For particles with known density and particle size, the moving speed and the time required to move to a specified position can be estimated based on formula (5).

[0100] The particle size, density and viscosity of the solution of the marker can also be known in advance. In addition, for the portion containing the marker, when the marker is large enough or heavy enough compared to the portion other than the marker, the portion containing the marker can be approximated by the marker. Thus, the moving speed of the portion containing the marker and the time to reach the specified position (detection area) of the tubular channel can be derived theoretically or experimentally based on formula (5). It should be noted that formula (5) is a theoretical formula, so when it deviates from the premise of formula (5), the moving speed can be calculated on the basis of correcting formula (5).

[0101] As described above, the time when the portion containing the marker passes through the detection area can be estimated based on the derived moving speed. In addition, the portion containing the marker passes through the detection area within a specified time period from the start of centrifugation. Therefore, the scattered light detected in the detection area within the specified time period can be determined as scattered light originating from the target detection object.

[0102] Furthermore, since the object to be detected moves in the tubular passage, its scattered light is an intermittent signal. Therefore, the scattered light that is continuously generated can be identified as background noise generated for some reason, thereby enabling the scattered light of the object to be detected specifically.

[0103] Therefore, the detection method of the present invention can appropriately detect a tiny detection target. The marker (the portion containing the marker) provided to the tubular channel originates from a complex formed with the target substance in the reaction field, so the presence of the target substance can be detected by detecting the marker.

[0104] In addition, the number of scattered light generated intermittently within the specified time can be counted. Based on the count, the number of parts containing the marker provided to the tubular channel, that is, the number of target substances, can be quantitatively evaluated. In particular, when the number of target substances in the sample is small, that is, in microanalysis, the quantitative performance is excellent.

[0105] Furthermore, in the detection method of the present invention, a portion containing a marker is separated from a solid phase and the separated portion containing a marker is introduced into a detection field. Therefore, compared with a previously proposed scheme of using a disk-like microreactor and counting beads that form an immune complex and performing an immunoassay, the size of the substance passing through the flow path can be greatly reduced, and the detection object can be smoothly and easily introduced into the detection field.

[0106] <Target substance>

[0107] In the determination method of the present invention, the target substance as the object of analysis is combined with a specific biomolecule and can be combined with a specific biomolecule through a biochemical reaction. For example, it can be detected by an immunological method or a genetic method, without particular limitation. As the target substance, specifically, various compounds such as pathogens such as bacteria and viruses, proteins, peptides, DNA, RNA, nucleic acids such as aptamers, exosomes, sugar chains, etc. can be exemplified. Furthermore, physiologically active substances such as hormones, their agonists, antagonists, alkaloids, etc. can be exemplified.

[0108] <First captured substance>

[0109] The first capture substance of the present invention binds to the target substance captured by the second capture substance, and sandwiches the target substance between the second capture substance fixed on the solid phase, forming a complex fixed on the solid phase, that is, a complex bound in the order of solid phase-second capture substance-target substance-first capture substance (first capture substance bound to a marker). When the target substance is an antigen, the first capture substance uses an antibody that specifically reacts with the target substance. On the other hand, when the target substance is an antibody, as the first capture substance, an antigen to which the antibody can specifically bind can be used, and an antibody that binds to the antibody and is different from the second capture substance can also be used, and an antibody produced by a host different from the host that produces the antibody can also be used. Therefore, when the target substance is an antibody, a complex of solid phase-antigen (second capture substance)-target substance-antigen (first capture substance) or antibody (first capture substance) and a complex of solid phase-antibody (second capture substance)-target substance-antigen (first capture substance) or antibody (first capture substance) can be formed.

[0110] Furthermore, as the first capture substance of the present invention, in addition to the above-mentioned antigens and antibodies, nucleic acids, aptamers, any one of biotin-avidin, any one of ligand-receptors, etc. can also be used according to the target substance. For example, when the target substance is DNA derived from bacteria, a nucleic acid molecule having a sequence complementary to a part of the DNA region can be used as the first capture substance. In addition, when the target substance is a sequence-specific DNA binding protein such as a transcription factor, a DNA having a specific sequence can be used as the first capture substance.

[0111] The antibody used as the first capture substance may be a polyclonal antibody or a monoclonal antibody, but is preferably a monoclonal antibody from the viewpoint of reaction specificity.

[0112] The antibody used as the first capture substance also includes antibodies and antibody fragments and modified antibodies having substantially the same reactivity as the antibody. Examples of antibody fragments include Fab fragments, F(ab')2 fragments, Fab' fragments, and scFv fragments.

[0113] <Second captured substance>

[0114] The second capturing substance of the present invention is a substance having the function of capturing the target substance on a solid phase, and is fixed on a solid phase for use. For example, when the target substance is an antigen, an antibody that specifically reacts with the target substance is used; when the target substance is an antibody, an antigen or antibody that can specifically react with the antibody is used.

[0115] 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, antagonists of target substances), and receptors can also be used according to the target substance. For example, when the target substance is DNA of bacterial origin, a nucleic acid molecule having a sequence complementary to a part of the DNA region 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, a DNA having a specific sequence can be used as the second capture substance.

[0116] The antibody used as the second capture substance may be a polyclonal antibody or a monoclonal antibody, but a monoclonal antibody is preferred from the viewpoint of reaction specificity.

[0117] The antibody used as the second capture substance also includes antibodies and antibody fragments and modified antibodies having substantially the same reactivity as the antibody. Examples of antibody fragments include Fab fragments, F(ab')2 fragments, Fab' fragments, and scFv fragments.

[0118] Here, if the size of the solid phase is compared with the size of the second capture substance, the solid phase is obviously larger. Therefore, it is easy to fix multiple second capture substances on the solid phase. In the previous method of detecting the target substance by capturing the target substance on a solid phase such as beads and counting the beads that captured the target substance, if multiple target substances are captured on one bead, it is likely to have a significant impact on the detection result. However, in the present embodiment, the detection object is the part containing the marker separated from the complex that is not constrained by the beads, so high-sensitivity detection can be achieved.

[0119] It should be noted that when the steric hindrance of the label significantly affects the first capture substance's access to the target substance captured on the solid phase, the concentration of the second capture substance can be appropriately adjusted when binding the second capture substance to the solid phase so that the second capture substance is sparsely distributed on the solid phase, i.e., in a sparse and dense phase.

[0120] <Marker>

[0121] The marker is a substance that marks the first capture substance, and there is no particular limitation as long as scattered light can be generated. For example, microparticles with inorganic substances such as glass, ceramics, metals, fluorescent substances or resins as the main components can be cited. As resin particles, particles of synthetic resins represented by latexes such as synthetic latexes such as polystyrene latex, natural rubber latex, polystyrene, etc. can be exemplified. In addition, the marker can be composed of one material or a variety of materials. Furthermore, it can be composed of inorganic-organic heterogeneous materials. As a combination of heterogeneous materials, it can be, for example, composite particles formed by loading metal particles on resin particles, ceramic particles, and composite particles formed by coating metal on resin particles. In addition, the resin particles can be loaded with fluorescent substances, but are not limited to this.

[0122] The particle size of these markers is preferably 80 nm to 3 μm from the perspective of scattered light intensity and mobility in the device. It should be noted that since scattered light intensity, mobility in the centrifugal field, reactivity with the first capture substance, etc. vary depending on the type and size of the marker, an appropriate particle size can be selected in consideration of these.

[0123] Here, considering the help of centrifugal force on the moving speed, as a marker, a substance with a specific gravity greater than the specific gravity of the liquid filling the tubular channel is preferred. Inorganic particles with a higher density than organic matter and resin particles with a relatively large particle size are suitable as markers. This is because the moving speed of the part containing the marker can be significantly different from the moving speed of the organic matter as the main inclusion in the sample in the detection field. Among them, metal particles and metal colloid particles that are expected to obtain good scattered light intensity are particularly suitable as markers.

[0124] As the metal colloid particles that can be used as a label, various metal colloid particles can be appropriately selected and used, and metal colloids such as gold colloid, silver colloid, platinum colloid, and their composite metal colloid particles can be listed. As the composite metal colloid, for example, gold colloid loaded with platinum, palladium colloid loaded with platinum, etc. can be listed.

[0125] Preferred metal colloid particles in the present invention include gold colloid particles, composite metal colloid particles thereof, and silver colloid particles, and gold colloid particles are more preferred.

[0126] The particle size of the metal colloid particles is usually 1nm to 500nm. The particle size of the metal colloid particles suitable for detection depends on the sensitivity, accuracy, measurement conditions, etc. of the selected optical detection system. For example, when a simple detection system that combines a laser light source and a light emitting diode to detect scattered light is used, the particle size of the metal colloid particles is 80nm or more, more preferably 150nm or more, and if it is 200nm or more, it can be more easily detected. Preferably, it is 80nm to 500nm, more preferably 100nm to 500nm, more preferably 120nm to 300nm, and particularly preferably 180nm to 250nm.

[0127] It should be noted that the case where the first capture substance is an antibody includes the case where the first capture substance, namely the antibody, is labeled and the case where an antibody that recognizes the antibody (eg, a secondary antibody) is labeled.

[0128] The method for binding the labeling substance to the first capture substance is not limited, and binding methods such as physical adsorption binding, chemical binding, affinity binding, and combinations thereof can be used. The labeling substance and the first capture substance can be bound by a known method.

[0129] If the size of the marker and the first capture substance is compared, the marker is relatively large. Therefore, it may happen that a plurality of first capture substances are bound to the marker. In the case of binding a plurality of first capture substances to the marker, the reactivity of the first capture substance with the target substance is improved. On the other hand, the higher the concentration of the target substance, the easier it is to form a state in which a plurality of target substances correspond to one marker. In this case, the concentration of the target substance will be evaluated as a concentration lower than the actual concentration. Therefore, in order to expand the concentration range that can be quantitatively detected to the higher concentration side, a process for optimizing the ratio of the marker to the first capture substance can be implemented. As such a process, for example, in the reaction in which the first capture substance bound to the marker is bound to the target substance, a process of adjusting the feeding ratio of the first capture substance bound to the marker to the target substance can be cited.

[0130] In addition, when introducing the sample (including the portion of the marker) into the detection field, a tubular channel as a hollow conduit is used, preferably a small-diameter tubular channel. As a small-diameter tubular channel, it can be, for example, a microchannel. When the marker is an insoluble substance such as metal colloid particles, in order to avoid blockage of the flow path, the smaller the particle size of the marker bound to the first capture substance, the more preferably. On the other hand, in view of the fact that the scattered light intensity decreases when the particle size is small, as described above, the marker can be appropriately preferably selected from a particle size range of 80nm to 3μm.

[0131] Here, when the label is a metal colloid particle, for example, a sensitization reaction can be performed to grow the colloid particles and increase the particle size. In this way, smaller metal colloid particles can be used when the first capture substance and the metal colloid particles of the label are combined, so the number of the first capture substance combined on one metal colloid particle can be suppressed. In addition, the particle size of the metal colloid particles is increased by a sensitization reaction before the label is introduced into the detection field, so the scattered light intensity can be increased. In the present invention, the label can be introduced into the detection field, and there is no need to introduce the target substance and the first capture substance combined therewith into the detection field. Therefore, even if the sensitization reaction causes the target substance and the first capture substance combined with the label to decompose and denature, there will be no problem.

[0132] Specifically, when the marker is metal colloid particles, for example, the particle size of the metal colloid can be increased by reducing and accumulating metal ions in a metal salt solution containing gold ions and silver ions that are separately added on the surface of the metal colloid of the marker by utilizing the catalytic activity of the metal colloid or using a reducing agent in combination.

[0133] As the metal colloid particles provided for the sensitization reaction, metal colloid particles having high stability of the metal species as the core relative to redox are preferred. It should be noted that the metal colloid particles as the core can be formed of a single metal or a composite metal. As the metal colloid particles, for example, gold colloid particles, platinum colloid particles, palladium colloid particles, etc. can be cited. In addition, as composite metal colloid particles, platinum-gold colloid particles formed by loading platinum colloid particles on gold colloid particles can be cited.

[0134] In the sensitization reaction of the metal colloid particles, the metal salt that serves as the metal ion supply source includes salts of metals belonging to the 4th, 5th, and 6th periods of the periodic table, preferably salts of gold, platinum, silver, palladium, nickel, cobalt, copper, etc. The precipitated metal may be the same metal as the metal colloid particles or a different metal, and there is no limitation as long as the reduction reaction can be carried out under simple conditions.

[0135] For example, palladium-platinum colloidal particles can be grown by using a solution containing any of metal ions such as nickel ions, cobalt ions, and copper ions as a sensitizer solution.

[0136] In addition, for example, for gold colloid particles, a sensitizer solution containing chloroauric acid can be used as a gold ion supply source, and gold colloids can be deposited on the surface of gold colloid particles as a marker to grow the colloid particles. In addition, for gold colloid particles, a sensitizer solution containing silver nitrate can be used to deposit silver colloids on the surface of gold colloid particles to grow in the form of composite metal colloid particles. Platinum-gold colloid particles can be grown using a sensitizer solution containing copper salts such as silver nitrate and copper sulfate using the catalytic activity of platinum.

[0137] The sensitizer solution contains various metal salts serving as a supply source of the above-mentioned metal ions as essential components, and may contain a complexing agent, a pH adjuster, a buffer, a stabilizer, etc. as necessary. As such a sensitizer solution, a conventionally known chemical plating solution may be used.

[0138] Examples of complexing agents include ammonia, citrates, tartrates, lactates, etc. Examples of pH adjusters include acetates, propionates, ammonium salts, etc. Examples of stabilizers include various surfactants, etc.

[0139] In order to promote the reduction reaction of metal ions, a solution containing a reducing agent can be used. As the reducing agent, citric acid, ascorbic acid, sodium hypophosphite, hydrazine hydrate, hydrazine sulfate, sodium borohydride, dimethylamine borane, formaldehyde, Rochelle salt, glucose, ethylene glycol, etc. can be listed, but it is not limited to these. The reducing agent used can be appropriately selected according to the metal ion to be reduced.

[0140] It should be noted that the sensitization reaction can be carried out using a sensitizer solution comprising a metal ion component and a reducing agent. In this case, a metal salt solution comprising a metal ion component and a reducing agent solution comprising a reducing agent can also be prepared separately, mixed before the sensitization reaction is about to be carried out, and the resulting solution is used for the reaction. Suitably, it is preferred to prepare a metal salt solution and a reducing agent solution separately and add them to the reaction system respectively. It should be noted that the catalytic activity of the metal colloid particles can also be used to implement the sensitization reaction without adding a reducing agent solution.

[0141] The sensitization reaction is preferably carried out after the complex is formed and before the portion containing the labeled substance is separated.

[0142] <Method for cutting out a portion including a marker>

[0143] 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 marker) is formed on the solid phase in the reaction field, the portion containing the marker is cut out (separated) from the complex to be freed from the solid phase into the liquid. The cutting out and separation are performed while the complex is immersed in the liquid. When the reaction field stores the liquid in addition to the tubular channel being filled with liquid and forms a state in which the solid phase is immersed in the liquid, the tubular channel and the reaction field are connected by the liquid. The portion containing the marker in the liquid of the reaction field separated from the solid phase by cutting out is intermittently supplied to the liquid filled in the tubular channel by centrifugal force. As described above, the separated portion only needs to contain the marker and does not need to be in the state of the first capture substance itself containing the marker. That is, as long as the marker or the portion containing the marker is separated, any bond in the complex can be cut off.

[0144] It should be noted that, from the viewpoint of transportability, the portion containing the marker after separation from the complex preferably does not contain a solid phase (part of the solid phase). Therefore, it is preferred that the complex is separated and the portion containing the marker is freed between the solid phase and the second capture substance, inside the second capture substance, between the second capture substance and the target substance, inside the target substance, between the target substance and the first capture substance, inside the first capture substance, or between the first capture substance and the marker.

[0145] As described above, the method for binding the label to the first capture substance is not limited, and binding methods such as physical adsorption binding, chemical binding, affinity binding, and combinations thereof can be used. The label can be bound to the first capture substance by a known method.

[0146] As means for cutting (separating) the portion containing the marker, examples include: a method of cutting by providing physical energy such as heat, light, vibration, etc. from the outside; a method of adding a cutting reagent containing an oxidant, a reducing agent, etc. The cutting reagent can cut the binding between the solid phase and the second capture substance, and can also cut other bindings in the complex. The cutting reagent can separate the portion containing the marker from the complex formed on the solid phase, and there is no particular limitation as long as it does not significantly damage the properties of the marker. It is preferred to cut the binding between the target substance and the second capture substance or the first capture substance, or the first capture substance and the marker.

[0147] Examples of the cleavage agent for cleaving the bond include pH adjusters, denaturants, reducing agents, enzymes, competitive agents, oxidants, and the like. Preferred examples include pH adjusters, denaturants, reducing agents, and enzymes, and more preferred examples include pH adjusters and denaturants.

[0148] pH regulators include acids, bases, and salts thereof, and are compounds or compositions that change pH. Acids may be any of inorganic acids and organic acids, and bases may be any of inorganic bases and organic bases. As the acid and base, as an example, Bronsted acid, bases, and salts thereof may be exemplified, for example, hydrochloric acid, boric acid, carbonic acid, nitric acid, acetic acid, boric acid, formic acid, citric acid, phosphoric acid, oxalic acid, lactic acid, malic acid, salicylic acid, glycine hydrochloride, glycine sodium hydroxide, sodium hydroxide, triethylamine, dimethylamine, methylamine, and other amines, ammonia, sodium bicarbonate, etc. may be exemplified, but are not limited to these. Among them, strong acids and strong bases are preferred, and as specific examples thereof, amines such as hydrochloric acid, nitric acid, formic acid, phosphoric acid, oxalic acid, glycine hydrochloride, glycine sodium hydroxide, sodium hydroxide, triethylamine, dimethylamine, and methylamine may be cited.

[0149] Denaturants are agents that change the higher-order structures of proteins and nucleic acids, and examples thereof include protein denaturants such as urea and guanidine salts, and surfactants such as sodium dodecyl sulfate. In addition, other denaturants include Lewis acids, alcohols or esters not included in the above-mentioned pH adjusters, and their derivatives, but are not limited to these.

[0150] As the reducing agent, those capable of cleaving a disulfide bond may be exemplified by 2-mercaptoethanol and dithiothreitol.

[0151] The enzyme is a substance that hydrolyzes a part of the constituent elements of the complex, and examples thereof include pepsin, papain, ficin, nuclease, and enzymes that specifically act on specific bonds of the constituent elements.

[0152] Examples of the competitor include agonists, antagonists, and competitive antibodies that compete for the binding of the target substance to the first or second capture substance.

[0153] Furthermore, peroxides including hydrogen peroxide and other oxidizing agents may be used as the cleavage agent as long as they do not affect the target substance.

[0154] The type and concentration of the cleavage agent can be appropriately selected according to the structure and binding strength of the target complex, and can be adjusted within a range that does not significantly impair the function and structure of the labeling substance.

[0155] Here, in order to specifically cut off any part of the complex, a joint can be introduced into a predetermined part of the complex in advance. For example, when the solid phase is combined with the second capture substance, or the label is combined with the first capture substance, the joint can be used to connect the two pairs of target substances. As long as the joint has a predetermined functional group or reactive group that can form a chemical bond at both ends, the solid phase can be connected to the second capture substance, or the label can be connected to the first capture substance with the functional groups at both ends of the joint. The joint can be used to connect the solid phase to the second capture substance, or the label to the first capture substance, but is not limited to this. For example, the joint can be introduced into the first capture substance and the second capture substance. As the functional groups or reactive groups at both ends of the joint, for example, covalent bonds such as amide bonds, disulfide bonds, and ester bonds can be exemplified. In this case, the terminal bond site of the joint can be hydrolyzed and treated with a reducing agent to release the terminal portion of the joint and cut the complex.

[0156] The binding mechanism between the linker and the target substance is not limited to the above-mentioned covalent bond, and may be, for example, physical adsorption, ionic bonding, binding by intermolecular affinity, or a combination thereof.

[0157] Furthermore, the molecular structure constituting the linker is not limited, and synthetic polymers such as proteins, peptides, sugar chains, nucleic acids, and polyethylene glycol can be used. In addition, biotin-avidin and the like that form affinity binding can also be used.

[0158] The cutting of the linker portion can be carried out by physical energy from an external field or a reagent. As the reagent, enzymes such as endopeptidase, saccharolytic enzyme, endonuclease, reducing agent, acid, base, surfactant, competitive agent, etc. can be used according to the type of the linker.

[0159] Specifically, for example, for disulfide bonds, a reducing agent having a thiol group in the molecule, such as 2-mercaptoethanol or dithiothreitol, can be used to cut the bonds. In addition, in the case of biotin-avidin binding, the above-mentioned binding can be cut by adding a pH adjuster to change the pH of the reaction system or using an elution buffer. In addition, when the linker molecule is composed of a sugar chain, a specific enzyme can be used to act on the bonds between monosaccharides constituting the sugar chain to cut specific bonds.

[0160] Furthermore, when a linker with a histidine tag is used, for example, a linker molecule with a histidine tag at the end is bound to the second capture substance, and nickel is fixed to the surface of the solid phase using a chelating agent, so that the second capture substance can be immobilized on the solid phase via the histidine tag. If histidine or imidazole, which is a competitor of the histidine tag, is added as a cleavage agent, the histidine tag can be dissociated from the nickel, so that the complex containing the marker can be separated from the solid phase.

[0161] The cleavage agent used to separate the portion containing the label from the complex can be removed or inactivated after the cleavage reaction. For example, when the reaction field is changed from neutral to acidic or alkaline by adding a pH adjuster, the treatment of further adding a pH adjuster to restore it to neutrality is a case in which the pH adjuster is further added.

[0162] The reagent of the present invention is configured to include the above-mentioned cleavage reagent. It should be noted that the reagent of the present invention can be configured as a reagent set. In the reagent set, in addition to the reagent (cleavage reagent) of the present invention, it can also include, for example, a cleaning solution, a dispersion liquid in which the first capture substance bound to the marker is dispersed, and a dispersion liquid of a solid phase loaded with the second capture substance.

[0163] <Solid phase>

[0164] The solid phase is an insoluble solid to which the second capture substance is bound, and is processed into a form, shape, and size that can be retained in the reaction field.

[0165] The form or shape of the solid phase is not particularly limited and may be any of the inner surface wall of the reaction container as the reaction field, a protrusion formed inside the reaction container and integrated with the reaction container, a structure such as a structure, or a member formed separately from the reaction container.

[0166] As the solid phase formed separately from the reaction vessel, a plate-like body, a granular body, a fibrous body, a woven fabric, a non-woven fabric, a film, a sheet, etc. that can be accommodated in the reaction vessel can be exemplified. The plate-like body, the granular body and the fibrous body can be any form such as hollow, solid, porous, etc. The shape of the granular body can be selected from any of a sphere, a ring, a flat body, a columnar body, an indefinite shape, etc. The granular body can be, for example, at least one of the resin particles formed by polymers such as magnetic particles, glass particles, ceramic particles, latex, and polystyrene. As the solid phase, glass particles, ceramic particles, magnetic particles, or resin particles are preferred.

[0167] The solid phase is preferably in the form of granules, fibers, woven fabric, or nonwoven fabric, more preferably in the form of granules or fibers, and still more preferably in the form of granules.

[0168] It should be noted that, as the binding between the solid phase and the second capture substance, binding modes such as physical adsorption, covalent bonds, ionic bonds, binding using affinity, and combinations thereof can be used. Preferably, the second capture substance is immobilized on the solid phase by a covalent bond or an ionic bond with a bond having a binding force stronger than that based on affinity or adsorption force. In addition, on the solid phase, the second capture substance is fixed to capture the maximum detectable amount of the target substance in the set detection range (detectable, set concentration range of the target substance).

[0169] Regarding the granules, if the capture of the target substance is considered, it is preferred that the specific surface area is relatively large. When introduced into a reaction chamber connected to a tubular channel, a granule of a size that will not flow into the tubular channel from the entrance of the tubular channel is selected. For example, if the minimum dimension of the length, width, and thickness of the granule is about 1.2 to 5 times the entrance diameter, the inflow into the tubular channel can be fully suppressed. It should be noted that when the entrance shape of the tubular channel is an irregular shape, the entrance diameter is set to the dimension in the short axis direction. As granules, various beads with a diameter of about 1 μm to 100 μm can be obtained in the form of commercial products. Therefore, a suitable one can be appropriately selected according to the size of the entrance. The granules used are 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, especially resin beads with a particle size of 15 μm to 30 μm.

[0170] <Reaction field>

[0171] In the present embodiment, in the method for detecting a target substance, a sample is introduced into a reaction field having a solid phase on which a second capture substance is pre-fixed, and the first capture substance is introduced after the second capture substance on the solid phase reacts with the target substance in the sample to form a complex sandwiching the target substance. Thereafter, the excess first capture substance is discharged outside the reaction system. Furthermore, the binding of the complex is cut off as described above to separate the portion containing the marker. The process until the portion containing the marker is separated is performed in the reaction field. The surface density of the fixed second capture substance on the solid phase can be adjusted in advance. Therefore, by introducing the first capture substance after the target substance reacts with the second capture substance in this way, the quantitativeness of the detection can be improved over a wide range of concentrations of the target substance contained in the specimen from low concentrations to high concentrations.

[0172] It should be noted that the reaction for forming a complex in the reaction field, i.e., the process of mixing the first capture substance, the solid phase loaded with the second capture substance and the sample containing the target substance, can be introduced into the reaction field in any order as long as a complex can be formed, and some or all of them can be introduced simultaneously.

[0173] That is, the process of forming a complex is not limited to reacting the target substance with the second capture substance fixed to the solid phase and then reacting the first capture substance bound to the marker to form a complex as described above. For example, the target substance may react with the first capture substance bound to the marker and then react with the second capture substance fixed to the solid phase to form a complex, or the first capture substance bound to the marker, the target substance, and the second capture substance fixed to the solid phase may react simultaneously to form a complex.

[0174] 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 marker) and separate the portion containing the marker from the complex. In order to implement the reaction process, only one reaction field can be used, or multiple reaction fields can be used. That is, the formation of the complex and the separation of the portion containing the marker can be implemented in the same reaction field (space) or in different reaction fields (spaces).

[0175] The reaction field can be formed, for example, using a divided container or chamber. As the reaction field, for example, a chamber or passage formed in a test tube, a microtube, a microplate, a microreactor, a bio disk, etc. can be used. In addition, the operation in the reaction field can be performed manually or automatically. When the operation in the reaction field is performed automatically, for example, a device in which the reaction field is installed in the form of a chamber can be used, and in addition, the form of a disk that enables the device to rotate around the central axis can be adopted.

[0176] Preferably, the device has at least one reaction field in the form of a reaction chamber. In the case of a disk-shaped device, the reaction chamber is arranged on the central axis side of the disk compared to the tubular channel, and the outer side is connected to the tubular channel. The reaction chamber is usually formed with a size larger than the inner diameter of the tubular channel in the depth direction and the width direction. Preferably, the reaction chamber can be formed into a shape that shrinks toward the tubular channel (mortar shape). The portion of the liquid in the reaction chamber that is separated from the solid phase by cutting off the reagent and contains the marker can be intermittently supplied from the reaction chamber with a large capacity to the liquid filled in the thin tubular channel when the disk rotates.

[0177] The communication port (entrance of the tubular channel) connecting the reaction chamber and the tubular channel needs to be formed with a size such that the portion containing the marker can pass through and the solid phase formed separately from the reaction chamber will not flow out when the solid phase is introduced into the reaction chamber (for example, when the solid phase is a solid such as beads and has fluidity in the reaction chamber). If the usual processing accuracy is taken into consideration, the communication port is, for example, 5 μm or more, preferably 10 μm or more, and more preferably 15 μm or more, and the solid phase is designed to be of a size that is easy to process without flowing into the tubular channel.

[0178] It is ideal that the portion containing the marker as the detection object is introduced into the detection field in a state where inclusions are reduced. The method of the present invention detects the marker that moves in the tubular channel by centrifugal force, so if the sample introduced into the tubular channel is mixed with excess markers, it will significantly affect the detection accuracy. Therefore, it is important to remove the excess first capture substance (the first capture substance bound to the marker, including the one that does not capture the target substance and the free marker) from the sample to be supplied to the tubular channel.

[0179] As a method for removing the excess first capture substance from the sample, a washing treatment can be selected. Specifically, if a washing treatment is performed after forming a complex sandwiching the target substance, the excess first capture substance that does not participate in the reaction can be removed. On the other hand, the first capture substance that captures the target substance is fixed to the solid phase with the help of the target substance and the second capture substance, and is therefore maintained in the reaction field. If a cutting reagent is added in the above state, the separated portion containing the marker can be introduced into the detection field under the condition that the excess first capture substance is removed and reaches a level that will not affect the detection result.

[0180] In addition, as a method for removing the excess first capture substance from the sample, it is possible to choose to exclude it by centrifugation. When using a disk, it is easy to implement to exclude the excess first capture substance by centrifugation. Specifically, for example, when a complex is formed (or introduced) in a reaction chamber, the first capture substance that captures the target substance is retained in the reaction chamber in a state fixed to the solid phase. On the other hand, since the excess first capture substance is not fixed, if a centrifugal force is applied in a state where the above-mentioned tubular channel is filled with liquid and the solid phase of the reaction chamber is immersed in the liquid, and the reaction chamber and the tubular channel are connected by liquid, it will move in the tubular channel due to the centrifugal force and can be discharged out of the reaction chamber. After that, a cutting reagent is added to the reaction chamber to separate the portion containing the marker from the complex, and the portion containing the marker can be introduced into the detection field without the coexistence of the excess first capture substance. Therefore, when the excess first capture substance is removed by centrifugal force, a cleaning treatment using a cleaning solution such as a cleaning agent may not be required. In addition, from the viewpoint of improving the cleaning power, the cleaning treatment may be performed by combining the removal of the excess first capturing material by centrifugal force and the cleaning with a cleaning liquid.

[0181] It should be noted that, in order to fix the excess first capture substance discharged from the reaction chamber with a binding agent that specifically binds to the first capture substance, the binding agent can be maintained in the movement path of the excess first capture substance. The binding agent is loaded at a location that does not hinder the passage of the portion containing the marker through the tubular channel. Thus, the excess first capture substance discharged from the reaction chamber is fixed at a predetermined location in the device.

[0182] As the binding agent, for example, an antigen or an antibody that specifically binds to the first capture substance is used, and the binding agent may be the same substance as the target substance of the first capture substance.

[0183] In addition to the above-mentioned antigens and antibodies, nucleic acids, aptamers, ligands (substrates, inhibitors, antagonists), and receptors to which the first capture substance can bind may also be used as binding agents.

[0184] The antibody used as the binding agent may be a polyclonal antibody or a monoclonal antibody, but is preferably a monoclonal antibody from the viewpoint of reaction specificity.

[0185] In addition, antibodies used as binding agents also include antibodies and antibody fragments and modified antibodies having substantially the same reactivity as the antibodies. Examples of antibody fragments include Fab fragments, F(ab')2 fragments, Fab' fragments, and scFv fragments.

[0186] <Testing site>

[0187] The detection field (second reaction field) is a field into which the portion containing the marker separated in the reaction field (first reaction field) is introduced and scattered light caused by the portion containing the marker is detected. A tubular channel is arranged in the detection field so that the portion containing the marker can move, and a light source for irradiating light toward the tubular channel and a light receiving element for detecting scattered light emitted by the marker in the tubular channel are provided.

[0188] After the complex is formed in the reaction field (first reaction field), the elements other than the complex are discharged out of the system by performing the above-mentioned washing treatment and centrifugation treatment. After that, if the portion containing the marker is cut, the sample can be introduced into the detection field in a state where the excess first capture material and other inclusions that affect the detection are reduced.

[0189] The tubular channel is provided in a device formed in a rotatable manner. The tubular channel is formed of a light-transmitting material so that the irradiation light irradiated from the light source can reach the inside thereof and the scattered light generated by the detection object can be emitted.

[0190] The tubular channel is preferably formed into a hollow cylindrical shape with both ends open, and is extended outwardly from the rotation center side 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 channel is communicated with the reaction field (the first reaction field) that maintains the solution containing the portion containing the marker. The tubular channel can be a hollow tube, and its cross-sectional shape is not limited. It can be a round tube or a square tube, or it can be a flat shape. In addition, it can be composed of one seamless component, or it can be composed of a combination of multiple components.

[0191] The inner diameter of the tubular channel is in the range of about 3 to 150 times the detection object to avoid clogging, and is designed in a shape that the solid phase in the reaction field (reaction chamber) will not 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 channel at the communication port (the entrance of the tubular channel) of the reaction chamber is smaller than the size of the solid phase, for example, it is set to a size of about 1 / 1.2 to 1 / 5 times the solid phase. In detail, for example, the minimum value of the maximum values ​​of the vertical, horizontal, and high values ​​of the solid phase is taken as a reference value, and the entrance diameter of the tubular channel is made smaller than the reference value. When the solid phase is a regular shape, the vertical, horizontal, and high values ​​are equal, so they can all be used as reference values. The inner diameter of the tubular channel can be formed uniformly with the same width as the implementation entrance diameter, or it can be formed with a width different from the entrance diameter, for example, it can be formed in a tapered or stepped manner to widen or narrow.

[0192] Specifically, for example, the inner diameter of the tubular channel is 1 to 100 μm, preferably 5 to 50 μm, and more preferably 10 to 20 μm. The smaller the inner diameter, the less the amount (absolute amount) of inclusions in the tubular channel, thereby reducing scattered light noise caused by inclusions.

[0193] It should be noted that when the cross section of the tubular channel is an irregular shape such as an ellipse or a rectangle, the short side of the cross section is formed in such a way that the channel height of the tubular channel and the long side is the channel width. In addition, the tubular channel is designed in such a way that the height direction is the above-mentioned inner diameter.

[0194] If the height of the tubular channel is specified to avoid the inflow of the solid phase, the size of the solid phase can be ignored in the design of the flow path width. The minimum value of the flow path width of the tubular channel is the size of the portion containing the marker, and the upper limit is the width of the abutment surface of the connected reaction chamber. The larger the width of the tubular channel, the smoother the liquid flow of the tubular channel, but the abutment portion with the reaction chamber is enlarged, and as a result, the inclination of the mortar shape from the reaction chamber toward the tubular channel becomes shallower, which inevitably affects the accumulation range of the solid phase. From this point of view, the narrower flow path width of the tubular channel is preferred. Therefore, the flow path width of the tubular channel can be set to a suitable width considering the liquid flow and the solid phase accumulation state in the reaction chamber, for example, 100μm~1cm, preferably 100μm~5mm, more preferably 500μm~3mm, and further preferably 500μm~1mm.

[0195] The open end of the outer side of the tubular channel (hereinafter sometimes referred to as "the other open end") is connected to the drainage storage chamber as the second storage chamber. The drainage storage chamber is a partitioned chamber for storing the liquid that has passed through the tubular channel. When the cleaning process is performed in the reaction field (first reaction field) formed in the device, the waste liquid of the cleaning process is discharged through the tubular channel and stored in the drainage storage chamber. The drainage storage chamber can be arranged at the downstream side of the tubular channel, for example, it can be arranged partially on the side of the rotation center along the rotation radius relative to the tubular channel, or it can be arranged entirely on the outside.

[0196] Here, in the method of the present invention, in order to implement the detection of the marker, it is necessary to form a state in which the tubular channel is filled with liquid. In other words, it is necessary that the liquid in the tubular channel does not flow out from the open end when the centrifugal force is applied. Therefore, a limiting mechanism for limiting the flow of the liquid in the tubular channel is provided. The limiting mechanism can be realized by, for example, extending a thin flow path from the end of the discharge storage chamber toward the rotation center side. Thus, under the action of centrifugal force, the liquid supplied to the tubular channel is stored in the discharge storage chamber and the overflowed liquid enters the thin flow path toward the rotation center side. Here, the liquid level is formed at the same distance from the rotation center, so if the liquid is supplied until the open end (the above-mentioned one open end) on the rotation center side of the tubular channel is reached, the liquid level is also formed at the same position as the open end (the above-mentioned one open end) on the rotation center side of the tubular channel in the flow path toward the rotation center side. At this time, when subjected to centrifugal force, even if the supply of liquid is interrupted, the backflow of liquid to the upstream side can be suppressed. Thus, the tubular channel is filled with liquid.

[0197] In addition, as a limiting mechanism, a drainage storage chamber can be used. For example, when at least a portion of the drainage storage chamber is arranged on the rotation center side relative to the tubular channel, the end portion of the side that is not connected to the tubular channel can be arranged in a manner that reaches the open end (the above-mentioned one open end) on the rotation center side of the tubular channel or is closer to the rotation center side than the open end on the rotation center side of the tubular channel. When the liquid is supplied until it reaches the open end (the above-mentioned one open end) on the rotation center side of the tubular channel, the liquid level of the drainage storage chamber and the liquid level of the tubular channel are formed at an equal distance from the rotation center. Thus, the tubular channel is filled with liquid. It should be noted that in this case, the drainage storage chamber plays a role in preventing the liquid in the tubular channel from flowing out from the open end, so it is not necessary to separately set a limiting mechanism for limiting the flow of liquid in the tubular channel.

[0198] It should be noted that when the cleaning process is not performed in the device, the drainage storage chamber may not be provided, and a thin flow path may be provided extending from the end of the tubular channel toward the rotation center side.

[0199] In addition, as other examples of the limiting mechanism, it can be illustrated that, for example, a switch valve is provided at the other open end of the tubular channel or in a space connected to the open end. Thus, if the switch valve is closed, the front of the tubular channel is closed, so that the liquid can be filled in the tubular channel. As the switch valve, not only can an electric, electromagnetic, or mechanical actuator be used, but also a capillary passive valve described later (because liquid will not flow out from the open end before a centrifugal force exceeding a specified value is given, so a mechanism that can function as a valve) etc. can be selected and used, and a suitable one can be selected from these.

[0200] During detection, centrifugal force is applied while the tubular channel is filled with liquid supplied to the tubular channel, so that the portion containing the labeled substance moves from the reaction field (first reaction field) on the rotation center side to the outside of the device through the tubular channel.

[0201] Then, by introducing the portion containing the labeled substance to be detected into the tubular channel, the irradiated light is scattered to generate scattered light, and the target substance can be detected by detecting the scattered light with a light receiving element such as a photodiode.

[0202] When the particle size of the marker is within a specified range, there is a correlation between the scattered light intensity and the particle size. In addition, the scattered light intensity in a specific direction will change according to the particle size. Since the particle size of the particle used as a marker can be grasped in advance, a light receiving element can be set at a position where scattered light can be detected, and the scattered light can be appropriately detected. It should be noted that when scattered light is generated in all directions, a light receiving element can be configured at an arbitrary position. The detected scattered light can be forward scattered light or back scattered light. The light receiving element is appropriately configured according to the direction in which the detected scattered light is scattered.

[0203] In addition, reflected light that becomes noise is generated at the interface where the irradiated light enters. By adjusting the positions and angles of the light source and the light receiving element, the reflected light noise can be reduced and scattered light from the marker can be appropriately detected.

[0204] Here, if an optical system or a rotating mechanism using a general-purpose laser light source such as that used in an optical pickup is used, a small and inexpensive detection device can be realized. However, when the part of the detection object including the marker passes through the detection field at high speed, it is difficult for the detection system using inexpensive general-purpose components to accurately detect the scattered light based on the tiny marker.

[0205] Here, 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 revolution: 60 / N seconds), the portion containing the marker moves at a moving speed of v μm / s in such a way that the marker of the detection object enters the laser spot at least once. In this case, the following formula holds true.

[0206]

[0207] For this formula, the moving speed v is solved as shown in the following formula.

[0208]

[0209] According to this formula, for example, when the laser spot diameter is 10 μm and the disk is rotated at 2000 rpm, the moving speed of the marker must be 333 μm / sec or less.

[0210] Also contemplated is a method in which, different from the method of the present invention, the portion containing the marker is passed through a tubular channel as the liquid passes through it and the scattered light from the marker moving in the tubular channel is detected. To this end, the moving speed of the liquid flowing by centrifugal force in the device is calculated by the following formula.

[0211]

[0212] U is the velocity (m / s), Q is the flow rate (m 3 / s), A is the flow path cross-sectional area (m 2 ), d H is the hydraulic diameter (m), ρ is the density of the fluid (kg / m 3 ), ω is the angular velocity (rad / s), r1 is the average rotation radius of the liquid in the flow path (m), Δr is the difference in rotation radius between the inlet and outlet of the flow path, η is the viscosity of the fluid (kg / m·s), and L is the length of the flow path (m).

[0213] When the medium is water at 20°C and rotated at a speed of 2000 rpm, the moving speed of the medium flowing by centrifugal force at a specified position in the radial direction of the disk is calculated based on formula (8), and the moving speed of the 200nm gold colloid particles moving in the medium by the generated centrifugal force is calculated based on the Stokes formula, the former is 60mm / sec, and the latter is 84μm / sec. In addition, it is actually confirmed that the liquid in the disk moves at a high speed due to rotation. Therefore, when the movement of the gold colloid particles (marker) is dominated by the flow of the liquid, it is difficult to detect scattered light.

[0214] Therefore, in the present invention, the tubular channel is filled with liquid and functions like a centrifuge tube, and the portion containing the marker moves in the liquid due to centrifugal force, thereby being able to move in the tubular channel at a speed that enables detection of scattered light.

[0215] In the present invention, in which the movement of the portion containing the marker in the tubular channel is not subject to the flow of the liquid, the movement speed of the portion containing the marker in the tubular channel changes according to the density and particle size of the portion containing the marker (the marker) and the centrifugal force, as long as the liquid filled in the tubular channel is the same. Therefore, the movement speed can be within the desired range by appropriately selecting the rotation speed of the device and the marker.

[0216] <Device>

[0217] The device is also called a bio-disc or microreactor, and at least comprises: a reaction chamber (corresponding to the reaction field) for holding the complex and reacting as required, and a tubular channel (corresponding to the detection field) for moving the detection object, a small-diameter flow path for liquid circulation as required, and a storage chamber for storing reagents and liquids, and is used for biological sample analysis. In addition, in order to impart centrifugal force, the device is formed in a rotatable manner.

[0218] The device can be made by known methods using known materials such as acrylic resin, polycarbonate resin, silicone resin and other plastics or glass, silicon and other inorganic materials. The material is preferably light-transmissive so that the detection object can be optically detected, and more preferably transparent.

[0219] The device can be made using known methods. For example, it can be made by laminating a sheet or plate having a flow path or chamber pattern with a flat sheet or plate. The internal structure such as the reaction chamber can also be formed by mechanical processing, etching using photolithography, or other conventional molding techniques. Furthermore, in order to increase the amount of light incident on the light-receiving element, a reflective layer can be arranged on the back of the tubular channel.

[0220] In order to supply liquid to the reaction chamber by centrifugal force, the storage chamber for storing reagents and liquids provided in the present device is arranged on the rotation center side relative to the reaction chamber. In the detection method of the target substance of the present invention, in a state where the liquid fills the tubular channel, the detection object is moved in the liquid in the tubular channel by centrifugal force. Therefore, when the liquid filling the tubular channel is pre-stored in the storage chamber, the liquid stored in the storage chamber is filled into the tubular channel at a specified time. In addition, in the present embodiment, when the complex held in the reaction chamber is subjected to a cleaning process, a cleaning liquid storage chamber as the first storage chamber and a drainage storage chamber as the second storage chamber are provided, and the cleaning liquid is stored in the cleaning liquid storage chamber. The drainage storage chamber can be arranged on the downstream side of the tubular channel, or at least a part of it can be arranged on the rotation center side relative to the tubular channel, or all of it can be arranged on the outside relative to the tubular channel. It should be noted that the cleaning liquid may also serve as the liquid filled in the tubular channel. In this case, the storage chamber for storing the liquid filled in the tubular channel and the cleaning liquid storage chamber may be one chamber.

[0221] When the tubular channel is filled with the cleaning liquid stored in the cleaning liquid storage chamber, when at least a portion of the drainage storage chamber is disposed on the rotation center side relative to the tubular channel, for example, the end portion of the side not connected to the tubular channel can be disposed in a manner that the distance between the open end (the above-mentioned one open end) on the rotation center side of the same tubular channel and the rotation center is equal to or shorter than the distance between the open end (the above-mentioned one open end) and the rotation center. In addition, the cleaning storage chamber can store a volume of cleaning liquid that can reach the drainage storage tank and the open end of at least the tubular channel on the rotation center side of the tubular channel. When the drainage storage chamber is disposed outside as a whole relative to the tubular channel, the cleaning liquid storage chamber can store a volume of cleaning liquid that exceeds the total content of the drainage storage chamber and the tubular channel and is less than the total content of the drainage storage chamber, the tubular channel, and the reaction chamber. By storing this amount of cleaning liquid, after cleaning, the tubular channel is filled with cleaning liquid, the reaction chamber also stores liquid, and the remaining space of the reaction chamber can accommodate the cutting reagent containing the part of the marker.

[0222] In addition, when the tubular channel is filled with the cleaning liquid stored in the cleaning liquid storage chamber, the cleaning process can be performed until the drainage storage chamber and the tubular channel are filled with the cleaning liquid, and as long as the remaining space in the reaction chamber can accommodate the cutting reagent containing the portion of the marker, the cleaning process can be performed until the cleaning liquid exceeds the capacity of the tubular channel and flows into the reaction chamber. When the cleaning liquid flows into the reaction chamber in addition to the tubular channel and the cleaning liquid is stored in the reaction chamber, the tubular channel and the reaction chamber are connected by the liquid.

[0223] Furthermore, in this embodiment, a cutting process for separating the portion containing the label from the complex is performed. When the cutting process is performed in the device, a cutting reagent storage chamber is provided as a third storage chamber to store the required cutting reagent. In addition, as described above, when the sensitization reaction of the metal colloid particles is performed, a sensitizer storage chamber is provided to store the sensitizer solution required for the sensitization reaction.

[0224] The reagent stored in the storage chamber can be stored in a liquid state or a solid state. When stored in a solid state, the reagent is sent from the storage chamber to the reaction chamber after a dissolving liquid for dissolving the reagent is introduced into the storage chamber.

[0225] It should be noted that when a cleavage reaction is performed to separate the portion containing the labeled substance by applying energy from an external field such as heat or electromagnetic waves, a cleavage reagent reservoir may not be provided.

[0226] <Control system>

[0227] Each device arranged in the detection field for scattered light detection is electrically controlled by the information processing device. In addition, signal processing is performed based on the received scattered light, and the marker (the portion including the marker) is detected using the scattered light as a signal.

[0228] In the detection method of the present invention, the centrifugal force generated by the rotating device causes the detection object, i.e., the marker, to move in the tubular channel. If the marker, the liquid filled in the tubular channel, and the centrifugal force applied are known, the moving speed can be calculated. In addition, the acceptable marker moving speed (the above formula (7)) can also be calculated from the rotation speed and the spot diameter of the laser light source. Based on these, the settable range of the rotation speed can be derived.

[0229] The light receiving element detects scattered light from a predetermined rotation radius position. In other words, the irradiation light from the laser light source is irradiated to the predetermined rotation radius position, and the spot diameter becomes the detection area. The light receiving element is arranged at a position where scattered light from the detection area can be detected. Examples of the light receiving element include, but are not limited to, photodiodes, avalanche photodiodes, photomultipliers, CMOS, and CCD.

[0230] If the marker enters the spot diameter of the laser light source, the scattered light is incident on a photodiode, for example, as a light receiving element. Then, the incident light is photoelectrically converted, output from the photodiode, and a voltage value corresponding to the intensity of the incident scattered light is input to the A / D converter. Then, the A / D converter is used to sample and digitize the signal at a specified sampling time, and 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 analog signals into digital signals, and can be, for example, an integrated circuit or an oscilloscope. The sampling time can be designed based on the resolution of the flow path width. For example, it is set to be able to obtain data at intervals of 1 / 10 or more and less than 1 / 1 relative to the spot diameter of the laser light source. For example, for a tubular channel rotating at 2000 rpm, when the spot diameter of the laser light source is 10 μm at a position of a rotation radius of 50 mm, in order to obtain data at intervals of about 1 μm, the sampling time can be set to about 100 ns.

[0231] In the information processing device, blank data is stored in association with a specified position in the width direction of the flow path of the tubular channel, for example, just after the tubular channel is filled with liquid and before the marker is supplied to the tubular channel, as needed. Afterwards, at each specified sampling time, the input voltage value is stored in association with the same position, and the difference with the blank data is calculated. When the difference between the two is below the specified threshold, it is determined that the marker is not detected at the position or it is determined to be scattered light of noise, and the generation of scattered light is not counted (the count is canceled). On the other hand, when the calculated difference exceeds the specified threshold, it is determined to be scattered light based on the marker. It should be noted that the marker is smaller than the spot diameter of the laser light source, so it will pass through it multiple times before passing through the spot diameter (detection area) and the result of detecting scattered light is obtained. Therefore, it can be configured as follows: based on the voltage value drop after multiple measurements (the difference between the blank and the input voltage value reaches below the specified threshold), the number of times the scattered light is generated is counted as 1.

[0232] Furthermore, according to the detection method of the present invention, the marker (the portion containing the marker) and the inclusions can be fully separated and detected, but for some reason, the state of the tubular channel such as foreign matter other than the marker, scratches, etc. may be mixed into the tubular channel at the time of detecting the marker (a predetermined time range from the start of detection based on the moving speed of the marker) and generate scattered light. In this case, the signal pattern of the scattered light caused by the foreign matter and the tubular channel is different from the signal pattern of the marker, so the two can be distinguished, and the target detection object can be accurately detected. When the target detection object can be distinguished and detected, the above-mentioned step of obtaining blank data before detecting the marker is not necessary.

[0233] Furthermore, in the present detection method, the marker and the liquid filled in the tubular channel, as well as the laser light source (spot diameter) can be pre-specified, and the rotation speed (rotation speed) of the device suitable for detection can also be pre-derived. Therefore, these values ​​can be stored in the information processing device by default. It should be noted that it can also be configured so that: during detection, the user can appropriately input the necessary values. Furthermore, in the case where it is pre-determined that specific scattered light is generated due to scratches on the tubular channel, etc., the signal pattern can be stored as blank data by default.

[0234] <Target substance detection method 1 (antibody test)>

[0235] An exemplary detection method 1 of the target substance (antibody) of this embodiment is described. Figure 1The outline of the detection method to be described is embodied as an antibody test. In this method, the antigen (second capture substance) for the target substance is first fixed to the surface of the polystyrene beads (solid phase) according to the conventional method. Then, after sealing, the polystyrene beads (the second capture substance fixed to the solid phase) are introduced into a container of a specified capacity. The polystyrene beads are introduced in a state dispersed in a buffer solution, and then the solvent is removed by appropriate filtration, centrifugation, etc. After that, a sample such as serum containing the target substance (antibody) is added, and after contacting and reacting with the beads for a certain period of time, it is washed with a buffer solution if necessary, and then a second antibody solution (the first capture substance bound to the marker) is added and reacted for a certain period of time. It should be noted that here, the marker is a metal colloidal particle. Thus, an antigen-antibody-secondary antibody sandwich-like immune complex is formed on the beads. After washing with a buffer solution, a cutting reagent (pH adjuster) is introduced to reach pH 0-4 or pH 10-14, so that the part containing the metal colloidal particles is freed from the immune complex formed on the solid phase.

[0236] It should be noted that in the antibody test, the user can arbitrarily select the stage from which the reaction chamber of the device is used as the reaction field after the antigen is fixed on the polystyrene beads (solid phase). In addition, when the reaction chamber of the device is used, the cleaning process can be performed only once after the antigen-antibody-secondary antibody immune complex is formed on the polystyrene beads.

[0237] After the cutting process, the separated part containing metal colloid particles is detected. In this method, the part containing metal colloid particles separated by the cutting process is introduced into the tubular channel of the device and detected. In the reaction chamber connected to the tubular channel, there is a connecting port (inlet) formed with a height smaller than the diameter of the polystyrene beads, and the part containing metal colloid particles is introduced or stored in the reaction chamber as the front chamber of the tubular channel. Then, the tubular channel is introduced through the connecting port (inlet) by centrifugal force, and moves outward along the tubular channel. Here, the tubular channel is filled with liquid, and the liquid does not move substantially. Therefore, the part containing metal colloid particles moves in the liquid phase by centrifugal force. The tubular channel is irradiated with light, and scattered light is generated when the metal colloid particles pass through the tubular channel. The scattered light is generated intermittently each time the metal colloid particles pass through. The signal corresponding to each metal colloid particle is not continuous, and is obtained in the form of a unique signal. Therefore, by measuring it, the metal colloid particles can be detected, and as a result, the antibody as the target substance can be detected.

[0238] As described above, according to the method, a target substance detection method for detecting a target substance can be implemented.

[0239] <Target substance detection method 2 (antigen test)>

[0240] An exemplary detection method 2 for a target substance (antigen) according to this embodiment is described. Figure 1 The immunoassay method called sandwich method, which is generally described in the text, is further specified. In this method, the antibody (second capture substance) against the target substance is first diluted to an appropriate concentration with phosphate-buffered saline (hereinafter referred to as PBS) or a buffer solution, and the antibody is fixed to the surface of polystyrene beads (solid phase) according to a conventional method. Then, after blocking, a container of a specified capacity is filled with a specified volume of polystyrene beads (the second capture substance fixed to the solid phase). A sample such as serum containing the target substance (antigen) is added, and after contacting and reacting with the beads for a certain period of time, it is washed with a buffer solution, and then a primary antibody labeled with metal colloidal particles (the first capture substance bound to the marker) is added to bind to the antigen. Thus, a sandwich-like immune complex of antibody-antigen-antibody is formed on the polystyrene beads.

[0241] It should be noted that, similarly to the above-mentioned detection method 1, the user can arbitrarily select from which stage the reaction chamber of the device is used in this method. In addition, when the reaction chamber of the device is used, the cleaning process can 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 the above-mentioned detection method 1, thereby implementing an immunoassay for detecting the target substance.

[0242] An example of the device of the present embodiment is further described in detail below using FIG. 2 . FIG. 2 is a diagram showing an overview of a disk 1 for implementing detection of a target substance, FIG. 2 (a) shows an overall view of the disk, and FIG. 2 (b) shows a partially enlarged view thereof. The disk 1 is formed in a disk-shaped plastic disk such as a compact disk (CD), a DVD, or a Blu-ray disk (BD) by forming a small-diameter flow path or chamber in which a liquid can flow. In order to impart centrifugal force, a disk shape suitable for rotation is adopted, and the center of the disk becomes the center of rotation. The disk 1 is constructed as follows: 6 units 2 are provided, and each unit 2 can independently detect a target substance.

[0243] Each unit 2 includes a reaction chamber 10 for holding the complex, a tubular channel 11 connected to the reaction chamber 10, a drainage storage chamber 12 connected to the tubular channel 11, a cleaning liquid storage chamber 13 for storing a cleaning liquid, and a cutting reagent storage chamber 14 for storing a cutting reagent.

[0244] The reaction chamber 10 has a supply port 10a on its upper surface that passes through the inside and outside of the chamber, and is formed in a manner that a solution, a reagent, etc. containing a solid phase can be introduced from the supply port 10a. The reaction chamber 10 becomes a reaction field in the disk 1. When a centrifugal force is applied, the solid phase introduced into the reaction chamber 10 is deposited on the inner wall (including any of the upper surface, the bottom surface, and the side surface) on the side of the tubular channel 11. Here, in order to suppress the spreading of the solid phase in the width direction (the length in the direction intersecting the rotation radius) of the reaction chamber 10 and achieve deposition, the outer side of the reaction chamber 10 is formed in a mortar-shaped manner when viewed from above. In addition, the bottom surface of the inside of the tubular channel 11 side of the reaction chamber 10 is formed into a slope in a manner that the bottom gradually becomes shallower when it faces the tubular channel 11 side.

[0245] The disk 1 can be Figure 1 The steps shown are performed in order to implement necessary processes. The disk 1 can be processed to separate the portion containing the marker from the complex in the reaction chamber 10, and the formed complex (complex consisting of solid phase-second capture substance-target substance-first capture substance bound to the marker, etc.) is held in the reaction chamber 10.

[0246] The tubular channel 11 is a hollow tube with both ends open, extending along the rotation radius of the disk 1, and 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. The tubular channel 11 is formed of a light-transmitting material. The flow path width of the tubular channel 11 is formed to be narrower than the width of the reaction chamber 10. In addition, in order to intercept the solid phase introduced into the reaction chamber 10, its depth is formed to be smaller than the minimum diameter of the outer shape of the introduced solid phase. In the disk 1, the tubular channel 11 abuts against the upper side of the reaction chamber 10 and communicates with the reaction chamber 10. The tubular channel 11 has a structure that intercepts the solid phase introduced into the reaction chamber 10 by defining its depth direction, so the solid phase size can be ignored in the design of the flow path width.

[0247] The drain storage chamber 12 is connected to the outer side of the disk 1 in the direction of the rotation radius relative to the tubular channel 11, and stores the waste liquid generated in the reaction chamber 10. The tubular channel 11 and the drain storage chamber 12 are connected by the communication port (outlet) 11b, and the waste liquid of the reaction chamber 10 is discharged to the drain storage chamber 12 through the tubular channel 11. In addition, the drain storage chamber 12 is spread out to both sides with the communication port 11b as the center, and is connected to the thin flow path 17 extending from both ends toward the rotation center side. The flow path 17 is connected to the cleaning liquid storage chamber 13 at its front end. The liquid supplied in excess of the capacity of the drain storage chamber 12 invades the flow path 17 and also invades the tubular channel 11.

[0248] The cleaning liquid storage chamber 13 is a chamber designed to store the cleaning liquid added to the reaction chamber 10. In order to introduce the cleaning liquid, an introduction port 13a is formed through the upper surface side of the disk. In addition, the cleaning liquid storage chamber 13 is arranged on the rotation center side relative to the reaction chamber 10, and is connected to the reaction chamber 10 via a flow path 15. It should be noted that only one cleaning liquid storage chamber 13 is provided in the unit 2 to store a single amount of cleaning liquid. In the disk 1, the cleaning liquid storage chamber 13 stores a cleaning liquid in an amount exceeding the total content of the storage chamber 12 and the tubular channel 11 and less than the total content of the storage chamber 12, the tubular channel 11 and the reaction chamber 10.

[0249] The cutting reagent storage chamber 14 is a chamber designed to store the cutting reagent added to the reaction chamber 10, and has an introduction port 14a formed through the upper surface of the disk for introducing the reagent. The cutting reagent storage chamber 14 is arranged on the rotation center side relative to the reaction chamber 10, and is connected to the reaction chamber 10 via a flow path 16.

[0250] The cutting reagent is adjusted to a high concentration and stored in the cutting reagent storage chamber 14 so that a desired final concentration is achieved when the total amount stored in the cutting reagent storage chamber 14 is added to the reaction chamber 10 .

[0251] The flow paths 15 and 16 are hollow tubes of small diameters that are open at both ends and can be freely switched. Here, "free switch" means a state in which the movement of the fluid inside and outside the flow path is controllable. That is, the open state and the closed state can be switched according to the rotation speed. It should be noted that in the present embodiment in which the rotation speed is increased in stages, when it is in the open state, until a series of treatments are completed, the flow paths 15 and 16 remain in an open state. Although they have the function of being freely switchable, they are physically formed in an openable manner. That is, in the detection method of the present invention, as an alternative to the flow path of free switch, the reagent storage chamber 14 and the reaction chamber 10 can be disconnected by the flow path that can be opened. The flow paths 15 and 16 have a function called a capillary passive valve, which forms an open mode by applying a centrifugal force above a specified value, and the circulation of liquid does not occur when the centrifugal force is below this value. The inner diameter of the flow path 15 is designed to be larger than the inner diameter of the flow path 16, and can be opened with a low centrifugal force (low rotation speed) compared to the open flow path 16. Thus, the cleaning liquid is supplied from the cleaning liquid reservoir 13 to the reaction chamber 10 . Then, when the rotation speed is increased, the flow path 16 is opened, the flow of the cutting reagent in the reagent reservoir 14 is cut off, and the cutting reagent is added to the reaction chamber 10 .

[0252] It should be noted that the rotation speed of supplying the cleaning liquid to the reaction chamber 10 can be determined based on the ease of bursting. The ease of bursting can be changed according to the shape of the flow path and the characteristics of the liquid flowing in 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 (rotation radius) and shape of the flow path, the surface tension, contact angle, and viscosity of the liquid. Therefore, the rotation speed can be selected by theory or experiment considering these factors. It should be noted that, starting from the mechanical load, the preferred rotation speed is less than 10,000 rpm, which is ideal.

[0253] The disk 1 is configured to clean once. When the cleaning frequency is multiple times, a plurality of cleaning liquid storage chambers 13 can be separated and provided with flow paths. Each flow path is connected to the reaction chamber 10. In addition, each flow path is designed to be opened at a lower speed than the opening flow path 16 and at a different rotation speed.

[0254] When the disk 1 is used to detect a target substance, a solution in which beads (solid phase) immobilized with a second capture substance 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 channel 11, and a solid phase having a size that cannot pass through the communication port 11a is selected. Therefore, the solid phase in the introduced solution is retained in the reaction chamber 10.

[0255] Then, the sample diluted to a suitable concentration is supplied from the supply port 10a to the reaction chamber 10, and the target substance in the sample is captured by the second capture substance on the beads. After a predetermined time, a solution containing the first capture substance is supplied from the supply port 10a to the reaction chamber 10, and the first capture substance and the target substance are combined to form a complex. Thus, the complex is retained in the reaction chamber 10. It should be noted that the solvent in the reaction chamber 10 is discharged from the communication port 11a as it rotates, and is stored in the drainage storage chamber 12 through the tubular channel 11.

[0256] When the disk 1 is rotated at a predetermined rotation speed to open the flow path 15, the cleaning liquid in the cleaning liquid reservoir 13 is supplied to the reaction chamber 10 to perform cleaning. Thus, the excess first capture material and other impurities in the reaction chamber 10 are transferred to the drain reservoir 12 by the cleaning liquid.

[0257] As the cleaning liquid is discharged from the cleaning liquid storage chamber 13, the cleaning liquid is gradually stored in the drain storage chamber 12. Since the cleaning liquid storage chamber 13 stores a cleaning liquid exceeding the internal volume of the drain storage chamber 12, the cleaning liquid overflows from the drain storage chamber 12 and enters the flow path 17, and the liquid level in the tubular channel 11 also rises. Here, since the centrifugal force acting toward the outside acts on the disk 1, the cleaning liquid does not flow to the reaction chamber 10 side in a disorderly manner. In addition, the flow path 17 is discharging air, and the air in the reaction chamber 10, the tubular channel 11, and the drain storage chamber 12 flows to the flow path 17 and is pressed toward the cleaning liquid storage chamber 13. When the cleaning liquid is completely discharged from the cleaning liquid storage chamber 13, the drain storage chamber 12 and the tubular channel 11 are filled with liquid, and liquid is also accumulated in the reaction chamber 10, forming a state in which a gap is left on the rotation center side and the solid phase is impregnated. It should be noted that most of the first capturing material transferred to the waste liquid storage chamber 12 along with the flow of the cleaning liquid is precipitated to the bottom surface (outside) of the waste liquid storage chamber 12 by the centrifugal force acting thereon.

[0258] Next, the rotation speed is increased to a predetermined rotation speed to open the flow path 16, and the cutting reagent stored in the cutting reagent storage chamber 14 flows into the reaction chamber 10. As a result, the concentration of the cutting reagent in the reaction chamber 10 gradually increases, and the part containing the marker is cut and separated from the complex. The separated part containing the marker passes through the tubular channel 11 and moves to the drainage storage chamber 12.

[0259] The predetermined position of the tubular channel is irradiated with irradiation light from a light source. Therefore, the portion containing the marker generates scattered light when passing through the tubular channel 11. By detecting it with a light receiving element, the portion containing the marker can be appropriately detected, and as a result, the target substance can be detected.

[0260] Figure 3 FIG. 1 is a modified example of the disk 1 of the first embodiment, and is a diagram showing another example of the disk. Specifically, the modified example changes the unit 2 of the disk 1. Figure 3 The unit 2 of this modified example is shown, and the same parts as those of the above-mentioned disk 1 are denoted by the same reference numerals, and the description thereof is omitted.

[0261] In the unit 2, a first storage chamber 101 for holding a sample, a flow path 102 connected to the first storage chamber 101, a second storage chamber 103 for storing a liquid filling the tubular channel 11, and the tubular channel 11 are provided outwardly from the rotation center side, and a thin flow path 17 branching from the front end of the tubular channel 11 and extending from the end of the branch flow path toward the rotation center side is also provided.

[0262] This unit 2 is used to introduce a sample that has been subjected to separation of a portion containing a labeled substance in a reaction field outside the disk 1 into the disk 1 and to detect the portion containing the labeled substance. Therefore, a washing process is not performed on the disk 1.

[0263] In the unit 2, first, a predetermined amount of sample solution is introduced into the first storage chamber 101 from the opening 101a on the upper surface of the first storage chamber 101. Here, the flow path 102 is configured in a manner having the function of a capillary passive valve, so that the sample solution introduced in a static state does not flow to the second storage chamber 103 through the flow path 102. In addition, a predetermined amount of liquid filling the tubular channel 11 is introduced into the second storage chamber 103 from the opening 103a on the upper surface of the second storage chamber 103. In the second storage chamber 103, an introduction portion is provided protruding toward the rotation center side at the end portion on the rotation center side and at a position deviated from the extension line of the flow path 102. The opening 103a is formed through the upper surface of the introduction portion. As a result, when the sample solution held in the first storage chamber 101 is introduced into the second storage chamber 103, it is possible to suppress the liquid from leaking out from the opening 103a.

[0264] The tubular channel 11 abuts against the upper portion of the second storage chamber 103 in the depth direction to form a communicating structure. Although the second storage chamber 103 stores a sufficient amount of liquid to fill the tubular channel 11, in order to fill the tubular channel 11 with the liquid, it is necessary to fill the tubular channel 11 with the liquid to the position corresponding to the communication port (inlet) 11a of the tubular channel 11 in the tubular channel 11, the branched flow path before it, and the flow path 17 communicating therewith, so the second storage chamber 103 is designed based on the total volume of the corresponding parts of these tubular channel 11, the branched flow path before it, and the flow path 17. In addition, the structure of the second storage chamber 103 is designed in such a way that the liquid level of the stored liquid is lower than the bottom surface of the communication port 11a of the tubular channel 11 when the liquid is stored. When the device 1 is formed of a general-purpose resin material, the interiors of the first and second storage chambers 101 and 103 become hydrophobic surfaces, thereby playing the role of keeping the introduced aqueous sample solution and the liquid for filling the tubular channel in the chambers.

[0265] Then, the device 1 is rotated at a predetermined rotation speed without opening the flow path 102, and the liquid in the second storage chamber 103 is introduced into the tubular passage 11. Here, the flow path 17 has a function of removing air, so when the disk 1 is rotated at a predetermined rotation speed, the liquid in the second storage chamber 103 can be smoothly introduced into the tubular passage 11.

[0266] Next, the rotation speed is increased to a speed that opens the flow path 102, and the sample solution flows from the first reservoir 101 to the second reservoir 103. In addition, the portion containing the marker moves outward in the tubular channel 11 by centrifugal force. Thus, when the cutting process is performed in the reaction field outside the disk 1, the target substance can be detected with a simple device configuration.

[0267] It should be noted that, during the rotation of the disk 1, for example, Figure 4The device shown. The device comprises a motor and a rotating shaft connected to the motor, on which a mounting table for rotating the disk 1 in the horizontal direction is axially supported. When the motor is driven, the disk 1 placed on the upper surface of the mounting table is rotated, imparting centrifugal force. The motor is connected to a microcontroller not shown in the figure, and is connected to an information processing device (personal computer (PC)) through the microcontroller. The information processing device sends and receives signals to and from each connected device, thereby controlling each device. It should be noted that 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 is input from the information processing device to the microcontroller, the microcontroller controls the motor in such a way that it rotates for a specified time at a predetermined speed set in advance.

[0268] When the disc 1 is placed on the stage, a window penetrating in the thickness direction is provided at a position opposite to the tubular passage 11. A laser light source (LD) is provided above the stage, and the laser light source (LD) is adjusted so that the irradiation light is incident on the upper surface of the stage at an inclined angle. A focusing lens (Lens) is provided in front of the laser light source, and the irradiation light from the laser light source is adjusted so that it is focused inside the tubular passage 11. The laser light source is connected to the PC via the above-mentioned microcontroller, and its output is controlled by the microcontroller receiving instructions from the PC.

[0269] A focusing lens (Lens) and a photodiode (PD) are arranged below the window of the mounting table to receive scattered light. Through the photoelectric conversion of the photodiode, the scattered light intensity is converted into a voltage and input into the signal detector (Oscilloscope). In the signal detector, sampling is performed at a specified sampling time, and the input analog signal (voltage value) is converted into a digital signal and input into the PC. Then, the measurement result of the scattered light is output in the PC. Therefore, the scattered light of the marker can be obtained and the target substance can be detected.

[0270] The present invention has been described above based on the embodiment, but the present invention is not limited to the above embodiment at all, and it is easily presumed that various improvements and modifications can be made without departing from the gist of the present invention.

[0271] For example, the disk 1 can be appropriately provided with a mechanism for controlling the movement of the fluid. As such a mechanism, a valve or the like can be exemplified. The valve switches the communication port of the flow path or the chamber between a closed state and an open state. In addition to the capillary passive valve provided in the flow paths 15 and 16, various valves that are operated by electricity, magnetism, electromagnetic force, mechanical mechanism, inertia, centrifugal force, or heat can also be used.

[0272] Furthermore, the disk 1 is described with a circular disk, for example, but in the present invention, it is not limited to a circular disk and can be in other shapes. That is, in the method of the present invention, the device can be in a form that can impart centrifugal force, and there is no obvious restriction on its shape. Therefore, for example, a chip-type device having only one unit 2 and a fan-shaped and rectangular shape can be exemplified. In this case, a mechanism and a fixture for holding the chip are provided on the mounting table of the device that rotates the device, and the centrifugal force can be imparted by embedding the chip in these and rotating it.

[0273] As modified examples of the present invention, the following inventions can be exemplified.

[0274] A method for detecting a target substance is shown, wherein a sample in which a complex formed by a first capture substance bound to a marker capturing a target substance and the first capture substance that does not capture the target substance are mixed is brought into contact with a binding agent that specifically binds to the first capture substance, the first capture substance that does not capture the target substance is bound to the binding agent, the binding agent is retained in a chamber for storing the sample, centrifugal force is applied, the complex is guided out of the chamber and moved in a tubular channel filled with liquid, and the complex is detected by scattered light generated by scattering irradiated light by the complex moving in the tubular channel.

[0275] In this variant, the detection object is a complex formed by the combination of the target substance and the first capture substance. Here, the excess first capture substance that is not bound to the target substance is also bound to the marker, so if it is introduced into the detection field, scattered light will also be detected from the unreacted first capture substance, and the detection result will be erroneous. In the first embodiment described above, the target substance is fixed to the solid phase and the first capture substance is loaded on the solid phase. In addition, the excess first capture substance is removed by appropriate cleaning treatment. In this variant, the excess first capture substance is bound to the binding agent. In addition, the binding agent is retained in the chamber, so that the excess first capture substance can be retained in the chamber, inhibiting its introduction into the detection field, and can omit cleaning and improve the accuracy of complex detection.

[0276] The formation of the complex and the binding of the excess first capture substance with the binding agent are carried out in a state where a liquid is stored. For example, if a tubular channel and an antechamber (chamber) for supplying a sample to the tubular channel are provided in a device and a binding agent is retained in the antechamber, the excess first capture substance contained in the sample introduced into the antechamber can be bound to the binding agent and retained in the antechamber. It should be noted that the chamber in which the binding agent is retained does not need to be formed integrally with the device, and other chambers provided outside the device can be used to retain the excess first capture substance. In the case of retaining the excess first capture substance in the chamber, a method such as pre-fixing the binding agent to the inner wall of the chamber or fixing the binding agent to a solid and introducing the solid into the chamber can be appropriately selected.

[0277] In addition, when the binding agent is introduced into the front chamber, the excess first capture substance can be retained as long as the solid to which the binding agent is fixed is larger than the tubular channel. Furthermore, it can also be configured as follows: a valve is provided in the chamber, and after the liquid is filled into the tubular channel, the valve is opened to introduce the complex formed by the combination of the target substance and the first capture substance into the tubular channel.

[0278] <Binder>

[0279] The binding agent is a substance that specifically binds to the first capture substance, and an antigen is preferably exemplified. In addition, the antigen can be directly loaded on a solid phase such as an inner wall or a solid, or can be indirectly fixed to the solid phase with the aid of an antibody, etc. In addition, for example, when an antigen is used as a binding agent, a label such as biotin can be added to the antigen, and if avidin is pre-bound to the solid phase, the binding agent can be fixed to the solid phase by the combination of biotin-avidin.

[0280] It should be noted that the binding between the solid phase and the binding agent can be achieved by physical adsorption, covalent bonding, ionic bonding, binding using affinity, and combinations thereof. Preferably, the binding agent is fixed to the solid phase by covalent bonding or ionic bonding with a stronger binding force than that based on affinity or adsorption.

[0281] It should be noted that when the marker is larger than the first capture substance, multiple first capture substances may be bound to the marker. In this variation, the smaller the number of first capture substances bound to the marker, the better the effect can be achieved. Ideally, it is expected that there is only one first capture substance bound to the marker.

[0282] Example

[0283] The present invention is further described in detail by way of examples. It should be noted that the present invention is not limited by the examples.

[0284] (Reference Example 1) Preparation of platinum-gold colloidal suspension

[0285] The glassware used is all cleaned with aqua regia. 390mL of ultrapure water is added to a flask to make it boil, chloroauric acid aqueous solution (per 1 liter of aqueous solution, 1g in gold, Katayama Chemical Industry Co., Ltd. system) 30mL is added to the boiling water, then 1 wt% sodium citrate aqueous solution 60mL is added. After adding sodium citrate aqueous solution, chloroplatinic acid aqueous solution (per 1 liter of aqueous solution, 1g in platinum, Wako Pure Chemical Industries, Ltd. system) tens of mL are added. 1 wt% sodium citrate aqueous solution 60mL is added after 5 minutes, refluxed for 4 hours, and platinum-gold colloidal suspension is obtained. It should be noted that, in order to reliably detect the part comprising the label, reagent, reducing agent are suitably added and the particle size of the colloid is adjusted to more than 100nm.

[0286] (Reference Example 2) Preparation of monoclonal antibodies

[0287] The commercially available SARS-CoV-2 nucleocapsid protein (hereinafter referred to as "SARS-CoV-2NP") (CUSABIO) was used as an immunization antigen, and anti-SARS-CoV-2NP monoclonal antibodies (hereinafter referred to as "anti-NP antibodies") were obtained according to conventional methods.

[0288] Specifically, mice (BALB / c, 5 weeks old, Japanese SLC) were immunized 3 times with 100 μg of SARS-CoV-2NP, and their spleen cells were used for cell fusion. For cell fusion, Sp2 / 0-Ag14 cells (Shulman et al., 1978), which are mouse myeloma cells, were used. During cell culture, a culture medium was used in which 0.3 mg / ml of L-glutamine, 100 units / ml of penicillin G potassium, 100 μg / ml of streptomycin sulfate, and 40 μg / ml of gentamicin were added to Dulbecco's modified Eagle's medium (Gibco) and fetal bovine serum (JRH) was added at 10%. Cell fusion was performed by mixing spleen cells and Sp2 / 0-Ag14 cells of immunized mice and adding a polyethylene glycol solution (Sigma) thereto. The fused cells were cultured with HAT-RPMI [RPMI supplemented with serum containing 0.1 mM hypoxanthine sodium, 0.4 μM aminopterin, and 0.1 mM thymidine (Gibco)], and the antibody production in the culture supernatant was confirmed by enzyme-linked immunosorbent assay (ELISA). Cells positive for antibody production were cultured with HT-RPMI [RPMI supplemented with serum containing 0.1 mM hypoxanthine sodium and 0.1 mM thymidine] to proliferate.

[0289] The cloned cells were inoculated into the peritoneal cavity of mice (BALB / c, retired, Japan SLC) inoculated with 2,6,10,14-tetramethylpentadecane (Kishida Chemical Co., Ltd.), and ascites was collected. The ascites was supplied to a protein G column to purify monoclonal antibodies. Finally, 83 clones of cells producing monoclonal antibodies against SARS-CoV-2NP were obtained.

[0290] (Reference Example 3) Preparation of gold colloidal suspension

[0291] Gold colloid particles are prepared by conventional methods using an aqueous solution of chloroauric acid (1 g of gold per liter of aqueous solution, manufactured by Katayama Chemical Industry Co., Ltd.) as a raw material. Specifically, 1 ml of a 1% (v / w) aqueous solution of chloroauric acid is added to 99 ml of pure water that is heated and boiled, and then 1.5 ml of a 1% (v / w) aqueous solution of sodium citrate is added after 1 minute, and the mixture is heated and boiled for 5 minutes and then cooled at room temperature. Subsequently, a 200 mM aqueous solution of potassium carbonate is added to the solution to adjust the pH to 9.0, and ultrapure water is added thereto to make the total amount 100 ml to obtain a gold colloid suspension in which gold colloid particles are suspended. It should be noted that when the desired particle size is not obtained, the amount of reducing agent added relative to chloroauric acid is appropriately adjusted to produce gold colloid particles of the desired particle size.

[0292] (Example 1) Separation of the fraction containing the marker (platinum-gold colloid particles) using a pH adjuster

[0293] (1) Preparation of Platinum-Gold Colloidal Labeled Antibody Solution

[0294] For one anti-NP antibody selected from the anti-NP antibodies obtained in Reference Example 2, platinum-gold colloid labeling was performed according to the following steps. An anti-NP antibody having a protein conversion weight of 1 μg (hereinafter, when the protein conversion weight is shown, the weight value based on the weight analysis of the purified protein is shown) and 1 mL of a platinum-gold colloid suspension having a particle size of 120 nm prepared in Reference Example 1 were mixed and allowed to stand at room temperature for 2 minutes to allow the anti-NP antibody to bind to the surface of the platinum-gold colloid particles. Then, a 10% bovine serum albumin (hereinafter referred to as "BSA") aqueous solution was added in a manner to a final concentration of 1.0% relative to the total liquid volume, and the residual surface of the platinum-gold colloid particles was blocked with BSA, thereby preparing a platinum-gold colloid-labeled anti-NP antibody solution. The solution was then 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), thereby preparing a platinum-gold colloid-labeled antibody solution.

[0295] (2) Cutting treatment using pH adjuster

[0296] The anti-NP antibody obtained in Reference Example 2, which is different from the anti-NP antibody used in the above-mentioned platinum-gold colloid-labeled anti-NP antibody, is prepared into 20 μg / mL with Tris hydrochloric acid buffer, thereby making an antibody dilution, and 100 μL of the antibody dilution is added to each well of a 96-well microplate, and the antibody is fixed by standing for 15-18 hours. Then, after the blocking treatment is implemented, 50 μL of SARS-CoV-2NP solution prepared into 10 ng / mL and 50 μL of platinum-gold colloid-labeled antibody solution are added, and the solution is left to stand at 37 ° C for 10 minutes. Then, it is washed with TBS-T (Tris Buffered Saline with Tween 20), and various reagents with different pH values ​​of 100 μL are added after the cleaning solution is discharged. After standing for 2 minutes at room temperature, the solution in the hole is recovered. The types and pH values ​​of the reagents used are shown in Table 1. The pH value is measured using a pH meter (made by LAQUA Horiba Co., Ltd.).

[0297] It should be noted that the pH of the citric acid solution is adjusted by mixing citric acid and sodium citrate to obtain a solution of the desired pH (pH 2.94 to pH 6.96). In addition, the phosphoric acid solution is prepared as follows: sodium dihydrogen phosphate and disodium hydrogen phosphate are mixed to obtain a desired pH near neutral (pH 6.99 to 8.56); disodium hydrogen phosphate and sodium phosphate are mixed to obtain a desired alkaline pH (pH 11.33 to 12.85). For glycine hydrochloride buffer, glycine NaOH buffer and triethylamine solution, hydrochloric acid or sodium hydroxide is added to adjust to the desired pH. In addition, the pH of the hydrochloric acid solution and the sodium hydroxide solution is adjusted by changing the dilution ratio.

[0298] [Table 1]

[0299]

[0300] (3) Determination of platinum-gold colloidal particles

[0301] The amount of platinum-gold colloidal particles contained in the solution recovered by the above operation was measured. Specifically, the recovered solution was introduced into a sample cell, a 650 nm laser beam was irradiated to the sample cell, and the resulting scattered light was measured using a light emitting diode installed at a predetermined position.

[0302] Since the scattered light intensity increases in proportion to the concentration of the platinum-gold colloid particles in the solution, the concentration of the platinum-gold colloid particles in the solution can be quantitatively analyzed by measuring the scattered light intensity. That is, by treating the immune complex with a pH adjuster, the "part containing the platinum-gold colloid particles" separated from the solid phase or the immune complex can be detected.

[0303] It should be noted that when the platinum-gold colloid-labeled anti-NP antibody is non-specifically adsorbed (in a place other than the antigen) and is not removed by a cleaning operation, it will be separated by a pH adjusting agent and mixed into the assay sample. Therefore, only 100 μL of a platinum-gold colloid-labeled anti-NP antibody solution is added to the hole treated with the same antibody fixation as above and left to stand at 37 ° C for 10 minutes, followed by adding 100 μL of a pH adjusting agent solution after washing with TBS-T, and the solution is recovered from the hole after standing for 2 minutes at room temperature, and scattered light is measured. The same operation is performed on each pH adjusting agent, and the obtained scattered light intensity is used as the blank value of each pH adjusting agent. It should be noted that when the processing capacity of the cutting complex of the pH adjusting agent is weak, the difference between the scattered light intensity of the sample and the scattered light intensity of the blank becomes smaller, so in the result of the difference between the two, the scattered light intensity of the measurement result of the sample is sometimes expressed as a negative value.

[0304] The blank value can evaluate the scattered light intensity of the platinum-gold colloid particles derived from the platinum-gold colloid-labeled anti-NP antibody non-specifically adsorbed in the wells, so the blank value is subtracted from the measured value to obtain the scattered light intensity of the "part containing the platinum-gold colloid particles" of the formed immune complex. The results are shown in Figure 5 .

[0305] Figure 5 This is a graph showing the relationship between the pH value of each reagent and the shutdown characteristics. Figure 5 The pH value of each reagent and the corresponding scattered light intensity value are displayed below. Figure 5 The values ​​are shown graphically on the upper side of the graph. The horizontal axis of the graph is pH value, and the vertical axis is scattered light intensity. Figure 5 It can be seen that when the pH value is near neutral, the scattered light intensity is low. That is, it shows that the added neutral reagent has low cutting performance of the "part containing platinum-gold colloid particles" of the complex. On the other hand, when the added reagent is acidic or alkaline, 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 cut by pH adjusters such as acids and bases.

[0306] In particular, it is shown that a pH adjuster (acid) having a pH lower than 4 and a pH adjuster (base) having a pH of 10.6 or higher have good shutdown performance, and a pH adjuster (base) having a pH of 11.6 or higher has excellent shutdown performance.

[0307] (Example 2) Separation of the fraction containing the marker (platinum-gold colloid particles) using sodium hydroxide

[0308] The results of Example 1 show that the cutting ability of the sodium hydroxide solution is particularly good, so the concentration of sodium hydroxide was changed to further evaluate the performance of cutting the portion containing the marker from the complex bound to the solid phase (solid phase-anti-NP antibody-SARS-CoV-2NP-platinum-gold colloid-labeled anti-NP antibody).

[0309] The concentration of sodium hydroxide was set to 0.01 mM, 0.1 mM, 1 mM, 10 mM, 100 mM, and 1 M. The same treatment as in Example 1 was performed except that 100 μL of the sodium hydroxide aqueous solution prepared to each concentration was used. The results are shown in Figure 6 .

[0310] Figure 6 This is a graph showing the cutting performance of the composite body by sodium hydroxide. Figure 6 The pH value and scattered light intensity values ​​at various concentrations of sodium hydroxide are shown below. Figure 6 The values ​​are shown in a graph on the upper side. The horizontal axis of the graph is the molar concentration of sodium hydroxide, and the vertical axis is the scattered light intensity.

[0311] Depend on Figure 6 It is known that sodium hydroxide separates (liberates) the part of the immune complex containing platinum-gold colloidal particles at a concentration of 10 mM or more. The pH of a 10 mM sodium hydroxide solution is 11.87.

[0312] (Example 3) Separation of the fraction containing the marker (platinum-gold colloid particles) using a denaturant

[0313] A microplate having holes to which anti-NP antibodies were fixed and blocked was prepared by the same method as in Example 1. Then, a denaturant was used instead of a pH adjuster to act on the complex formed with the holes of the microplate as a solid phase (solid phase-anti-NP antibody-SARS-CoV-2NP-platinum-gold colloid-labeled anti-NP antibody). Otherwise, the solution in the holes was recovered in the same manner as in Example 1, and the amount of platinum-gold colloid particles was measured in the same manner as in Example 1 (3). It should be noted that 8M urea, 6M guanidine hydrochloride, or 1% SDS was used as the denaturant. The results are shown in Figure 7 It should be noted that, for comparison, the results obtained by the same treatment using 1M-NaOH are shown together. Figure 7 .

[0314] Figure 7 This is a graph showing the effect of a denaturant on an immune complex bound to a solid phase using the scattered light intensity of separated (free) platinum-gold colloid (part containing platinum-gold colloid particles). As comparative data, the case of using 1M-NaOH is shown. Figure 7 It was found that any denaturant liberated the portion containing the platinum-gold colloidal particles.

[0315] (Example 4) Sensitization treatment of gold colloidal particles

[0316] The gold colloidal particle suspension (particle size of about 12 nm) prepared in Reference Example 3 is prepared with pure water to a concentration of 4.36 nM, and 2 μL thereof is added to 100 μL of 1M NaOH and mixed. The treatment with 1M NaOH represents the cutting treatment of the complex with a pH adjuster. 100 μL of 1M HCl is added thereto for neutralization. 6.35 μL of the neutralized solution is added to a mixture of 972 μL of 10mM Walpole buffer (pH 2.0) and 1.65 μL of 12.1M chloroauric acid solution, and 20 μL of 375mM hydroxylamine hydrochloride is added and mixed, and the mixture is allowed to stand at room temperature for 10 minutes for sensitization reaction. The gold colloidal particles in the solution are then centrifuged to settle, the supernatant is removed, and the mixture is washed with ultrapure water. 1 μL of the gold colloidal particle dispersion thus obtained was dripped onto a sample holder attached with a carbon tape, the atmosphere was removed, and the sample was photographed using an electron microscope TM4000Plus (manufactured by Hitachi, Ltd.). The applied voltage was set to 15 kV and the magnification was set to 1000. Five or more fields of view were photographed and image analysis was performed to calculate the average particle size and standard deviation. It should be noted that ultrapure water without gold colloidal particles was used instead of the 4.36 nM gold colloidal particle dispersion and the same sensitization treatment was performed as a comparison object. The results are shown in Figure 8 .

[0317] Figure 8 This is a graph showing changes in the particle size of gold colloid caused by the sensitization reaction. Figure 8 The vertical axis represents the particle size. Figure 8 It can be seen that through the sensitization reaction, the gold colloid particles in the 4.36nM gold colloid particle dispersion can grow into particles with a particle size of about 3 to 5μm. On the other hand, when there are no gold colloid particles, the precipitation of gold colloid particles is also observed, but the particles are significantly smaller than when the sensitization treatment is performed, so the two can be clearly distinguished in the detection. It should be noted that the particle size can of course be adjusted by adjusting the reaction conditions, so it can be seen that gold colloid particles with a particle size suitable for the detection system can be prepared.

[0318] This result shows that the sensitization reaction can increase the particle size of tiny gold colloid particles, and the sensitized gold (colloid) particles can be fully detected by optical means.

[0319] (Example 5) Changes in scattered light intensity due to the particle size of metal particles

[0320] (1) Preparation of gold and platinum colloid particles of various particle sizes

[0321] Platinum-gold colloidal particles with a particle size of 120 nm were prepared by Reference Example 1. In addition, gold colloidal particles with particle sizes of 12 nm and 200 nm were prepared by Reference Example 3, and commercially available gold (colloidal) particles with particle sizes of 500 nm and 1000 nm (NANOPARTz, A11-500-CIT-DIH-1-50, A11-1000-NPC-DIH-1-100) were used. For each particle, particles dispersed in a solution were prepared.

[0322] (2) Determination of scattered light intensity

[0323] The gold colloidal particle dispersions of various particle sizes prepared above were diluted to a concentration within the range in which scattered light could be measured, and the scattered light measurement was performed in the same manner as in Example 1. The scattered light intensity value obtained 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 colloidal particles increased. The results are shown in Fig. 9 .

[0324] Fig. 9 Graph showing the relationship between the particle size of metal colloid particles and the scattered light intensity. The horizontal axis represents the particle size (nm), and the vertical axis represents the scattered light intensity in logarithmic terms. Fig. 9 It can be seen that the scattered light intensity increases significantly from 120 nm, indicating that metal colloidal particles can be detected from about 100 nm using a simple scattered light detection system of a laser source and a light emitting diode.

[0325] (Example 6) Antigen detection based on a portion containing a marker (platinum-gold colloidal particles)

[0326] The anti-NP antibody used for fixation in Example 1 was prepared to 20 μg / mL with Tris-HCl buffer, thereby preparing an antibody dilution solution. It was mixed with magnetic particles (manufactured by JSR LIFE SCIENCES) to fix the anti-NP antibody to the magnetic particles by chemical bonds. The anti-NP antibody fixed to the magnetic particles, each SARS-CoV-2NP solution prepared at a concentration of 10, 100, 1000, and 10000 pg / mL, and the platinum-gold colloid-labeled anti-NP antibody prepared in Example 1 were then reacted at 37°C for 30 minutes. Each of the solutions after the reaction was centrifuged using a 0.45 μm centrifugal filter (Merck), the magnetic particles were recovered, and washed 5 times with TBS-T. 1M-NaOH was then added to the magnetic particles, and the complex bound to the magnetic particles (magnetic particles-anti-NP antibody-SARS-CoV-2NP-platinum-gold colloid-labeled anti-NP antibody) was cut (eluted), and the scattered light intensity of the recovered eluate was measured. The measurement results are shown in Fig.10It should be noted that, regarding the dissolution in the examples, it is sufficient to cut off the portion containing the marker as a part of the complex, so that this portion is separated from the solid phase and released into the liquid, and it should not be construed as limiting the separation to dissolving in the liquid.

[0327] Fig.10 This is a graph showing the concentration of dissolved platinum-gold colloid particles (parts containing platinum-gold colloid particles) in terms of scattered light intensity. The vertical axis shows the scattered light intensity in logarithmic form, and the horizontal axis shows the antigen concentration. Fig.10 It is known that the scattered light intensity based on the platinum-gold colloid particles is proportional to the antigen concentration. That is, when the antigen concentration increases, the dissolved platinum-gold colloid particles also increase, thus confirming that the complex formed by the antigen-antibody reaction on the solid phase is effectively cut, and the cut part containing the marker is separated, dissolved and properly detected.

[0328] (Example 7) Detecting scattered light from a rotating disk

[0329] (1) Experimental evaluation of the outflow of gold colloid particles

[0330] This embodiment evaluates the outflow of gold colloidal particles by experiment. In order to conduct the experiment, a disk is prepared with a chamber separated at the center side of a circular plate and a flow path connected to the chamber and extending outward in the radial direction of the circular plate. On the upper surface of the circular plate where the chamber and the flow path are set in the recess of the disk, a polypropylene sheet (hereinafter simply referred to as "PP sheet") with a thickness of 50 μm is adhered by means of an adhesive layer, and the upper surface of the chamber and the flow path is formed by the PP sheet.

[0331] Fig.11 This is a diagram schematically showing a cross section of a portion of the flow path portion of the disk used in this embodiment. The inner side of the chamber is formed to have a depth of 1000 μm in the thickness direction, and the upper part of the wall on the opposite side of the rotation center in the chamber is connected to the flow path. The flow path is formed in a manner of 230 μm in width and 15 μm in depth, and has a rectangular flow path cross section, forming a capillary passive valve that opens when the disk rotates at a speed exceeding a specified speed and allows liquid to flow. In addition, as Fig.11 As shown, the wall of the chamber connected to the flow path is inclined and rises obliquely from the bottom surface. Therefore, the depth of the chamber gradually becomes shallower toward the flow path. In the prepared tray, a through hole is pierced through the PP sheet on the upper surface of the chamber to allow the inside and outside of the chamber to pass through.

[0332] With the help of the through hole formed by drilling, a dispersion of polystyrene beads (PS beads) (micromod company, particle size 20μm, 25mg / ml aqueous dispersion) was injected into the chamber. The injected dispersion was a small amount compared to the capacity of the chamber, so a liquid pool was formed near the through hole due to surface tension. Then, the solvent in the chamber was rotated at 2000rpm for 1 minute to discharge it into the flow path. For the solvent after passing through the flow path, a hole was opened on the PP sheet on the upper surface of the chamber in front of the flow path, and all of the solvent was sucked in and recovered with a pipette.

[0333] Afterwards, 10 μL of the gold colloid particle dispersion prepared in Reference Example 3 was injected into the chamber from the through-hole. As a result, a liquid pool of the gold colloid particle dispersion was formed near the through-hole due to surface tension. Next, the solution in the chamber was rotated at 2000 rpm for 1 minute to allow the solution to flow out to the flow path, and the liquid after passing through the flow path was similarly recovered from the chamber in front of the flow path. For the recovered liquids of the gold colloid particle dispersion and the PS bead dispersion prepared in Reference Example 3, the absorbance or scattered light intensity was measured using a micro-spectrophotometer or a scattered light intensity measuring device, and the two were compared. As a result, none of the recovered liquids contained PS beads, while on the other hand, the recovered liquid derived from the gold colloid particle dispersion contained gold colloid particles exceeding 60% of the initial concentration. It can be confirmed that the gold colloid particles pass through the PS beads used for interception and flow out of the chamber to the flow path.

[0334] (2) Specific detection of markers by scattered light

[0335] Then, scattered light detection of the marker is performed when the biological material coexists. BSA is used schematically as an inclusion. The gold colloid particles use a dispersion of gold colloid particles with a particle size of 200 nm prepared in the same way as in Reference Example 3. Prepare a 10% BSA solution (manufactured by ORIENTAL YEAST Co., Ltd., Cat 47408903) or water, and dilute the 2pM gold colloid particle dispersion to 2fM as a solvent. Add the diluted gold colloid particle dispersion and 10% BSA solution to 300μL colorimetric tubes, respectively, and measure the scattered light intensity using the scattered light detection device used in Example 1.

[0336] In order to conduct the experiment, a disk was prepared with a chamber separated on the center side of rotation and a flow path connected to the chamber and extending outward in the radial direction of the disc. In the disk, the chamber and the flow path are recessed on the resin disc, and a 50μm thick PP sheet is tightly attached to the upper surface of the disc with the help of an adhesive layer. The upper surface of the chamber and the flow path is formed by the PP sheet. The chamber and the flow path are both formed with a depth of 15μm, and the bottom surfaces are continuous with each other. The width of the flow path is 1mm, and the front end of the extended flow path is connected to the branch path of the straight flow path, and the branch path is connected to the thin flow path at both ends toward the rotation center side. The thin flow path is connected to the end of the rotation center side of the chamber and is connected in the end of the rotation center side of the chamber. The chamber is formed wider than the flow path. In addition, a through hole is pierced on the upper surface of the chamber.

[0337] Into the chambers of the disk, BSA solution or a gold colloidal particle dispersion diluted to 2 fM with BSA solution was injected. Figure 4 The stage of the device shown was rotated at 2000 rpm for 2 minutes, and the number of scattered lights generated was counted.

[0338] [Table 2]

[0339]

[0340] When calibrated with water = 0

[0341] In Table 2, the measurement results of gold colloid particle dispersion ("GNP"), BSA solution ("BSA"), and gold colloid particle dispersion diluted with BSA solution ("GNP+BSA") are shown in order from the upper section. The middle column shows the scattered light intensity measurement results obtained using the scattered light detection device, and the rightmost column shows the counting results of the number of scattered light generated from the rotating disk. It can also be seen that in the scattered light intensity measurement using a colorimetric tube, the scattered light is measured separately in the case of gold colloid particle dispersion and BSA solution, and the sample mixed with the two measures stronger scattered light. According to the results, in the measurement method of detecting scattered light by irradiating irradiation light in a static state using a cuvette with a larger capacity such as a 300μL colorimetric tube, it is difficult to specifically detect the detection object such as gold colloid particles by scattered light in the presence of inclusions. On the other hand, when using a disk, the measurement with water as the blank and the measurement of the BSA solution are the same results, and the generation of scattered light from BSA is not counted (detected). Furthermore, when the gold colloidal particle dispersion is measured, the generation of scattered light originating from the gold colloidal particles is counted, indicating that the gold colloidal particles are detected.

[0342] Fig.12 Graphs showing the results of gold colloidal particle detection using a disk. Fig.12The horizontal axis of represents the position in the flow channel width direction in the tubular channel, and the vertical axis represents the intensity of scattered light in terms of voltage value. In addition, the peak of the counted gold colloid particles is indicated by ▼. Fig.12 The change of the detection peak over time is shown from top to bottom in the figure. According to the result, there are peaks that are continuously observed without changing over time and intermittent peaks that appear only for a short time. Peaks that do not change over time can be judged as noise. Therefore, the scattered light intensity (voltage value) of the blank at the same position of the tubular channel is obtained, and the noise can be eliminated by taking the difference between the intensity (voltage value) of the scattered light obtained in any cycle during the measurement. On the other hand, the intermittent peaks that are generated intermittently can be judged as peaks of gold colloid particles based on the dynamics of the flow path. Therefore, it is shown that even if there are inclusions, the target detection object can be separated and specifically detected.

[0343] (Example 8) Using a rotating chip-type device to separate a portion containing a label (gold colloid particles) and detect scattered light

[0344] (1) Preparation of gold colloid-labeled antibody solution

[0345] As a label, the gold colloid particles with a particle size of 200 nm prepared in Reference Example 3 were used instead of the platinum-gold colloid particles, and the gold colloid-labeled anti-NP antibody was prepared by the same steps as the preparation of the platinum-gold colloid-labeled antibody in Example 1. The prepared gold colloid-labeled anti-NP antibody was suspended in the sample extract to prepare a gold colloid-labeled antibody solution.

[0346] (2) Preparation of polystyrene beads immobilized with antibodies

[0347] Among the anti-NP antibodies prepared in reference example 2, an antibody different from the anti-NP antibody used in the above-mentioned gold colloid-labeled anti-NP antibody is selected, and it is bound to polystyrene beads (manufactured by Micromod, particle size 20 μm, 25 mg / ml aqueous dispersion) by the maleimide method, thereby preparing polystyrene bead-fixed anti-NP antibodies.

[0348] (3) Formation of antibody-antigen-antibody sandwich-like immune complex

[0349] Using SARS-CoV-2NP as an antigen, the antigen, the gold colloid-labeled antibody solution, and the polystyrene bead-immobilized anti-NP antibody are adjusted to a predetermined concentration using the sample extract. These are then mixed and reacted at room temperature for 10 minutes to generate an antibody-antigen-antibody sandwich-like immune complex.

[0350] (4) Cleaning

[0351] A chip-type device having one unit formed independently is prepared. Figure 2bSimilarly, it has a reaction chamber, a flow path, a tubular channel, a drainage storage chamber, a cleaning liquid storage chamber and a reagent storage chamber (cut-off reagent storage chamber).

[0352] Then, the solution containing the immune complex prepared in the above step is introduced into the chip-type device. Specifically, the solution containing the immune complex prepared in the above step is injected into the reaction chamber, and the chip-type device is placed in Figure 4 The mounting table of the device shown in the figure rotates at 2000 rpm for 10 seconds to discharge the liquid from the reaction chamber, then stops rotating, and injects the cleaning liquid into the reaction chamber. After that, the chip-type device is rotated at 2000 rpm for 9 minutes to perform a cleaning process. After the cleaning process, the tubular channel is filled with liquid. In addition, the cleaning liquid is filled and stored to a position exceeding the entrance of the tubular channel and roughly immersing the polystyrene beads in the reaction chamber.

[0353] (5) Separation of the fraction containing the marker (gold colloid particles) and specific detection of the marker by scattered light

[0354] After the cleaning treatment, 1M NaOH as a cutting agent is injected into the reaction chamber. Then, the chip-type device is rotated at 2000 rpm for 3 minutes to separate the gold colloid particles (the part containing the gold colloid particles) from the complex, that is, to dissolve them into the liquid stored in the reaction chamber. The gold colloid particles dissolved into the liquid in the reaction chamber pass through the tubular channel due to centrifugal force, and the scattered light generated by irradiating the irradiation light at this time is detected. The detected scattered light is converted into an electrical signal and obtained in the form of a voltage value. Fig.13a and Fig.13b The results of scattered light detection when the antibody concentration was adjusted to 0.5 pg / mL to form an immune complex are shown.

[0355] Fig.13a , Fig.13b This is a diagram showing the result of detecting gold colloid particles (a portion including gold colloid particles) by scattered light in this example. Fig.13a In the figure, the horizontal axis represents the rotation time of the chip-type device (time (revolution)), and 1000time is equivalent to 30 seconds. The vertical axis represents the position in the width direction of the tubular channel (position (smp: sampling)), and 100smp is equivalent to 492μm. In addition, the voltage value corresponding to the detected scattered light is output and displayed in the form of grayscale image data. The depth of the grayscale corresponds to the height of the voltage value, and the higher the voltage value, the lower the brightness. Fig.13b for Fig.13a A local enlarged view of the image, where the horizontal axis represents the range of 1000 to 2000 rounds, and the vertical axis represents the range of 200 to 300 smp.

[0356] Here, a solution of anti-NP antibody fixed to polystyrene beads and gold colloid-labeled antibody was mixed without adding antigen as a sample, and the results of the above-mentioned steps (4) of the cleaning treatment and (5) of the separation (elution) of the part containing the marker (gold colloid particles) and the specific detection of the marker by scattered light in this embodiment are shown in FIG. Fig.14a , Fig.14b . Fig.14a , Fig.14b The vertical axis, horizontal axis, and display data are respectively Fig.13a , Fig.13b same, Fig.14b for Fig.14a A partial enlarged view of .

[0357] The gold colloid-labeled anti-NP antibody introduced into the reaction chamber is sometimes adsorbed (non-specifically adsorbed) on substances other than antigens. The non-specifically adsorbed gold colloid-labeled anti-NP antibody will also separate the portion containing the gold colloid particles from the adsorbate when NaOH (cutting reagent) is added. This phenomenon is caused by the addition of the cutting reagent, so the gold colloid particles that are not the detection object are introduced into the tubular channel together with the gold colloid particles that are the detection object (the gold colloid particles that form a complex) and will be accidentally detected. However, the amount of non-specifically adsorbed gold colloid-labeled anti-NP antibody can be used to correct the results by using the above method to grasp in advance.

[0358] It should be noted that Fig.13a and Fig.14a In the images, a continuous strong signal that does not change over time is observed near both ends of the tubular channel in the width direction. Fig.14a Therefore, it is difficult to believe that the signal originates from the gold colloid particles, and it can be judged that it is caused by the noise of light refraction, scattering, strain, etc. occurring near the ends of the tubular channel. When such a signal is detected, the vicinity of the two ends of the tubular channel is excluded from the effective detection range.

[0359] In addition, the signal detected continuously at the same location ( Fig.13a , Fig.14a ) is scattered light from a substance that does not move in the tubular channel, and can therefore be judged to be noise based on scratches or the like in the tubular channel.

[0360] Furthermore, only gold colloid particles were flowed into the tubular channel of the chip-type device and scattered light was detected. The shape of the signal representing the scattered light of the gold colloid particles was confirmed from the obtained image data. As a result, it was found that Figure 13a to Figure 14b The roughly circular tiny spots in the image correspond to the signals of the gold colloid particles. Fig.14aThe irregular shapes and slightly larger spots observed in the image are different from the shapes of the signals originating from the gold colloid particles, so they can be judged to be scattered light (noise) from foreign matter. Therefore, these noise signals can be excluded from the detection results.

[0361] On the other hand, the intermittently generated roughly circular tiny spots can be judged to be scattered light originating from the dynamic gold colloid particles passing through the tubular channel. Fig.13a and Fig.14a In the effective detection range except the vicinities of both ends in the width direction of the tubular channel, the spots corresponding to the gold colloidal particles are counted.

[0362] Then, from Fig.13a Subtract the count value from Fig.14a The count value of the gold colloid particles corresponding to the antigen is taken as the count value. The relationship between the count value (count(-blank)) and the antigen concentration when the immune complex is formed is shown in Fig.15 The horizontal axis shows the antigen concentration in a common logarithmic manner, and the vertical axis shows the count value of scattered light corresponding to the antigen.

[0363] Depend on Fig.13a and Fig.14a By comparison, we can see that Fig.14a The spots shown represent gold colloid particles, which are much more numerous than Fig.13a Furthermore, if Fig.15 As shown in the figure, the count value detected (calculated (-blank)) is a "positive value" in thousands, which is significantly different from the cells in the absence of antigen. Therefore, it is shown that by using the device to wash the immune complex fixed to the solid phase and cutting it with a cutting agent, it is possible to separate the part containing the marker from the complex, make the separated part containing the marker move in the tubular channel by centrifugal force, and detect the marker by scattered light from the moved marker.

[0364] Furthermore, Fig.13b , Fig.14b As shown, spots corresponding to the gold colloid particles can be clearly confirmed. That is, the signal based on the scattered light of each gold colloid particle can be counted, indicating that quantitative evaluation of gold colloid particles can be achieved.

[0365] In particular, according to Fig.15 , it can be observed that when the antigen concentration is low, the count value of the scattered light based on the gold colloid particles tends to be well correlated with the antigen concentration. This indicates that, especially when the antigen contained in the sample is at a low concentration, the gold colloid particles corresponding to the antigen concentration can be more quantitatively detected.

[0366] In order to study whether this method can detect lower concentrations of antigens compared to previous immunochromatography, the above results are compared with the results of previous immunochromatography. The previous immunochromatography used IMMUNOACE SARS-CoV-2II (TAUNS Laboratories, Inc.), and the antigen detection results were visually evaluated. It should be noted that as an antigen, SARS-CoV-2NP adjusted to a specified concentration was used. The results are shown in Table 3.

[0367] [Table 3]

[0368]

[0369] 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 using the conventional immunochromatography method. The rightmost column shows the results using the method of Example 8. Regarding "+", it means that the antigen is detected in the immunochromatography method, and in the method of this embodiment, it means that the scattered light is counted. Regarding "ND", it means that the antigen is not detected in the immunochromatography method, and in the method of this embodiment, it means that the scattered light is not counted. In the method of this embodiment, it is confirmed that the minimum detectable antigen concentration is 0.5pg / mL, while in the conventional immunochromatography method it is 50pg / mL. Therefore, it can be said that the method of this embodiment can detect lower concentrations of antibodies than the conventional immunochromatography method, and has excellent sensitivity.

[0370] It should be noted that in the method of the present embodiment described above, during the cleaning process, the cleaning liquid is directly injected into the reaction chamber from the outside. Instead of this method, the cleaning liquid can also be stored in the cleaning liquid storage chamber, and the cleaning liquid can be supplied from the cleaning liquid storage chamber to the reaction chamber by rotating the chip-type device. In this regard, in another experiment, 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 cleaning liquid in the cleaning liquid storage chamber decreased and the cleaning liquid in the reaction chamber increased. This result 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 is performed.

[0371] In addition, in the chip-type device, the reagent storage chamber (cutting reagent storage chamber) is arranged on the rotation center side relative to the reaction chamber, and is connected to the reaction chamber by a flow path. The reagent storage chamber is connected to the reaction chamber and the tubular channel by the same flow path as the cleaning liquid storage chamber. Therefore, according to the above-mentioned experiment of transferring the cleaning liquid by rotation, it can be said that if the cutting reagent is stored in the reagent storage chamber and the chip-type device is rotated, the cutting reagent can be supplied to the reaction chamber by the centrifugal force generated, and the cutting process can be performed in the reaction chamber in the same way as the cleaning liquid.

[0372] Industrial Applicability

[0373] 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 method and apparatus for detecting substances in various industrial fields in addition to the medical field.

[0374] Description of Reference Numerals

[0375] 1 plate

[0376] Unit 2

[0377] 10 Reaction chamber

[0378] 11 Tubular channel

[0379] 12 Drainage storage room

[0380] 13 Cleaning fluid storage room

[0381] 14 Cut off reagent storage room

Claims

1. A method for detecting a target substance, comprising: The target substance is sandwiched between a first capture substance bound to a label and a second capture substance immobilized on a solid phase to form a complex, and a portion containing the label is separated from the formed complex. The separated portion containing the marker moves in the liquid filled in the tubular channel by centrifugal force. The portion containing the marker is detected by scattered light generated by scattering light irradiated into the tubular channel by the portion containing the marker.

2. The method for detecting a target substance according to claim 1, comprising: Before separating the portion containing the labeling substance from the complex, a washing process is performed to wash the complex while the inflow of the complex into the tubular channel is suppressed.

3. The method for detecting a target substance according to claim 2, wherein: When the cleaning process is performed, the tubular channel is filled with a cleaning liquid for cleaning.

4. The method for detecting a target substance according to claim 2, wherein: The cleaning liquid used in the cleaning process is stored downstream of the tubular channel.

5. The method for detecting a target substance according to claim 2, wherein: By holding the complex in a predetermined chamber, the inflow of the complex into the tubular channel is suppressed.

6. The method for detecting a target substance according to claim 5, wherein: In the device formed in a rotatable manner, a first storage chamber storing the cleaning liquid, the chamber, and a second storage chamber storing the liquid flowing out of the chamber are sequentially arranged from the rotation center side of the device toward the outside, the first storage chamber and the chamber are connected by a flow path that can be opened and closed freely, and the chamber and the second storage chamber are connected by the tubular channel. The target substance detection method includes rotating the device to deliver the cleaning solution from the first storage chamber into the chamber and then discharge the cleaning solution into the second storage chamber through the tubular channel to perform the cleaning process.

7. The method for detecting a target substance according to claim 6, wherein: The first storage chamber and the cavity are communicated with each other via an openable flow path.

8. The method for detecting a target substance according to claim 6, wherein: The cleaning process is performed until the second storage chamber and the tubular channel are filled with the cleaning liquid.

9. The method for detecting a target substance according to claim 8, wherein: A third storage chamber is arranged on the rotation center side of the chamber in the device, the third storage chamber stores a liquid containing a reagent for separating the portion containing the label from the complex, and the third storage chamber is connected to the chamber via a flow path that can be opened and closed freely. The method for detecting the target substance comprises: rotating the device, introducing the liquid containing the reagent into the chamber after the cleaning process to separate the portion containing the marker, and moving the separated portion containing the marker in the tubular channel.

10. The method for detecting a target substance according to claim 9, wherein: The third storage chamber is communicated with the cavity via an openable flow path.

11. The method for detecting a target substance according to any one of claims 1 to 10, wherein: The scattered light intermittently generated from the tubular passage within a predetermined time is detected.

12. The method for detecting a target substance according to any one of claims 1 to 10, wherein: The marker is a substance having a specific gravity greater than the specific gravity of the liquid filling the tubular channel.

13. The method for detecting a target substance according to claim 12, wherein: The labeling substance is a fine particle having one selected from the group consisting of metal, ceramic, glass and resin as a main component.

14. The method for detecting a target substance according to claim 13, wherein: The marker is metal colloidal particles.

15. The method for detecting a target substance according to any one of claims 1 to 10, wherein: The solid phase is at least one kind of granular body selected from the group consisting of glass particles, ceramic particles, magnetic particles, and resin particles.

16. The method for detecting a target substance according to any one of claims 1 to 10, wherein: The solid phase is a structure inside a container used to form the complex.

17. The method for detecting a target substance according to any one of claims 1 to 10, wherein: The separation of the portion containing the label from the complex is performed by at least one treatment selected from the group consisting of heat treatment, pH adjustment treatment, denaturation treatment, oxidation treatment, reduction treatment, enzyme treatment and competitive reaction treatment.

18. The method for detecting a target substance according to any one of claims 1 to 10, 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.

19. The method for detecting a target substance according to any one of claims 1 to 10, 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 the target substance. 20 . A reagent used in the method for detecting a target substance according to claim 1 , which has a function of separating the portion containing the label from the complex. The reagent according to claim 20 , comprising at least one selected from the group consisting of a pH adjuster, a denaturant, a reducing agent, an oxidizing agent, an enzyme, and a competitive agent.

22. The reagent according to claim 21, comprising a pH adjuster.

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