JPWO2019159958A1 - Measuring method - Google Patents

Abstract

To provide a measurement method that makes it possible to improve the efficiency of measurement work. The measuring method is a measuring method for measuring a measurement target contained in a sample, and in the reaction vessel, the measurement target and the magnetic particles are identified by a predetermined method having a larger particle size than the measurement target and the magnetic particles. The reaction between the production step of producing the composite 74 by reacting with possible non-magnetic particles and the production step of the measurement target and the non-magnetic particles housed in the reaction tank during or after the production step. The removal step of removing the missing measurement target and non-magnetic particles, the measurement step of measuring the number of non-magnetic particles contained in the reaction vessel after the removal step, and the number of non-magnetic particles measured in the measurement step A specific step of identifying the number of measurement targets based on the above is included.

Description

The present invention relates to a measuring method.

Conventionally, as one of the techniques for measuring the concentration of biomolecules contained in a fluid sample at the single molecule level, a plurality of biomolecules (specifically, target analyze molecules linked to a capture substance via a capture component) are used. , The number (concentration) of biomolecules in the fluid sample by arranging them separately in a plurality of wells formed on the substrate and detecting signals (fluorescent signals) indicating the presence of biomolecules from the plurality of wells. ) Has been proposed (see, for example, Patent Document 1).

Special Table 2013-521500

However, in the above-mentioned conventional method, since a plurality of biomolecules are separately arranged in a plurality of wells as described above, for example, the number of all biomolecules contained in a fluid sample is measured. In this case, the work of arranging the biomolecule in the well may take a long time, and the complicated work is performed by using a device having a complicated structure for arranging the biomolecule. Since it is necessary, there is room for improvement from the viewpoint of improving the efficiency of measurement work.

An object of the present invention is to solve the above-mentioned problems in the prior art, and to provide a measurement method capable of improving the efficiency of measurement work.

In order to solve the above-mentioned problems and achieve the object, the measurement method according to claim 1 is a measurement method for measuring a measurement target contained in a sample, and the measurement target and magnetic particles are contained in a container. And the production step of producing a composite by reacting the measurement target and the non-magnetic particles having a particle size larger than that of the magnetic particles and identifiable by a predetermined method, and during or during the production step. After the step, of the measurement target and the non-magnetic particles contained in the container, the removal step of removing the measurement target and the non-magnetic particles that did not react in the production step, and after the removal step, the said It includes a measuring step of measuring the number of the non-magnetic particles contained in the container and a specific step of specifying the number of measurement targets based on the number of the non-magnetic particles measured in the measuring step.

The measuring method according to claim 2 is the measuring method according to claim 1, wherein at least a part of the composite housed in the container is placed on a substrate in the measuring step, and the lower part of the substrate. The previously placed composite is collected at a predetermined position on the substrate by a magnet provided in the above, the collected composite is imaged by an imaging means, and an image captured by the imaging means. Based on, the number of the non-magnetic particles is measured.

The measuring method according to claim 3 is the measuring method according to claim 2, wherein the particle size of the magnetic particles is set to a size invisible from the captured image, and the particle size of the non-magnetic particles is set to the imaging. The size is visible from the image.

The measuring method according to claim 4 is the measuring method according to claim 1, wherein at least a part of the complex contained in the container is flowed in a predetermined direction in the measuring step. The number of the non-magnetic particles is measured based on the magnitude of the reflected light when the laser light is irradiated from the laser light source to a part of the flow path through which the composite flows.

The measuring method according to claim 5 is the measuring method according to claim 1, wherein at least a part of the complex contained in the container is flowed in a predetermined direction in the measuring step. The number of the non-magnetic particles is measured based on the electrical change when the complex flowing through the flow path passes through the pores provided in the flow path.

The measuring method according to claim 6 is the measuring method according to claim 1, wherein at least a part of the complex housed in the container is flowed in a predetermined direction in the measuring step. The complex flowing through the flow path is imaged by the imaging means, and the number of the non-magnetic particles is measured based on the captured image captured by the imaging means.

The measuring method according to claim 7 is the measuring method according to any one of claims 1 to 6, wherein the particle size of the non-magnetic particles is 1.5 times or more the particle size of the magnetic particles. ..

The measuring method according to claim 8 is the measuring method according to any one of claims 1 to 7, wherein the shape, color, fluorescence, enzyme labeling, isotope labeling, or particle size of the non-magnetic particles is determined. Two or more types were used.

According to the measuring method according to claim 1, by reacting a measurement target with magnetic particles and non-magnetic particles having a particle size larger than that of the magnetic particles and identifiable by a predetermined method in a container. , A generation step of forming a composite, a removal step of removing measurement targets and non-magnetic particles that did not react in the production step during or after the production step, and a non-magnetic housed in a container after the removal step. It includes a measuring step of measuring the number of particles and a specific step of specifying the number of measurement targets based on the number of non-magnetic particles measured in the measuring step. According to this method, by allowing a sufficiently large number of non-magnetic particles having a particle size sufficiently large with respect to the target substance to exist in the reaction system with respect to the amount to be measured, in principle, with respect to one molecule of the target substance. One non-magnetic particle can be bonded together. Then, by counting the non-magnetic particles bound to the measurement target, the measurement target can be detected at the single molecule level. Compared with the conventional technique (a technique for measuring the number of a plurality of biomolecules separately arranged in a plurality of wells), this method does not require complicated work and can easily and quickly reduce the number of measurement targets. It is possible to measure the efficiency of the measurement work.

According to the measurement method according to claim 2, in the measurement step, the composite mounted on the substrate is collected at a predetermined position on the substrate by a magnet provided below the substrate, and is collected by the imaging means. Since the composite is imaged and the number of non-magnetic particles is measured based on the image captured by the imaging means, a large number of non-magnetic particles can be efficiently measured, and the efficiency of the measurement work can be improved. It becomes possible to plan.

According to the measurement method according to claim 3, the particle size of the magnetic particles is set to a size invisible from the captured image, and the particle size of the non-magnetic particles is set to a size visible from the captured image. The particles can be accurately identified, and the number of non-magnetic particles can be accurately measured in the measuring process.

According to the measurement method according to claim 4, in the measurement step, at least a part of the complex contained in the container is flowed in a predetermined direction in the flow path, and the complex flows with respect to a part of the flow path. Since the number of non-magnetic particles is measured based on the magnitude of the reflected light when the laser light is irradiated from the laser light source, the number of non-magnetic particles can be measured relatively accurately, and the measurement accuracy is improved. It becomes possible to make it.

According to the measurement method according to claim 5, at least a part of the complex contained in the container is flowed in a predetermined direction in the flow path, and the complex flowing through the flow path is provided in the flow path. Since the number of non-magnetic particles is measured based on the electrical change when passing through the pores, the number of non-magnetic particles can be measured relatively accurately, and the measurement accuracy can be improved.

According to the measurement method according to claim 6, at least a part of the complex housed in the container is flowed in a predetermined direction in the flow path, and the complex flowing through the flow path is imaged by an imaging means. Since the number of non-magnetic particles is measured based on the captured image captured by the imaging means, the number of non-magnetic particles can be measured relatively accurately, and the measurement accuracy can be improved.

According to the measuring method according to claim 7, since the particle size of the non-magnetic particles is 1.5 times or more the particle size of the magnetic particles, the particle size of the non-magnetic particles is set to the particles of the magnetic particles in the measurement step. Compared with the case where the diameter is less than 1.5 times, the non-magnetic particles can be identified more accurately, and the number of non-magnetic particles can be accurately measured in the measuring step.

According to the measurement method according to claim 8, since the shape, color, fluorescence, enzyme label, isotope label, or particle size of the non-magnetic particles are set to two or more types, for example, measurement targets having different types or conditions are used. When measuring, the number of measurement targets can be accurately measured for each type or condition, and the measurement can be performed according to the needs of the user.

It is a schematic diagram which illustrates the measurement system which concerns on Embodiment 1 of this invention. It is a schematic diagram which shows the outline of the 1st generation process. It is a schematic diagram which shows the outline of the 2nd production process, (a) is the figure which shows the state before adding the non-magnetic particle which was bound with the non-magnetic side antibody, and (b) is the figure which is bound with the non-magnetic side antibody. It is a figure which shows the state after adding the non-magnetic particles. It is a schematic diagram which shows the outline of the measurement process, (a) is a figure which shows the state before collecting a complex, (b) is a figure which shows the state after collecting a complex. It is a figure which shows the captured image of Test Example 1, (a) is a figure which shows a latex particle, (b) is a figure which shows a magnetic particle, (c) is a figure which shows a latex particle and a magnetic particle. It is a figure which shows the photograph image of the test example 2, (a) is a figure which shows the sample prepared using a positive solution, (b) is a figure which shows the sample prepared by using a negative solution. It is a figure which shows the captured image of Test Example 3, (a) is the figure which shows the latex particle of the particle size 1.9 μm, (b) is the figure which shows the latex particle of the particle size 0.76 μm, (c) is the figure which shows the particle size It is a figure which shows the latex particle of 0.52 μm. It is a figure which shows the captured image of the test example 3 following FIG. 7, (d) is the figure which shows the latex particle of the particle diameter 0.33 μm, (e) is the figure which shows the latex particle of the particle diameter 0.20 μm, (f. ) Is a diagram showing latex particles having a particle size of 0.20 μm and latex particles having a particle size of 0.33 μm. It is a schematic diagram which illustrates the measurement system which concerns on Embodiment 2. FIG. It is a schematic diagram which illustrates the measurement system which concerns on Embodiment 3. FIG. It is a schematic diagram which shows the outline of the measurement process. It is a schematic diagram which illustrates the measurement system which concerns on Embodiment 4. FIG.

Hereinafter, embodiments of the measurement method according to the present invention will be described in detail with reference to the accompanying drawings. First, [I] the basic concept of the embodiment will be described, then [II] the specific content of the embodiment will be described, and finally, [III] a modified example of the embodiment will be described. However, the present invention is not limited to the embodiments.

[I] Basic concept of the embodiment First, the basic concept of the embodiment will be described. Embodiments generally relate to a measuring method for measuring a measurement object contained in a sample.

Here, the "sample" means a material to be inspected or analyzed, and in the embodiment, it is configured to include a measurement target and a solvent. Further, the “measurement target” means a substance to be measured among the substances contained in the sample, and is a concept including, for example, an antigen, a biomolecule, a protein, a nucleic acid, a small molecule, and the like. Further, the "solvent" means a substance having the largest amount (number of molecules) among the substances contained in the sample, and for example, it is possible to react the measurement target with the magnetic particles and the non-magnetic particles. Moreover, it is a concept including a reaction solution (for example, an antigen-antibody reaction solution) capable of passing electricity, magnetism, or light. Here, the "magnetic particle" means a particle having magnetism and capable of binding to a measurement target. Further, the "non-magnetic particles" mean particles having non-magnetism and capable of binding to a measurement target, and include, for example, particles made of a fluorescent dye, a resin, a carbon material, an inorganic substance, or the like. Further, the bond between the measurement target and the magnetic particle or the non-magnetic particle may be, for example, a direct bond between the measurement target and the particle, but preferably, a probe is previously attached to the magnetic particle (or non-magnetic particle). Indirect binding is desirable in which a molecule, an antibody, a binding protein or the like is immobilized, and the probe molecule, the antibody or the like is bound to the measurement target. Further, the type of this measurement method is arbitrary, and for example, a 1-step competition method, a 1-step sandwich method, a delay 1-step method, a 2-step sandwich method, a dilution 2-step method, or the like can be used. Hereinafter, in the embodiment, the measurement target is an antigen, the solvent is an antigen-antibody reaction solution, the magnetic particles are magnetic nanoparticles prebonded to an antibody (hereinafter, referred to as “magnetic side antibody”), and the non-magnetic particles are used. A case where a measurement target is measured by using a two-step sandwich method as latex particles pre-bonded to an antibody (hereinafter referred to as “non-magnetic side antibody”) will be described as an example.

In the present invention, the "particle size" refers to the average particle size. Specifically, when the shape of the particles used is spherical or substantially spherical and substantially uniform, it refers to the number average particle diameter. On the other hand, when the particle shape is irregular, it refers to the volume average particle diameter.

Further, the measuring method according to the embodiment is roughly described in 1) 1st generation step, 2) 1st removal step, 3) 2nd generation step, 4) 2nd removal step, 5) measurement step. And 6) The specific steps are sequentially performed. Of these, in the first production step, by adding magnetic particles bound to the magnetic side antibody to the sample contained in the container, the measurement target contained in the sample is reacted with the magnetic side antibody (antigen-antibody reaction). Then, this measurement target is bonded to the magnetic particles. Further, in the first removing step, the measurement target and the magnetic particles contained in the container that did not react in the first generation step are removed. Further, in the second production step, by adding the non-magnetic particles bound to the non-magnetic side antibody to the sample, the measurement target bound to the magnetic side antibody in the first production step reacts with the non-magnetic side antibody (antigen). By (antibody reaction), this measurement target is bound to the non-magnetic particles. Further, in the second removal step, the non-magnetic particles that did not react in the second generation step are removed from the measurement target and the non-magnetic particles contained in the container. Further, in the measuring step, after the second removing step, the number of non-magnetic particles contained in the container is measured. Further, in the specific step, the number of measurement targets is specified based on the number of non-magnetic particles measured in the measurement step. The above-mentioned "first generation step" and "second generation step" correspond to the "generation step" in the claims, and the above-mentioned "first removal step" and "second removal step" are patented. Corresponds to the "removal process" in the claims.

3) In the second generation step, the non-magnetic particles added to the container have a sufficiently large particle size with respect to a measurement target such as a biomolecule having a size of nano-order or less, and can be visually recognized by, for example, an optical microscope. It has a particle size exceeding 0.2 μm. It is impossible to physically bond a plurality of non-magnetic particles to a single molecule of measurement target. In addition, the amount of non-magnetic particles added to the container is excessive with respect to the number of objects to be measured. As a result, it is difficult for two or more molecules to be measured to be bound to one non-magnetic particle. As described above, in the second generation step, it is possible in principle to bind one non-magnetic particle to one molecule to be measured.

[II] Specific contents of the embodiment Next, the specific contents of the embodiment will be described.

[Embodiment 1]
First, the first embodiment will be described. The first embodiment is an image captured by an imaging device described later, and the number of non-magnetic particles is measured based on an image captured including a composite mounted on a substrate.

(Constitution)
First, the configuration of the measurement system according to the first embodiment will be described. FIG. 1 is a schematic diagram illustrating a measurement system according to a first embodiment of the present invention. In the following description, the X direction in FIG. 1 is the left-right direction of the measurement system (-X direction is the left direction of the measurement system, the + X direction is the right direction of the measurement system), and the Y direction in FIG. 1 is the up-down direction of the measurement system (+ Y). The direction is the upward direction of the measurement system, the −Y direction is the downward direction of the measurement system), the direction orthogonal to the X and Y directions is the front-back direction (the direction leading to the front side of the paper in FIG. 1 is the front direction of the measurement system, and the figure). The direction leading to the back side of the paper surface of 1 is referred to as the rear direction of the measurement system). As shown in FIG. 1, the measurement system 1 is roughly composed of a reaction tank 10, a mounting table 20, a substrate 30, a first magnet (not shown), a second magnet 40, an image pickup device 50, and an image pickup device 50, which will be described later. The control device 60 is provided. Further, among these components, the image pickup device 50 and the control device 60 are electrically connected via the wiring 2.

(Construction-Reaction tank)
The reaction vessel 10 of FIG. 2, which will be described later, reacts the measurement target 71 of FIG. 2, which will be described later, the magnetic particles 72 of FIG. 2, which will be described later, with the non-magnetic particles 73 of FIG. 2, which will be described later, to form the complex 74. It is a container for producing. The reaction vessel 10 is configured by using, for example, a known reaction vessel formed of a glass material, a resin material, ceramics (silicon nitride, silicon chloride, etc.), a metal material, an inorganic material, an organic material, or the like. , Is provided near the substrate 30.

(Configuration-Mounting stand)
Returning to FIG. 1, the mounting table 20 is a table on which the substrate 30 is mounted, and is configured by using, for example, a known mounting table or the like, and is provided on an installation surface (not shown).

(Configuration-Board)
The substrate 30 is a plate on which the complex 74 is placed. As shown in FIG. 1, the substrate 30 is formed of a substantially flat plate-like body, and is mounted on the upper surface of the mounting table 20. Further, the specific shape and size of the substrate 30 are arbitrary, but in the first embodiment, as shown in FIG. 1, the measurement target 71 is efficiently measured for the planar shape of the substrate 30. A desired amount of the composite 74 and the solvent 75 are set in a long rectangular shape having an area that can be placed on the substrate 30 so that the composite 74 and the solvent 75 can be placed on the substrate 30. Further, the thickness of the substrate 30 is set to a thickness that can secure a predetermined strength. Further, the material of the substrate 30 can be arbitrarily formed as long as it has excellent chemical resistance and is capable of conducting magnetism. For example, even if it is formed of a glass material, a resin material, or the like. Good.

(Construction-First magnet)
Of the measurement target 71 of FIG. 2 and the non-magnetic particles 73 of FIG. 2 described later, which were housed in the reaction tank 10, the first magnet did not react during the formation of the complex 74, and the measurement target 71 and the non-reaction target 71 were not reacted. It is a removing means for removing the magnetic particles 73. This first magnet is configured by using, for example, a known magnet (for example, a ferrite magnet or the like), and is provided in the vicinity of the reaction tank 10 of FIG. 2, which will be described later.

(Construction-Second magnet)
Returning to FIG. 1, the second magnet 40 is a magnet for collecting the complex 74 mounted on the substrate 30 at a predetermined position on the substrate 30. The second magnet 40 is configured by using, for example, a known magnet (for example, a ferrite magnet or the like), and is provided below the substrate 30 as shown in FIG. The specific shape and size of the second magnet 40 are arbitrary, but in the first embodiment, the planar shape of the substrate 30 is larger than the planar shape of the substrate 30 as shown in FIG. It is set in a small rectangular shape. Further, the thickness of the second magnet 40 is set to a thickness that can secure a predetermined magnetic force.

(Configuration-Imaging device)
The imaging device 50 is an imaging means for imaging the complex 74 collected by the second magnet 40. The image pickup apparatus 50 is configured by using, for example, a known microscope (an optical microscope as an example), and is provided above the substrate 30 as shown in FIG.

(Configuration-Control device)
The control device 60 is a device for controlling the measurement system 1, and as shown in FIG. 1, includes an operation unit 61, a communication unit 62, an output unit 63, a power supply unit 64, a control unit 65, and a storage unit 66. ing. Since the control device 60 can be configured by, for example, a known personal computer or the like, detailed description thereof will be omitted.

(Configuration-Control device-Operation unit)
The operation unit 61 is an operation means for receiving operation inputs related to various types of information. The operation unit 61 is configured by using a known operation means such as a touch pad, a remote operation means such as a remote controller, or a hard switch.

(Configuration-Control device-Communication unit)
The communication unit 62 is a communication means for communicating with the image pickup apparatus 50 via the wiring 2.

(Configuration-Control device-Output unit)
The output unit 63 is an output means for outputting various information based on the control of the control unit 65, and is a known display means such as a flat panel display such as a liquid crystal display or an organic EL display, or a known audio output such as a speaker. It is configured by means and the like.

(Configuration-Control device-Power supply unit)
The power supply unit 64 is a power supply means for supplying the electric power supplied from a commercial power source (not shown) or the electric power stored in the power supply unit 64 to each unit of the image pickup device 50 and the control device 60.

(Configuration-Control device-Control unit)
The control unit 65 is a control means for controlling each unit of the control device 60. Specifically, the control unit 65 includes a CPU, various programs that are interpreted and executed on the CPU (including a basic control program such as an OS, and an application program that is started on the OS and realizes a specific function). It is a computer configured with an internal memory such as a RAM for storing programs and various data.

Further, as shown in FIG. 1, the control unit 65 includes a first measurement unit 65a and a specific unit 65b in terms of functional concept.

The first measuring unit 65a is a first measuring means for measuring the number of non-magnetic particles 73 of FIG. 2, which will be described later, based on the captured image captured by the imaging device 50.

The specific unit 65b is a specific means for specifying the number of measurement targets 71 in FIG. 2 to be described later based on the number of non-magnetic particles 73 in FIG. 2 to be described later measured by the first measurement unit 65a. The details of the processing executed by the control unit 65 will be described later.

(Configuration-Control device-Storage unit)
Returning to FIG. 1, the storage unit 66 is a recording means for recording a program and various data necessary for the operation of the control device 60, and is configured by using, for example, a hard disk (not shown) as an external recording device. .. However, in place of or with a hard disk, other media including magnetic recording media such as magnetic disks, optical recording media such as DVDs and Blu-ray discs, or electrical recording media such as Flash Rom, USB memory, and SD cards. Any recording medium can be used.

(Measuring method)
Next, a measurement method performed using the measurement system 1 configured in this way will be described. FIG. 2 is a schematic diagram showing an outline of the first generation step. 3A and 3B are schematic views showing an outline of the second production step, FIG. 3A is a diagram showing a state before adding the non-magnetic particles 73 bound to the non-magnetic side antibody 73a, and FIG. 3B is a non-magnetic diagram. It is a figure which shows the state after adding the non-magnetic particle 73 bound with the magnetic side antibody 73a. 4A and 4B are schematic views showing an outline of a measurement process, FIG. 4A is a diagram showing a state before collecting the complex 74, and FIG. 4B is a diagram showing a state after collecting the complex 74. Is. As described above, the measurement method according to the first embodiment includes a first generation step, a first removal step, a second generation step, a second removal step, a measurement step, and a specific step. The detailed contents of each step will be described below.

Here, the specific shapes and sizes of the measurement target 71, the magnetic particles 72, and the non-magnetic particles 73 used in this measurement method are arbitrary, but in the first embodiment, they are as follows. That is, first, the shapes of the magnetic particles 72 and the non-magnetic particles 73 are set to one type (for example, a spherical shape). Further, the particle size of the measurement target 71 is set to a size that cannot be visually recognized from the captured image captured by the imaging device 50, and is set to less than 0.05 μm as an example. Further, the particle size of the magnetic particles 72 is set to a size larger than that of the measurement target 71 and invisible from the image captured by the imaging device 50. Although it is set to 0.2 μm, the present invention is not limited to this, and for example, it may be set to 0.05 μm or more and less than 0.2 μm. Further, the particle size of the non-magnetic particles 73 is set to a size larger than that of the measurement target 71 and the magnetic particles 72 and visible from the image captured by the imaging device 50. In the first embodiment, the particle size is set to 0.3 μm, which is 1.5 times the particle size of the magnetic particles 72, but the present invention is not limited to this, and for example, the size exceeds 1.5 times the particle size of the magnetic particles 72. (As an example, it may be set to 0.4 μm or more and 5 μm or less). Further, the color of the magnetic particles 72 and the color of the non-magnetic particles 73 are set to one type each. With such a setting, the non-magnetic particles 73 can be accurately identified, and the number of non-magnetic particles 73 can be accurately measured in the measurement step.

(Measurement method-first generation step)
First, the first generation step will be described. The first generation step is performed in the following procedure in order to bond the measurement target 71 contained in the sample 70 to the magnetic particles 72. That is, first, as shown in FIG. 2, after adding the magnetic particles 72 bound to the magnetic side antibody 72a into the reaction tank 10 containing the sample 70 composed of the measurement target 71 and the solvent 75. , The sample 70 to which the magnetic particles 72 are added is stirred. The amount of the magnetic particles 72 added is arbitrary, but for example, all of the measurement targets 71 contained in the sample 70 are set to an amount capable of reacting with the magnetic side antibody 72a (antigen antibody reaction). As an example, in order to improve the efficiency of the reaction, the amount of the magnetic particles 72 is set to an excessive amount (for example, 100 times the amount of the measurement target 71) with respect to the amount of the measurement target 71. (The same applies to the amount of the non-magnetic particles 73 added in the second generation step described later). Next, the stirred sample 70 is left at a predetermined temperature (for example, 37 ° C.) until a predetermined time elapses after the magnetic particles 72 are added. This predetermined time is set to, for example, a time longer than the time generally assumed to be required for the measurement target 71 to react with the magnetic side antibody 72a.

(Measurement method-first removal step)
Next, the first removal step will be described. The first removal step is performed in the following procedure in order to remove the measurement target 71 that did not react in the first production step from the measurement target 71 contained in the sample 70. That is, a first magnet is attached to a part of the reaction tank 10 (for example, a side portion of the reaction tank 10), and the magnetic particles 72 that have reacted with the measurement target 71 react with the measurement target 71 in a part of the reaction tank 10. In a state where the magnetic particles 72 that did not react with the magnetic particles 72 are collected, the sample 70 and the measurement target 71 that did not react with the magnetic particles 72 are washed away with a solvent 75 or the like to remove the sample 70 (the second removal described later). The same applies to the process).

(Measurement method-2nd generation step)
Next, the second generation step will be described. The second production step is performed in the following procedure in order to bond the measurement target 71 reacted in the first production step to the non-magnetic particles 73. That is, first, as shown in FIG. 3A, the non-magnetic particles 73 bound to the non-magnetic side antibody 73a are added to the reaction vessel 10, and then the sample 70 to which the non-magnetic particles 73 are added is added. Stir. Next, the stirred sample 70 is left at a predetermined temperature (for example, 37 ° C.) until a predetermined time elapses after the non-magnetic particles 73 are added. This predetermined time is set to, for example, a time longer than the time generally assumed to be required for the measurement target 71 to react with the non-magnetic side antibody 73a. As a result, as shown in FIG. 3B, the complex 74 (magnetic particles 72, magnetic side antibody 72a, measurement target 71, non-magnetic side antibody 73a, and non-magnetic particles 73) is generated.

(Measurement method-second removal step)
Next, the second removal step will be described. The second removal step is performed in substantially the same manner as the first removal step in order to remove the non-magnetic particles 73 that did not react in the second generation step among the non-magnetic particles 73 contained in the sample 70.

(Measurement method-Measurement process)
Next, the measurement process will be described. The measuring step is performed in the following procedure in order to measure the number of non-magnetic particles 73 housed in the reaction vessel 10 after the second removing step.

That is, first, as shown in FIG. 4A, at least a part of the complex 74 housed in the reaction vessel 10 is placed on the substrate 30. Specifically, a predetermined amount of the complex 74 and the solvent 75 are taken out from the reaction vessel 10 by a liquid feeding tube (for example, a pipette or the like) (not shown), and the taken out complex 74 and the solvent 75 are dropped onto the substrate 30. By doing so, it will be placed.

Next, as shown in FIG. 4B, the composite 74 placed above is placed at a predetermined position on the substrate 30 (for example, the central portion of the substrate 30) by the second magnet 40 provided below the substrate 30. Collect in. This collection method is arbitrary, but for example, in a state where the substrate 30 and the second magnet 40 are separated (or a state where the lower surface of the substrate 30 and the second magnet 40 are in contact with each other), the composite portion of the substrate 30 is formed. Collection may be performed by moving the second magnet 40 so that the magnetism of the second magnet 40 flows over the entire portion where the body 74 and the solvent 75 are dropped.

Next, the image pickup device 50 images the collected composite 74 at a predetermined magnification (for example, the magnification of the objective lens of the image pickup device 50 = 100 times, the magnification of the eyepiece lens of the image pickup device 50 = 10 times), and the image is taken. The captured image is transmitted from the image pickup device 50 to the control device 60. This imaging method is arbitrary, but for example, an image may be taken for each area in which a predetermined portion of the substrate 30 is divided into a predetermined number (for example, 10 to 1000 divisions, etc.). This makes it possible to image the entire complex 74 mounted on the substrate 30.

Subsequently, the first measuring unit 65a of the control device 60 measures the number of non-magnetic particles 73 based on the captured image received by the communication unit 62. The method for measuring the number of the non-magnetic particles 73 is arbitrary, but for example, the particle size of the particles included in the captured image is equal to or larger than the threshold value (one example) by a known image analysis method (or artificial intelligence processing). , 0.3 μm or more) is extracted, and the number of the extracted particles is measured as the number of non-magnetic particles 73. Alternatively, the number is not limited to this, for example, the number of the received captured images displayed on the output unit 63 (display means) and input by the user via the operation unit 61, which is the number counted by the user. May be measured as the number of non-magnetic particles 73.

In the same manner thereafter, the above-mentioned series of operations is repeated until the complex 74 and the solvent 75 in the reaction vessel 10 are visually eliminated so that all the non-magnetic particles 73 contained in the reaction vessel 10 can be measured (in this case). In the above, when the complex 74 is mounted on the substrate 30, it is desirable to mount it on a new substrate 30). Even if the complex 74 and the solvent 75 in the reaction tank 10 disappear in appearance, a new solvent may be left in the reaction tank 10 or the liquid feed tube. By washing the reaction vessel 10 and the liquid feed tube with 75, the remaining complex 74 is taken into the solvent 75 (so-called co-washing is performed), and these complexes 74 and the solvent 75 are taken out by the liquid feed tube. It is desirable to place the removed composite 74 and the solvent 75 on the substrate 30 (note that this work may be repeated if necessary). Then, after the series of operations described above is repeated, the total value of the number of non-magnetic particles 73 measured for each number of repetitions is measured as the number of non-magnetic particles 73 to be measured.

By such a measurement step, a large number of non-magnetic particles 73 can be efficiently measured, and the efficiency of the measurement work can be improved.

(Measurement method-Specific process)
Subsequently, the specific process will be described. The specific step is performed in the following procedure in order to specify the number of measurement targets 71. That is, first, the number of non-magnetic particles 73 measured in the measurement step is specified as the number of measurement targets 71 to be specified by the identification unit 65b of the control device 60. Next, the control unit 65 of the control device 60 causes the output unit 63 to output the number of the specified measurement targets 71. The method of outputting the number of measurement targets 71 is arbitrary, but for example, the number of measurement targets 71 may be displayed on the screen of the output unit 63 (display means). Alternatively, the output unit 63 (voice output means) outputs voice data indicating the number of measurement targets 71 (for example, voice data including a standard message that "the number of measurement targets contained in the sample was XXX"). ) May be output as audio.

By the above-mentioned measurement method, the measurement target 71 can be measured without requiring complicated work as compared with the conventional technique (a technique for measuring the number of a plurality of biomolecules separately arranged in a plurality of wells). The number can be measured easily and quickly, and the efficiency of the measurement work can be improved.

(Test results)
Next, the results of various tests conducted by the applicant will be described. Here, the test results of a test for verifying whether or not the non-magnetic particles 73 and the magnetic particles 72 according to the first embodiment of the present invention can be confirmed from the captured image will be described.

(Test Results-Test Example 1: Optical Microscopic Observation of Latex Particles and Magnetic Particles)
First, Test Example 1 will be described.

Latex particles (Invitrogen's Carboxyl latex, product number: C37274, particle size 1.0 μm) or magnetic particles (micromod's nanomag-D, COOH, product number: 09-02-132, particle size 0.13 μm) It was suspended in PBS at 100-fold dilution. Similarly, a solution in which latex particles and magnetic particles were suspended in PBS so as to be diluted 100-fold was also prepared. The solution prepared above was observed using an inverted microscope IX71 (CCD camera system: DP80) manufactured by Olympus Corporation with a combination of an objective lens of 100 times and an eyepiece lens of 10 times.

5A and 5B are views showing a captured image of Test Example 1, in which FIG. 5A shows latex particles, FIG. 5B shows magnetic particles, and FIG. 5C shows latex particles and magnetic particles. .. As shown in FIG. 5, it was confirmed that only the latex particles could be observed and the magnetic particles had a particle size that could not be observed.

(Test Results-Test Example 2: Measurement of Particle Complex Targeting IL-6)
Next, Test Example 2 will be described.

(1) Preparation of anti-IL-6 antibody-bound latex particles To 4 mg of latex particles (Carboxyl latex manufactured by Invitrogen, trade number: C37274, particle size 1.0 μm) suspended in 200 μL of 50 mM MES (pH = 5.5), 8 μL of 1M ethyl (dimethylaminopropyl) carbodiimide and 8 μL of 1M N-hydroxysuccinimide were added, and the mixture was inverted and mixed at room temperature for 30 minutes. After washing once with 200 μL of 50 mM MES (pH = 5.5), 200 μL of anti-IL-6 antibody (Clone HH61-2, manufactured in-house) dissolved in 50 mM MES (pH = 5.5) at 0.4 mg / mL. In addition, the anti-IL-6 antibody was bound onto the latex particles by inversion and mixing at room temperature for 60 minutes. Anti-IL-6 antibody-bound latex particles were obtained by washing twice with a carbonated buffer containing 200 μL of 2% BSA.

(2) Preparation of anti-PSA antibody-bound magnetic particles Magnetic particles (nanomag-D, COOH manufactured by micromod, trade number: 09-02-132, particle size 0.13 μm) and anti-IL-6 antibody (Clone HH61-10, Anti-IL-6 antibody-bound magnetic particles were prepared in the same manner as in (1) using (manufactured in-house).

(3) Measurement of IL-6 (measurement target) hIL-6 (Human Interleukin-6) antigen solution (manufactured by Cell Signaling, trade number: 8904) was diluted with TBS containing 2% BSA, and 0,1000 pg / mL of IL-6 solution (negative solution and positive solution, respectively) was prepared. A 25 μL negative or positive solution is reacted with 125 μL of a solution containing 0.015% anti-IL-6 antibody-bound magnetic particles at 37 ° C. for 30 minutes, and hIL- is placed on the anti-IL-6 antibody-bound magnetic particles. 6 antigens were captured. After washing the unbound material with 250 μL PBS-T three times, a solution containing 125 μL of 0.015% anti-IL-6 antibody-bound latex particles was added and reacted at 37 ° C. for 30 minutes. After washing 6 times with 250 μL PBS-T, suspend in 10 μL PBS, and prepare each with a combination of 100x objective lens and 10x eyepiece using an inverted microscope IX71 (CCD camera system: DP80) manufactured by Olympus. The sample was observed.

(4) Results FIG. 6 is a diagram showing a captured image of Test Example 2, (a) shows a sample prepared using a positive solution, and (b) shows a sample prepared using a negative solution. It is a figure. As shown in FIG. 6, in the sample prepared using the positive solution, a particle image having almost the same particle size as the latex particles (Carboxyl latex manufactured by Invitrogen, product number: C37274, particle size 1.0 μm) could be confirmed. On the other hand, in the sample prepared using the negative solution, the particle image of the same particle size could not be confirmed. From this, it was found that the presence or absence of the antigen was related to the number of latex particles, and the effectiveness of the measurement method according to the first embodiment was confirmed.

(Test result-Test example 3: Difference in optical microscope image depending on the particle size of latex particles)
Next, Test Example 3 will be described.

Latex particles (Carboxyl latex manufactured by invitrogen, particle size 1.9 μm, 0.76 μm, 0.52 μm, 0.33 μm, or 0.20 μm), magnetic particles (nanomag-D, COOH manufactured by micromod, product number: 09- 02-132, particle size 0.13 μm), and a mixture of latex particles (particle size 0.20 μm) and magnetic particles (particle size 0.13 μm) were suspended in PBS so as to be diluted 1000-fold each. The solution was also prepared. The solution prepared above was observed using an inverted microscope IX71 (CCD camera system: DP80) manufactured by Olympus Corporation with a combination of an objective lens of 100 times and an eyepiece lens of 10 times.

7A and 7B are views showing captured images of Test Example 3, in which FIG. 7A shows latex particles having a particle size of 1.9 μm, and FIG. 7B shows latex particles having a particle size of 0.76 μm, (c). ) Is a diagram showing latex particles having a particle size of 0.52 μm. 8A and 8B are views showing captured images of Test Example 3 following FIG. 7, where FIG. 8D shows latex particles having a particle size of 0.33 μm, and FIG. 8E shows latex particles having a particle size of 0.20 μm. FIG. (F) is a diagram showing latex particles having a particle size of 0.20 μm and latex particles having a particle size of 0.33 μm. From the captured images of FIGS. 7 and 8, it was confirmed that latex particles up to 0.33 μm could be observed, and that the latex particles of 0.20 μm had an unobservable particle size. Therefore, regarding the measurement method according to the first embodiment, it is considered that the two particle size ratios are 1.5 times or more.

As described above, according to the first embodiment, in the reaction vessel 10, the measurement target 71, the magnetic particles 72, and the non-magnetic particles 73 having a particle size larger than that of the magnetic particles 72 and being distinguishable by a predetermined method are used. After the production step of producing the complex 74, the removal step of removing the measurement target 71 and the non-magnetic particles 73 that did not react in the production step during or after the production step, and the removal step. , A measurement step of measuring the number of non-magnetic particles 73 housed in the reaction vessel 10, and a specific step of specifying the number of measurement targets 71 based on the number of non-magnetic particles 73 measured in the measurement step. Since it is included, the number of measurement targets 71 can be easily and quickly reduced without requiring complicated work as compared with the conventional technique (a technique for measuring the number of a plurality of biomolecules separately arranged in a plurality of wells). It is possible to improve the efficiency of the measurement work.

Further, in the measurement step, the composite 74 mounted on the substrate 30 is collected at a predetermined position on the substrate 30 by a magnet provided below the substrate 30, and the collected composite 74 is imaged by the imaging device 50. Then, since the number of non-magnetic particles 73 is measured based on the captured image captured by the image pickup apparatus 50, a large number of non-magnetic particles 73 can be efficiently measured, and the efficiency of the measurement work is improved. It becomes possible.

Further, since the particle size of the magnetic particles 72 is set to a size invisible from the captured image and the particle size of the non-magnetic particles 73 is set to a size visible from the captured image, the non-magnetic particles 73 can be accurately identified. This makes it possible to accurately measure the number of non-magnetic particles 73 in the measuring step.

Further, since the particle size of the non-magnetic particles 73 is 1.5 times or more the particle size of the magnetic particles 72, the particle size of the non-magnetic particles 73 is 1.5 times the particle size of the magnetic particles 72 in the measurement step. Compared with the case where it is less than, the non-magnetic particles 73 can be accurately identified, and the number of non-magnetic particles 73 can be accurately measured in the measuring step.

[Embodiment 2]
Next, the second embodiment will be described. The second embodiment is a mode in which the number of non-magnetic particles is measured based on the magnitude of the reflected light when a part of the flow path described later is irradiated with the laser light. However, the configuration of the second embodiment is substantially the same as the configuration of the first embodiment unless otherwise specified, and the configuration substantially the same as the configuration of the first embodiment is used in the first embodiment. The same code and / or name as the one used was added as necessary, and the description thereof will be omitted.

(Constitution)
First, the configuration of the measurement system according to the second embodiment will be described. FIG. 9 is a schematic diagram illustrating the measurement system according to the second embodiment. As shown in FIG. 9, the measurement system 100 according to the second embodiment generally includes a reaction tank 10, a flow path 110, a laser light source 120, a light receiving unit 130, and a control device 60. Further, among these components, each of the laser light source 120 and the light receiving unit 130 and the control device 60 are electrically connected via the wiring 2.

(Structure-flow path)
The flow path 110 is for flowing at least a part of the complex 74 housed in the reaction tank 10 in a predetermined direction (downward in FIG. 9). The flow path 110 is formed of, for example, a long tubular body (in FIG. 9, a tubular body having open ends at both ends), and as shown in FIG. 9, the longitudinal direction of the flow path 110 is in the vertical direction. (In addition, in the vicinity of the open end on the lower side of the flow path 110, the complex 74 and the solvent 75 that flowed out from the flow path 110 are fixed to a fixing member (not shown). A storage tank (not shown) for accommodating the flow path 110 is provided. Further, although the specific size of the flow path 110 is arbitrary, in the second embodiment, the flow path diameter of the flow path 110 and the flow path 110 The diameter of the open end on the lower side of the above is set to a size at which the flow velocity of the complex 74 flowing through the flow path 110 becomes a predetermined flow velocity. The flow velocity is set so that the complex 74 flowing through the flow path 110 can be specified one by one by the measuring unit 65c. The thickness of the flow path 110 is set to a thickness that can secure a predetermined strength. Further, the material of the flow path 110 can be arbitrarily formed as long as it is a material having excellent chemical resistance and allowing light to pass through, for example, a glass material, a resin material, or the like. May be formed.

(Structure-laser light source)
The laser light source 120 is an irradiation means for irradiating a part of the flow path 110 through which the composite 74 flows with laser light LL (for example, ultraviolet laser light, visible light laser light, etc.). The laser light source 120 is configured by using, for example, a known laser oscillator, and is arranged at a position near the flow path 110 (in FIG. 9, the left side of the flow path 110) as shown in FIG.

(Structure-light receiving part)
The light receiving unit 130 is a light receiving means that receives the reflected light RL when the laser light LL is irradiated from the laser light source 120. The light receiving unit 130 is configured by using, for example, a known light receiver, and is located near the flow path 110 and faces the laser light source 120 via the flow path 110 (as shown in FIG. 9). In FIG. 9, it is arranged on the right side of the flow path 110).

(Configuration-Control device)
As shown in FIG. 9, the control device 60 includes an operation unit 61, a communication unit 62, an output unit 63, a power supply unit 64, a control unit 65, and a storage unit 66. Further, as shown in FIG. 9, the control unit 65 includes a second measurement unit 65c and a specific unit 65b in terms of functional concept. Here, the second measuring unit 65c is a second measuring means for measuring the number of non-magnetic particles 73 based on the size of the reflected light RL received by the light receiving unit 130.

(Measuring method)
Next, a measurement method performed using the measurement system 100 configured in this way will be described. FIG. 9 is a schematic diagram showing an outline of the measurement process. The measurement method according to the second embodiment is performed in substantially the same manner as the measurement method according to the first embodiment. However, the measurement process has been devised as shown below.

(Measurement method-Measurement process)
The measuring step is performed in the following procedure in order to measure the number of non-magnetic particles 73 housed in the reaction vessel 10 after the second removing step.

That is, first, at least a part of the complex 74 housed in the reaction vessel 10 in the flow path 110 (specifically, in the flow path 110 containing the solvent 75) is placed in a predetermined direction (downward in FIG. 9). Shed towards. Specifically, a predetermined amount of the complex 74 and the solvent 75 are taken out from the reaction tank 10 by a liquid feeding pipe (not shown), and the taken-out complex 74 and the solvent 75 are flowed from the upper open end of the flow path 110. It is dropped into 110, and the dropped complex 74 is allowed to flow according to the flow of the solvent 75 in the flow path 110. Regarding the work related to the dropping, the work is repeated until the complex 74 and the solvent 75 in the reaction tank 10 disappear so that all the non-magnetic particles 73 contained in the reaction tank 10 can be measured.

Next, as shown in FIG. 9, the reflected light RL when the laser light LL is irradiated from the laser light source 120 to a part of the flow path 110 through which the composite 74 flows by the second measuring unit 65c of the control device 60. The number of non-magnetic particles 73 is measured based on the size of.

The method for measuring the number of the non-magnetic particles 73 is arbitrary, but for example, first, as shown in FIG. 9, the timing at which the complex 74 and the solvent 75 are dropped (or a timing slightly earlier than that). Then, a part of the flow path 110 is irradiated with the laser light LL from the laser light source 120, and the reflected light RL at the time of the irradiation is received by the light receiving unit 130. Next, the second measuring unit 65c of the control device 60 specifies the size of the reflected light RL received by the light receiving unit 130 at predetermined timings using a known optical measuring method, and the specified size is specified. Information (hereinafter, referred to as “reflected light information”) indicating the above is stored in the storage unit 66. Then, at the timing when a predetermined time elapses after the work related to the dropping is completed, the size of the reflected light RL equal to or larger than the threshold value among the reflected light information stored in the storage unit 66 by the second measuring unit 65c of the control device 60. The reflected light information indicating the above is extracted, and the number of the extracted reflected light information is measured as the number of non-magnetic particles 73 to be measured. The predetermined time is set to be longer than, for example, a time generally assumed to be required until the last dropped complex 74 is irradiated with the laser beam LL.

By such a measurement step, the number of non-magnetic particles 73 can be measured relatively accurately, and the measurement accuracy can be improved.

As described above, according to the second embodiment, in the measurement step, at least a part of the complex 74 housed in the reaction vessel 10 is flowed in the flow path 110 in a predetermined direction, and the flow path 110 through which the complex 74 flows. Since the number of non-magnetic particles 73 is measured based on the magnitude of the reflected light RL when the laser light LL is irradiated from the laser light source 120 to a part of the above, the number of non-magnetic particles 73 is relatively accurate. It can be measured and the measurement accuracy can be improved.

[Embodiment 3]
Next, the third embodiment will be described. The third embodiment is a mode in which the number of non-magnetic particles is measured based on the electrical change when the composite flowing through the flow path described later passes through the pore described later. However, the configuration of the third embodiment is substantially the same as the configuration of the first embodiment unless otherwise specified, and the configuration substantially the same as the configuration of the first embodiment is used in the first embodiment. The same code and / or name as the one used was added as necessary, and the description thereof will be omitted.

(Constitution)
First, the configuration of the measurement system according to the third embodiment will be described. FIG. 10 is a schematic diagram illustrating the measurement system according to the third embodiment. As shown in FIG. 10, the measurement system 200 according to the third embodiment generally includes a reaction tank 10, a detection mechanism 210, and a control device 60. Further, among these components, each of the first electrode portion described later, the second electrode portion described later, and the detection unit 214 of the detection mechanism 210 and the control device 60 are electrically connected via the wiring 2. There is.

(Configuration-Detection mechanism)
The detection mechanism 210 is a mechanism for detecting an electrical change (for example, a change in current) in the flow path 211, and includes a flow path 211, a passing portion (not shown), and a detecting portion 214.

(Configuration-Detection mechanism-Flow path)
The flow path 211 is for flowing at least a part of the complex 74 housed in the reaction tank 10 in a predetermined direction (downward in FIG. 10), and is abbreviated as the flow path 110 according to the second embodiment. It has the same structure (note that the material of the flow path 211 is made of a glass material, a resin material, or the like capable of conducting electricity). Further, as shown in FIG. 10, a partition plate 212 is provided in the flow path 211, and a pore 213 is formed in the partition plate 212. The pore 213 is a through hole for passing the complex 74, and is arranged in the central portion of the partition plate 212 as shown in FIG. The specific shape and size of the pore 213 are arbitrary, but in the third embodiment, the complex 74 can be reliably passed and the detection sensitivity of the detection unit 214 is set to a predetermined value or more. The circular shape is set to be substantially the same as or slightly larger than the maximum diameter of the complex 74 so that it can be maintained.

(Configuration-Detection mechanism-Passing part)
The passing portion is a passing means for passing the complex 74 through the pore 213. The passing portion preferably has a configuration in which the complex 74 is passed through the pore 213 by electrophoresis, and includes a first electrode portion and a second electrode portion (both are not shown).

Of these, the first electrode portion is configured by using, for example, a known electrode member or the like, is provided above the partition plate 212 in the flow path 211, and is fixed to the flow path 211. .. Further, the second electrode portion is a negative electrode or a ground pole when the first electrode portion is a positive electrode, a positive electrode or a ground pole when the first electrode portion is a negative electrode, and a ground pole when the first electrode portion is a ground pole. It is an electrode part that becomes a positive electrode or a negative electrode. The second electrode portion is configured by using, for example, a known electrode member or the like, is provided below the partition plate 212 in the flow path 211, and is fixed to the flow path 211.

(Configuration-Detection mechanism-Detection unit)
The detection unit 214 is a detection means for detecting an electrical change in the flow path 211 (change in current, etc. in the third embodiment). The detection unit 214 is configured by using, for example, a known current measurement sensor or the like, and is provided in the vicinity of the pore 213 outside the flow path 211 (or inside the flow path 211) as shown in FIG. , Is fixed to the flow path 211.

(Configuration-Control device)
The control device 60 includes an operation unit 61, a communication unit 62, an output unit 63, a power supply unit 64, a control unit 65, and a storage unit 66. Further, as shown in FIG. 10, the control unit 65 includes a third measurement unit 65d and a specific unit 65b in terms of functional concept. Here, the third measuring unit 65d is a third measuring means for measuring the number of non-magnetic particles 73 based on the electrical change when the complex 74 flowing through the flow path 211 passes through the pore 213.

(Measuring method)
Next, a measurement method performed using the measurement system 200 configured in this way will be described. FIG. 11 is a schematic diagram showing an outline of the measurement process. The measurement method according to the third embodiment is performed in substantially the same manner as the measurement method according to the first embodiment. However, the measurement process has been devised as shown below.

(Measurement method-Measurement process)
The measuring step is performed in the following procedure in order to measure the number of non-magnetic particles 73 housed in the reaction vessel 10 after the second removing step.

That is, first, at least a part of the complex 74 housed in the reaction vessel 10 in the flow path 211 (specifically, in the flow path 211 containing the solvent 75) is directed in a predetermined direction (downward in FIG. 11). Shed towards. Specifically, a predetermined amount of the complex 74 and the solvent 75 are taken out from the reaction tank 10 by a liquid feeding pipe (not shown), and the taken-out complex 74 and the solvent 75 are flowed from the upper open end of the flow path 211. It is dropped into 211, and the dropped complex 74 is allowed to flow according to the flow of the solvent 75 in the flow path 211. Regarding the work related to the dropping, the work is repeated until the complex 74 and the solvent 75 in the reaction tank 10 disappear so that all the non-magnetic particles 73 contained in the reaction tank 10 can be measured.

Next, as shown in FIG. 11, the dropped complex 74 is moved downward by passing a current C through the flow path 211 by the first electrode portion and the second electrode portion (that is, electricity). By electrophoresis), the complex 74 is passed through the pore 213. Further, at the timing when the current C starts to flow, the detection unit 214 detects the current C flowing in the flow path 211 at predetermined timings by using a known electric detection method (for example, an electric nanopulse method or the like). Do.

Subsequently, the third measuring unit 65d of the control device 60 measures the number of non-magnetic particles 73 based on the electrical change (change in current C) detected by the detection unit 214 at predetermined timing intervals. The method for measuring the number of the non-magnetic particles 73 is arbitrary. For example, first, information indicating the magnitude of the current C detected by the detection unit 214 by the third measurement unit 65d of the control device 60 ( Hereinafter referred to as "current information") is stored in the storage unit 66. Then, at the timing when a predetermined time elapses after the work related to the dropping is completed, the third measuring unit 65d of the control device 60 determines the magnitude of the current C equal to or larger than the threshold value among the current information stored in the storage unit 66. The indicated current information is extracted, and the number of the extracted current information is measured as the number of non-magnetic particles 73 to be measured. The predetermined time is set, for example, to be longer than the time generally assumed to be required for the last dropped complex 74 to pass through the pore 213.

By such a measurement step, the number of non-magnetic particles 73 can be measured relatively accurately, and the measurement accuracy can be improved.

As described above, according to the third embodiment, at least a part of the complex 74 housed in the reaction tank 10 is flowed in the flow path 211 in a predetermined direction, and the complex 74 flowing through the flow path 211 is the flow path. Since the number of non-magnetic particles 73 is measured based on the electrical change when passing through the pore 213 provided in 211, the number of non-magnetic particles 73 can be measured relatively accurately, and the measurement accuracy is high. Can be improved.

[Embodiment 4]
Next, the fourth embodiment will be described. The fourth embodiment is the captured image, in which the number of non-magnetic particles is measured based on the captured image including the complex flowing through the flow path. However, the configuration of the fourth embodiment is substantially the same as the configuration of the second embodiment unless otherwise specified, and the configuration substantially the same as the configuration of the second embodiment is used in the second embodiment. The same code and / or name as the one used was added as necessary, and the description thereof will be omitted.

(Constitution)
First, the configuration of the measurement system according to the fourth embodiment will be described. FIG. 12 is a schematic diagram illustrating the measurement system according to the fourth embodiment. As shown in FIG. 12, the measurement system 300 according to the fourth embodiment generally includes a reaction tank 10, a flow path 110, an image pickup device 310, and a control device 60. Further, among these components, the image pickup device 130 and the control device 60 are electrically connected via the wiring 2.

(Configuration-Imaging device)
The imaging device 310 is an imaging means for imaging the complex 74 flowing through the flow path 110. The image pickup device 310 has substantially the same configuration as the image pickup device 50 of the first embodiment, and is arranged near the flow path 110 as shown in FIG.

(Configuration-Control device)
As shown in FIG. 12, the control device 60 includes an operation unit 61, a communication unit 62, an output unit 63, a power supply unit 64, a control unit 65, and a storage unit 66. Further, as shown in FIG. 12, the control unit 65 includes a fourth measurement unit 65e and a specific unit 65b in terms of functional concept. Here, the fourth measuring unit 65e is a fourth measuring means for measuring the number of non-magnetic particles 73 based on the captured image captured by the imaging device 310.

(Measuring method)
Next, a measurement method performed using the measurement system 300 configured in this way will be described. The measurement method according to the fourth embodiment is performed in substantially the same manner as the measurement method according to the second embodiment. However, the measurement process has been devised as shown below.

(Measurement method-Measurement process)
The measuring step is performed in the following procedure in order to measure the number of non-magnetic particles 73 housed in the reaction vessel 10 after the second removing step.

That is, first, at least a part of the complex 74 housed in the reaction vessel 10 is flowed in the flow path 110 toward a predetermined direction (downward in FIG. 12).

Next, the imaging device 310 captures an image of the complex 74 flowing through the flow path 110 (for example, dynamic imaging), and the captured image is transmitted from the imaging device 310 to the control device 60.

Subsequently, similarly to the method of measuring the number of non-magnetic particles 73 according to the first embodiment, the non-magnetic particles 73 are not based on the captured image received by the communication unit 62 by the fourth measurement unit 65e of the control device 60. The number of magnetic particles 73 is measured.

By such a measurement step, the number of non-magnetic particles 73 can be measured relatively accurately, and the measurement accuracy can be improved.

As described above, according to the fourth embodiment, in the measurement step, at least a part of the complex 74 housed in the reaction tank 10 is flowed in the flow path 110 in a predetermined direction, and the flow path 110 is operated by the image pickup apparatus 310. Since the number of non-magnetic particles 73 is measured based on the image captured by the image pickup device 310, the number of non-magnetic particles 73 can be measured relatively accurately. , It is possible to improve the measurement accuracy.

[III] Modifications to Embodiments The embodiments of the present invention have been described above, but the specific configurations and means of the present invention are within the scope of the technical idea of each invention described in the claims. , Can be arbitrarily modified and improved. Hereinafter, such a modification will be described.

(About the problem to be solved and the effect of the invention)
First, the problem to be solved by the invention and the effect of the invention are not limited to the above-mentioned contents, and the present invention solves a problem not described above or an effect not described above. It can also be played, and may solve only some of the tasks described or play only some of the effects described. For example, even if the measurement work of the measurement method according to the present invention is speeded up and simplified to the same level as the conventional method, the measurement work can be speeded up and simplified to the same level as the conventional method by a method different from the conventional method. In some cases, the problems of the present invention have been solved.

(About distribution and integration)
Further, each of the above-mentioned electrical components is a functional concept, and does not necessarily have to be physically configured as shown in the figure. That is, the specific form of dispersion or integration of each part is not limited to the one shown in the figure, and all or part of them are functionally or physically dispersed or integrated in arbitrary units according to various loads and usage conditions. Can be configured. For example, the control device 60 is dispersedly configured in a plurality of devices configured to be able to communicate with each other, a control unit 65 is provided in a part of the plurality of devices, and the other part of the plurality of devices is provided. A storage unit 66 may be provided.

(About shape, numerical value, structure, time series)
With respect to the components illustrated in the embodiments and drawings, the shapes, numerical values, or the interrelationships of the structures or time series of the plurality of components shall be arbitrarily modified and improved within the scope of the technical idea of the present invention. Can be done.

(About magnetic particles and non-magnetic particles)
In the above embodiments 1 to 4, it has been explained that the particle size of the magnetic particles 72 is set to be larger than the particle size of the measurement target 71, but the present invention is not limited to this, and for example, it is abbreviated as the particle size of the measurement target 71. It may be set to be the same.

Further, in the first to fourth embodiments, it has been explained that the particle size of the magnetic particles 72 is set to a size that cannot be seen from the captured image, but the present invention is not limited to this, and for example, the magnetic particles 72 can be visually recognized from the captured image. And may be set to a size smaller than the particle size of the non-magnetic particles 73.

Further, in the above-described first to fourth embodiments, it has been explained that the particle size of the non-magnetic particles 73 is 1.5 times or more the particle size of the magnetic particles 72, but the present invention is not limited to this, and for example, the magnetic particles It may be larger than the particle size of 72 and less than 1.5 times the particle size of the magnetic particles 72.

In the above-described first to fourth embodiments, the shape, color, and particle size of the non-magnetic particles 73 have been described as one type, but the present invention is not limited to this. For example, the shape, color, fluorescence, enzyme label, isotope label, or particle size of the non-magnetic particles 73 may be two or more. Thereby, for example, when measuring the measurement target 71 having different types or conditions, the number of measurement targets 71 can be accurately measured for each type or condition, and the measurement can be performed according to the needs of the user. It will be possible.

(About the measurement system)
In the first embodiment, it has been described that the measurement system 1 includes the second magnet 40, but the present invention is not limited to this, and for example, the second magnet 40 may be omitted. In this case, in the measurement step of the measurement method, after at least a part of the complex 74 housed in the reaction vessel 10 is placed on the substrate 30, the above-described complex 74 is placed by the imaging device 50. The image is taken at a predetermined magnification.

(Additional note)
The measurement method of Appendix 1 is a measurement method for measuring a measurement target contained in a sample, in which the measurement target, the magnetic particles, the measurement target, and the magnetic particles have a larger particle size than the measurement target and the magnetic particles. A production step of producing a composite by reacting with identifiable non-magnetic particles by a predetermined method, and the measurement target and the non-magnetic particles contained in the container during or after the production step. Of these, a removal step of removing the measurement target and the non-magnetic particles that did not react in the production step, and a measurement step of measuring the number of the non-magnetic particles contained in the container after the removal step. , A specific step of specifying the number of measurement targets based on the number of non-magnetic particles measured in the measurement step.

In the measuring method of Appendix 2, in the measuring method of Appendix 1, at least a part of the composite housed in the container is placed on the substrate and provided below the substrate in the measuring step. The complex previously placed is collected at a predetermined position on the substrate by a magnet, the collected complex is imaged by an imaging means, and based on an image captured by the imaging means, the image is taken. The number of the non-magnetic particles is measured.

In the measuring method of Appendix 3, in the measuring method described in Appendix 2, the particle size of the magnetic particles is set to a size invisible from the captured image, and the particle size of the non-magnetic particles is visible from the captured image. I made it a big size.

In the measuring method of Appendix 4, in the measuring method described in Appendix 1, at least a part of the complex contained in the container is flowed in a predetermined direction in the flow path in the measuring step, and the complex is formed. The number of the non-magnetic particles is measured based on the magnitude of the reflected light when the laser light is irradiated from the laser light source to a part of the flowing flow path.

In the measuring method of Appendix 5, in the measuring method described in Appendix 1, at least a part of the complex contained in the container is flowed in a predetermined direction in the flow path in the measuring step, and the flow path is flowed. The number of the non-magnetic particles is measured based on the electrical change when the flowing composite passes through the pores provided in the flow path.

In the measuring method of Supplementary Note 6, in the measuring method of Supplementary Note 1, at least a part of the complex contained in the container is flowed in a predetermined direction in the flow path in the measuring step, and the imaging means is used. The complex flowing through the flow path is imaged, and the number of the non-magnetic particles is measured based on the image captured by the imaging means.

In the measuring method of Appendix 7, in the measuring method according to any one of Supplements 1 to 6, the particle size of the non-magnetic particles was set to 1.5 times or more the particle size of the magnetic particles.

In the measuring method of Appendix 8, in the measuring method according to any one of Supplements 1 to 7, the shape, color, fluorescence, enzyme labeling, isotope labeling, or particle size of the non-magnetic particles are set to two or more types. did.

(Effect of appendix)
According to the effect described in Appendix 1, the measurement target, the magnetic particles, and the non-magnetic particles having a larger particle size than the magnetic particles and identifiable by a predetermined method are reacted in the container to form a composite. The generation step of forming the body, the removal step of removing the measurement target and the non-magnetic particles that did not react in the production step during or after the production step, and the removal step of the non-magnetic particles contained in the container after the removal step. It includes a measuring step of measuring the number of particles and a specific step of specifying the number of objects to be measured based on the number of non-magnetic particles measured in the measuring step. According to this method, by allowing a sufficiently large number of non-magnetic particles having a particle size sufficiently large with respect to the target substance to exist in the reaction system with respect to the amount to be measured, in principle, with respect to one molecule of the target substance. One non-magnetic particle can be bonded together. Then, by counting the non-magnetic particles bound to the measurement target, the measurement target can be detected at the single molecule level. Compared with the conventional technique (a technique for measuring the number of a plurality of biomolecules separately arranged in a plurality of wells), this method does not require complicated work and can easily and quickly reduce the number of measurement targets. It is possible to measure the efficiency of the measurement work.

According to the effect described in Appendix 2, in the measurement step, the composite mounted on the substrate is collected at a predetermined position on the substrate by the magnet provided below the substrate, and the composite collected by the imaging means. Since the number of non-magnetic particles is measured based on the captured image captured by the imaging means, a large number of non-magnetic particles can be efficiently measured and the measurement work can be made more efficient. Is possible.

According to the effect described in Appendix 3, the particle size of the magnetic particles was set to a size invisible from the captured image, and the particle size of the non-magnetic particles was set to a size visible from the captured image. It can be identified accurately, and the number of non-magnetic particles can be accurately measured in the measuring process.

According to the effect described in Appendix 4, in the measurement step, at least a part of the complex contained in the container is flowed in a predetermined direction in the flow path, and a laser is applied to a part of the flow path through which the complex flows. Since the number of non-magnetic particles is measured based on the magnitude of the reflected light when the laser beam is irradiated from the light source, the number of non-magnetic particles can be measured relatively accurately, and the measurement accuracy can be improved. Is possible.

According to the effect described in Appendix 5, at least a part of the complex housed in the container is flowed in a predetermined direction in the flow path, and the composite flowing through the flow path is provided in the pore. Since the number of non-magnetic particles is measured based on the electrical change when passing through, the number of non-magnetic particles can be measured relatively accurately, and the measurement accuracy can be improved.

According to the effect described in Appendix 6, at least a part of the complex housed in the container is flowed in a predetermined direction in the flow path, and the image pickup means is used to image the complex flowing through the flow path. Since the number of non-magnetic particles is measured based on the captured image captured in the above, the number of non-magnetic particles can be measured relatively accurately, and the measurement accuracy can be improved.

According to the effect described in Appendix 7, the particle size of the non-magnetic particles was set to 1.5 times or more the particle size of the magnetic particles. Therefore, in the measurement step, the particle size of the non-magnetic particles was set to the particle size of the magnetic particles. Compared with the case where the value is less than 1.5 times, the non-magnetic particles can be accurately identified, and the number of non-magnetic particles can be accurately measured in the measuring step.

According to the effect described in Appendix 8, since the shape, color, fluorescence, enzyme label, isotope label, or particle size of the non-magnetic particles are set to two or more types, for example, measurement targets having different types or conditions are measured. In this case, the number of measurement targets can be accurately measured for each type or condition, and the measurement can be performed according to the needs of the user.

1 Measurement system 2 Wiring 10 Reaction tank 20 Mounting table 30 Board 40 Second magnet 50 Imaging device 60 Control device 61 Operation unit 62 Communication unit 63 Output unit 64 Power supply unit 65 Control unit 65a First measurement unit 65b Specific unit 65c Second measurement Part 65d 3rd measurement part 65e 4th measurement part 66 Storage part 70 Sample 71 Measurement target 72 Magnetic particles 72a Magnetic side antibody 73 Non-magnetic particles 73a Non-magnetic side antibody 74 Complex 75 Solvent 100 Measurement system 110 Flow path 120 Laser light source 130 Light receiving part 200 Measurement system 210 Detection mechanism 211 Flow path 212 Partition plate 213 Pore 214 Detection part 300 Measurement system 310 Imaging device C Current LL Laser light RL Reflected light

Claims (8)

It is a measurement method for measuring the measurement target contained in the sample.
A composite is produced by reacting the measurement target with magnetic particles in a container with non-magnetic particles having a particle size larger than that of the measurement target and the magnetic particles and which can be identified by a predetermined method. Generation process and
A removal step of removing the measurement target and the non-magnetic particles that did not react in the production step among the measurement target and the non-magnetic particles contained in the container during or after the production step.
After the removal step, a measuring step of measuring the number of the non-magnetic particles contained in the container, and a measuring step of measuring the number of the non-magnetic particles.
A specific step of specifying the number of measurement targets based on the number of non-magnetic particles measured in the measurement step, and a specific step of specifying the number of measurement targets.
Measurement method including. In the measurement step
At least a part of the complex contained in the container is placed on the substrate, and the complex is placed on the substrate.
The complex previously placed is collected at a predetermined position on the substrate by a magnet provided below the substrate.
The collected complex is imaged by an imaging means, and the complex is imaged.
The number of the non-magnetic particles is measured based on the captured image captured by the imaging means.
The measuring method according to claim 1. The particle size of the magnetic particles is set to a size invisible from the captured image.
The particle size of the non-magnetic particles was set to a size visible from the captured image.
The measuring method according to claim 2. In the measurement step
At least a part of the complex contained in the container is allowed to flow in a predetermined direction in the flow path.
The number of the non-magnetic particles is measured based on the magnitude of the reflected light when the laser light is irradiated from the laser light source to a part of the flow path through which the composite flows.
The measuring method according to claim 1. In the measurement step
At least a part of the complex contained in the container is allowed to flow in a predetermined direction in the flow path.
The number of the non-magnetic particles is measured based on the electrical change when the complex flowing through the flow path passes through the pores provided in the flow path.
The measuring method according to claim 1. In the measurement step
At least a part of the complex contained in the container is allowed to flow in a predetermined direction in the flow path.
The complex flowing through the flow path is imaged by the imaging means, and the complex is imaged.
The number of the non-magnetic particles is measured based on the captured image captured by the imaging means.
The measuring method according to claim 1. The particle size of the non-magnetic particles was set to 1.5 times or more the particle size of the magnetic particles.
The measuring method according to any one of claims 1 to 6. The shape, color, fluorescence, enzyme label, isotope label, or particle size of the non-magnetic particles were set to two or more types.
The measuring method according to any one of claims 1 to 7.

Images