JP2012172981A - Kit for detection of specific reaction, device for detection of specific reaction, and method for detection of specific reaction - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a kit for detection of specific reaction, a device for detection of specific reaction, and a method for detection of specific reaction, which are capable of reducing test burdens, and performing determination easily.SOLUTION: A device for detection of specific reaction 1 according to the present invention comprises: a plate for detection of specific reaction 10; and magnetic radiation means 20 for generating a magnetic field. A passage of the plate for detection of specific reaction 10 is in communication with an inlet into which a reagent sample 5 and a test sample 6 are introduced. The reagent sample 5 includes a magnetic particle complex 60 in which a magnetic particle is surface-modified with a substance for specific reaction. The presence or absence of another substance for specific reaction in the test sample 6 is determined. When specific reaction occurs, the test sample 6 forms an aggregate 8. In the passage, the specific reaction is detected by creating a difference between a magnetic aggregation pattern of the magnetic particle complex 60 and a magnetic aggregation pattern of the aggregate 8 using a magnetic force generated by the magnetic radiation means 20 and a flow pressure of a fluid of the passage.

Description

  The present invention relates to a specific reaction detection kit. The present invention also relates to a specific reaction detection device and a specific reaction detection method using the specific reaction detection kit.

  Antibodies that recognize antigens to form complexes play an important role in vertebrate defense mechanisms. The antigen and the antibody are in a relationship like a key and a keyhole, and an antigen-antibody reaction occurs when they completely match. Utilizing this property, antigen-antibody reaction tests have been put into practical use.

  Examples of methods that have been put into practical use include radioimmunoassay methods using isotope as a label, enzyme immunoassay methods using an enzyme as a label, and fluorescent immunoassay methods using a fluorescent substance as a label. In addition, a method of detecting a magnetic substance using magnetic particles as a label has been proposed (Patent Documents 1 to 4).

  In Patent Document 1, a magnetic particle is labeled by labeling an antigen or antibody with a magnetic particle, the magnetic label and the sample are reacted with an antigen-antibody, and then an unreacted magnetic label is separated and removed from the sample. A SQUID immunoassay method has been proposed in which the magnetization of a specimen is measured with a superconducting magnetic flux quantum interferometer (SQUID). From the measured value, it is possible to know the presence and amount of the magnetic label in the sample.

  Patent Document 2 proposes a method of analyzing by using magnetic particles as a label for antigen-antibody reaction and measuring the aggregation of magnetic particles with a magnetic sensor. In Patent Document 3, a high-frequency magnetic field is applied to magnetic particles remaining due to a biochemical reaction to generate heat, thereby detecting a change in physical quantity corresponding to the number of magnetic particles, and being contained in a sample solution. A method for detecting a substance has been proposed.

  Patent Document 4 proposes a labeled reagent comprising magnetic particles to which n types of antibodies are bound via an avidin / biotin bond. In addition, as an analysis method using this, a liquid sample is introduced into the above-mentioned labeled reagent held in a wet state so as to be movable in a wet state, and a complex is formed by an antigen-antibody reaction, and then installed downstream thereof. There has been proposed a method in which a complex is reacted specifically with a primary antibody immobilized on a reaction part, magnetic particles are immobilized on a substrate, and the state is detected by a magnetic detection device.

  Patent Document 5 proposes a detection method using the integration characteristics of magnetic beads and fluorescence. Specifically, a reaction circuit for performing an antigen-antibody reaction between the first monoclonal antibody and the second monoclonal antibody in the reagent of the reagent supply source and a biomarker in urine collected through the urinary catheter; A tube that guides the reagent of the reagent supply source to the reaction circuit, a magnetic mixture that takes in the mixed solution after the reaction in the reaction circuit, adsorbs the magnetic beads by magnetic force, and collects the biomarkers bound to the magnetic beads-first monoclonal antibody A collection unit and a fluorescence amount measurement unit that measures the amount of fluorescence generated by exciting the fluorescence dye of the second monoclonal antibody-fluorescent pigment bound to the collected biomarker with light of the excitation wavelength. A method is disclosed. Patent Document 6 and Non-Patent Document 1 will be described later.

JP-A-63-90765 JP 63-302367 A JP-A-2005-315677 JP 2007-139538 A JP 2010-256116 A Japanese Patent No. 4183407

"Efficient transfection reagent using magnetic particles", [online], April 21, 2008, Funakoshi Co., Ltd. [searched June 1, 2009], Internet (http: //www.funakoshi. co.jp/shiyaku/entry/539.php#top)

  In the spread of inspection, it is required that the inspection burden is small and the inspection determination method is simple. If a method for detecting an antigen-antibody reaction that realizes these can be provided, it has great significance in preventive medicine. In the above, the problems related to the detection method of antigen-antibody reaction have been described. However, there are other complementary relationships such as ligand / receptor, gene / complementary gene, etc. The problem may arise.

  The present invention has been made in view of the above-mentioned problems, and the object of the present invention is to provide a specific reaction detection kit, a specific reaction detection device, and a specific reaction that can be easily determined with a small examination burden. It is to provide a detection method.

  The specific reaction detection kit according to the present invention includes a specific reaction detection plate having a flow path for determining whether or not a substance in a test sample specifically reacts with a substance in a reagent sample. And magnetic irradiation means for generating a magnetic field in at least a part of the flow path of the specific reaction detection plate. The flow path of the specific reaction detection plate includes a reagent sample containing a magnetic particle complex obtained by surface-modifying one of substances to be subjected to a specific reaction on the magnetic particles, and the other target to be subjected to the specific reaction. For injecting a test sample that forms an aggregate between the magnetic particle complex and the other substance when the specific reaction occurs. By communicating with the inflow port and making the magnetic accumulation pattern of the magnetic particle complex and the aggregate different in the flow path by the magnetic force by the magnetic irradiation means and the fluid pressure of the fluid in the flow path, The reaction is detected.

  According to the specific reaction detection kit of the present invention, since the test sample and the reagent sample are mixed and the magnetic integration pattern is changed by the magnetic irradiation means to detect the specific reaction, the test burden is small. There is an excellent effect that a simple determination can be made.

  A specific reaction detection apparatus according to the present invention includes the specific reaction detection kit of the above aspect and an integrated pattern detection apparatus that detects a magnetic integrated pattern of the magnetic field generation flow path.

  The specific reaction detection method according to the present invention includes a reagent sample containing a magnetic particle complex in which one of substances targeted for a specific reaction is surface-modified on a magnetic particle, and the magnetic particle in the reagent sample is modified with the magnetic particle. A target for determining whether or not a specific reaction occurs with the substance to be subjected to the specific reaction, and when the specific reaction occurs, between the magnetic particle complex and the other substance. And injecting a test sample that forms an aggregate into the flow channel, mixing the reagent sample and the test sample, generating a magnetic field by at least a part of the flow channel by a magnetic irradiation means, and The specific reaction is detected by differentiating the magnetic accumulation pattern of the magnetic particle complex and the aggregate in the path by the magnetic force by the magnetic irradiation means and the fluid pressure of the fluid in the flow path.

  According to the present invention, there is an excellent effect that a specific reaction detection kit, a detection apparatus, and a specific reaction detection method that can be easily determined with a small examination burden can be provided.

The typical explanatory view of the specific reaction detecting device concerning a 1st embodiment. The typical perspective view of the specific reaction detection plate which concerns on 1st Embodiment. The top view of the 2nd board | substrate of the specific reaction detection plate which concerns on 1st Embodiment. 3B is a cross-sectional view taken along the line IIIB-IIIB in FIG. 3A. 3C is a cross-sectional view taken along the line IIIC-IIIC in FIG. 3A. Schematic explanatory drawing of an antibody-magnetic particle complex. Schematic explanatory drawing of an antigen. Schematic explanatory drawing which shows the antigen-antibody-magnetic particle aggregate in the case of positive. Schematic explanatory drawing of antibody-magnetic particle complex in the case of negative. The typical explanatory view of the specific reaction detecting device concerning a 2nd embodiment. The typical disassembled perspective view of the magnetic irradiation means which concerns on 2nd Embodiment. The typical explanatory view of the specific reaction detecting device concerning a 3rd embodiment. FIG. 9 is a schematic cross-sectional view of a specific reaction detection plate at the IX-IX cut portion in FIG. 8. The electron micrograph (after 45 second) of the magnetic integration pattern in the case of the negative which concerns on an Example. The electron micrograph (after 90 second) of the magnetic integration pattern in the case of the negative which concerns on an Example. The electron micrograph (after 135 second) of the magnetic integration pattern in the case of the negative which concerns on an Example. The electron micrograph (after 45 second) of the magnetic integration pattern in the case of positive which concerns on an Example. The electron micrograph (after 90 second) of the magnetic integration pattern in the case of positive which concerns on an Example. The electron micrograph (after 135 second) of the magnetic integration pattern in the case of being positive according to the example. The fluorescence macro imaging photograph of the magnetic integrated pattern which concerns on an Example.

  The detection of a specific reaction according to the present invention includes a reagent sample containing a magnetic particle complex in which one of substances to be subjected to a specific reaction is surface-modified to a magnetic particle, and a specific sample modified to a magnetic particle in the reagent sample. It is a target for determining whether or not a specific reaction occurs with a substance that is a target of a physical reaction, and when the specific reaction occurs, a test sample that forms an aggregate is used. More specifically, the test sample and the reagent sample are injected into the flow channel and mixed together, and a magnetic field is generated in at least a part of the flow channel by the magnetic irradiation means. The presence or absence of a specific reaction is detected from the magnetic accumulation pattern by differentiating the magnetic accumulation pattern of the magnetic particle composite and the aggregate according to the magnetic force and the fluid pressure of the fluid in the flow path. The detection of a specific reaction here is qualitative detection or / and quantitative detection.

  Hereinafter, an example of an embodiment to which the present invention is applied will be described. Needless to say, other embodiments are also included in the scope of the present invention as long as they meet the spirit of the present invention. Further, the sizes and ratios of the members in the following drawings are for convenience of explanation, and do not necessarily match the actual ones. In the following embodiments and examples, the same element members are denoted by the same reference numerals, and the description thereof is omitted as appropriate. In the following embodiments, an antigen-antibody reaction will be described as an example of a specific reaction. However, other complementary reactions such as a ligand-receptor reaction and a complementary internucleic acid reaction that is a gene / complementary gene are used. It is also possible to use for detection of substances that are related and react specifically.

[First Embodiment]
The specific reaction detection apparatus according to the first embodiment is a test system that detects a specific reaction such as an antigen-antibody reaction, and includes (a) a specific reaction detection kit and (b) an integrated pattern detection apparatus. (A) The specific reaction detection kit includes a specific reaction detection plate having a flow path for flowing a fluid, and magnetic irradiation means. (B) The integrated pattern detection device includes means for detecting a magnetic integrated pattern in the specific reaction detection plate.

  In FIG. 1, the typical explanatory drawing of the specific reaction detection apparatus which concerns on 1st Embodiment is shown. The specific reaction detection device 1 includes a specific reaction detection kit 2 and an integrated pattern detection device 3. The specific reaction detection kit 2 includes a specific reaction detection plate 10 and magnetic irradiation means 20. The integrated pattern detection device 3 includes a fluorescence microscope 30, an imaging device 40, and an image processing device 50.

  FIG. 2 shows a schematic perspective view of the specific reaction detection plate 10. The specific reaction detection plate 10 includes a first substrate 11, a second substrate 12, a first inlet 13, a second inlet 14, and an outlet 15. Inside the specific reaction detection plate 10, the first inlet 13, the second inlet 14, and the outlet 15 communicate with each other, and a flow path through which the test sample and the reagent sample can flow is formed. Yes. A part of one side surface of the specific reaction detection plate 10 is provided with a recess 16 for installing the magnetic irradiation means 20 (see FIG. 1).

  As shown in FIG. 1, the fluorescence microscope 30 includes a stage 31, a lens column 32, a lens barrel 33, an adjustment handle 34, an eyepiece lens 35, a revolver 36, an objective lens 37, a power supply terminal 38, and a planar light source sheet that functions as a light source. 39 etc. The fluorescence microscope 30 is connected to the imaging device 40 and the image processing device 50.

  The stage 31 of the fluorescence microscope 30 functions as a table on which the specific reaction detection plate 10 is placed. The stage 31 also functions as a mirror leg. The stage 31 may include a slide table mechanism that can move back and forth and right and left by a guide rail (not shown) or the like. Further, the stage 31 may be configured to be rotatable. Moreover, you may provide functions, such as a diagonal inclination function. It is preferable that the stage 31 includes an engaging portion or a fixed portion that can fix the planar light source sheet 39.

  The lens column 32 is attached to the stage 31. A lens barrel 33 that holds the objective lens 37 and the eyepiece lens 5 is attached to the upper part of the lens column 32. The height of the lens barrel 33 can be adjusted by an adjustment handle 34. Thereby, the distance between the specific reaction detection plate 10 and the objective lens 37 can be changed and the focus can be adjusted. A plurality of objective lenses 37 are attached to the revolver 36. The objective lens 37 to be used can be switched by rotating the revolver 36.

  The imaging device 40 is, for example, a CCD camera or the like. By connecting the CCD camera to the image processing unit 9, it is possible to perform image processing using a computer.

  In addition, a configuration may be adopted in which a lens leg is provided separately at the lower portion of the stage 31, the lens column 32 is fixed to the lens barrel 33, and the stage 31 is moved up and down with respect to the lens column 32 to focus. Good. The structure for moving the stage 31 is advantageous when an optical additional component such as an imaging device is attached to the lens barrel 33.

  The planar light source sheet 39 serves as a light source for irradiating the specific reaction detection plate 10 with excitation light of fluorescent molecules modified with magnetic particles, which will be described later, or as a white light transmitting light source. The planar light source sheet 39 is detachably attached to the stage 31 on the outermost surface on the stage 31. When a configuration in which the planar light source sheet 39 is installed on the uppermost part of the stage 31 and the planar light source sheet 39 is detachable from the stage 31 is desired to change the type of irradiation light or the life In the event of a failure, it can be easily replaced with a new planar light source sheet 39.

  The specific reaction detection plate 10 is placed immediately above the planar light source sheet 39 so as to be in contact therewith. The material of the 1st board | substrate 11 and the 2nd board | substrate 12 will not be specifically limited if it is the material which can be detected with the integrated pattern detection apparatus 3, and can detect the antigen antibody reaction based on this invention. For example, a glass substrate or a substrate made of resin can be used. From the viewpoint of easy visual observation or internal observation with a microscope, a transparent substrate is preferable. Moreover, a transparent resin is preferable from the viewpoint of ease of production. If a specific reaction detection plate 10 is made of resin, it is also suitable for disposable use.

  Although the thickness of the 1st board | substrate 11 and the 2nd board | substrate 12 is not specifically limited, Usually, it is about 2-5 mm. Moreover, although the vertical and horizontal sizes of the first substrate 11 and the second substrate 12 are not particularly limited, for example, they are about 30 mm × 70 mm.

  The first inlet 13, the second inlet 14, and the outlet 15 of the first substrate 11 are provided so as to penetrate the substrate from the surface of the first substrate 11. The first inflow port 13 and the second inflow port 14 are provided in the vicinity of one side of the first substrate 11, and the outflow port 15 is provided in the vicinity of one side opposite to the vicinity of one side where the first inflow port 13 and the like are provided. The first inlet 13 is an inlet for injecting a reagent sample, and the second inlet 14 is an inlet for injecting an antibody sample.

  3A is a schematic top view of the second substrate 12, FIG. 3B is a sectional view taken along the line IIIB-IIIB in FIG. 3A, and FIG. 3C is a sectional view taken along the line IIIC-IIIC in FIG. On the surface of the second substrate 12 that faces the first substrate 11, there are a first recess 17, a second recess 18, and a third recess 19 for forming an internal space structure. Further, a reagent sample channel 21, a test sample channel 22, a mixing channel 23, and a magnetic field generating channel 24, which are formed in a groove shape and are formed by joining to the first substrate, are formed. . These have a structure in which the first inlet 13 and the second inlet 14 communicate with the outlet 15.

  The first recess 17, the second recess 18, the reagent sample channel 21, the test sample channel 22, the mixing channel 23, and the magnetic field generation channel 24 have the same depth. On the other hand, the third recess 19 is deeper than the first recess 17 and the like so as to function as a liquid reservoir. In the first embodiment, an example is described in which the depths other than the third recess 19 are the same. For example, the bottom surface is inclined so as to become deeper toward the downstream side, or the magnetic field generation flow path 24 is provided. The depth and shape can be designed as appropriate so as to optimize the magnetic integration characteristics.

  The first recess 17 of the second substrate 12 and the first inlet 13 of the first substrate 11 form an integral space by joining the first substrate 11 and the second substrate 12. The same applies to the second recess 18 of the second substrate 12 and the second inlet 14 of the first substrate 11, the third recess 19 of the second substrate 12 and the outlet 15 of the first substrate 11.

  The first recess 17 communicates with the reagent sample channel 21, and the second recess 18 communicates with the test sample channel 22. The reagent sample channel 21 and the test sample channel 22 communicate with the mixing channel 23 on the downstream side. That is, in the mixing channel 23, the reagent sample that has passed through the reagent sample channel 21 and the test sample that has passed through the test sample channel 22 are mixed.

  The mixing channel 23 communicates with the magnetic field generating channel 24. The magnetic field generation flow path 24 is formed with a flow path that is folded with a folded portion. As shown in FIG. 1, a magnetic irradiation means 20 that is a magnetic field generation means is installed at one of the folded portions of the magnetic field generation flow path 24.

  With this configuration, the reagent sample passes from the first inlet 13 serving as the source flow through the first recess 17, passes through the reagent sample channel 21, the mixing channel 23, and the magnetic field generation channel 24, and further to the third recess 19. Up to. Similarly, the test sample passes from the second inlet 14 serving as the source flow through the second recess 18, passes through the test sample channel 22, the mixing channel 23, and the magnetic field generation channel 24, and further to the third recess. Up to 19.

  The flow path only needs to include at least the mixing flow path 23 and the magnetic field generation flow path 24, and the reagent sample flow path 21 and the test sample flow path 22 may not be provided. Further, the shape and forming method of the flow path are merely examples, and various modifications can be made without departing from the spirit of the present invention.

  The magnetic irradiation means 20 is disposed at one end of the side surface of the magnetic field generation flow path 24 of the specific reaction detection plate 10 and irradiates the magnetic field generation flow path 24 near the magnetic irradiation means 20 with magnetism. The magnetic irradiation means 20 has a magnet and a shield means. The shield means is constituted by a magnetic shielding member. Thereby, directivity can be imparted to the magnetic lines of force of the magnet. In addition, as the magnetic irradiation means 20, what is necessary is just to have a magnet, and when it is not necessary to provide directivity to a magnet, the shielding means does not need to be provided.

  The type and shape of the magnet are not particularly limited. For example, an electromagnet, a superconducting magnet, or a permanent magnet can be used. The magnetic field may be changed by varying the voltage or current using an electromagnet. Furthermore, you may give the device which provides a magnetic gradient. From the viewpoint of realizing miniaturization of the magnetic irradiation means 20, it is preferable to use a permanent magnet. The type of the permanent magnet is not particularly limited, but examples thereof include ferrite, Ne—Fe—B alloy, and samarium-cobalt alloy. Further, at least a part of the specific reaction detection plate itself may be composed of a magnet such as a rubber magnet. In the case where a strong magnetic force is required, a Ne—Fe—B alloy is preferable.

  As described above, the shield means has a shield function for imparting directivity in the magnetic field generation direction of the magnet. The shield means was constituted by a yoke. Of course, the material is not limited to this as long as it has a shielding function. By providing the shielding means, it is possible to increase the magnetic force from one direction of the magnet and to realize a significant attenuation of the magnetic force in other portions.

  Moreover, you may install so that a removable shield board can be attached between the specific reaction detection plate 10 and a magnet. Thereby, it becomes possible to control on / off of the magnetic irradiation to the specific reaction detection plate 10. Alternatively, a magnet made of an electromagnet may be fixed to the specific reaction detection plate 10 so that the generation of magnetic field lines can be turned on and off by turning on and off electricity.

  The reagent sample channel 21, test sample channel 22, mixing channel 23, and magnetic field generating channel 24 have channel widths and channel depths that allow the sample to be measured to flow smoothly and clog the sample due to drying. For the purpose of preventing or deactivation, it may be designed as appropriate according to the viscosity of the sample to be measured. Usually, the flow path width is in the range of 1 to 5000 μm and the depth is in the range of 1 to 1000 μm. From the viewpoint of easy generation of hydraulic pressure, it is preferable that the flow width is in the range of 50 to 5000 μm and the depth is in the range of 50 to 500 μm. Considering the flow velocity of magnetic nanoparticles having high magnetic adsorption efficiency, it is more preferable that the depth is 50 to 200 μm and the width is 50 to 200 μm.

  In the sample passage portions of the first substrate 11 and the second substrate, the antigen-antibody is not deactivated or the flow degree is adjusted, and the antibody-magnetic particle complex described later and the non-test sample In order to prevent specific adsorption, surface treatment can be performed as necessary. For the surface treatment, a known method can be used without limitation.

  The specific reaction detection plate 10 configured as described above is placed on the planar light source sheet 39. The specific reaction detection plate 10 only needs to be installed so that the light emitting region of the planar light source sheet 39 can irradiate the observation target region, and the sizes of the planar light source sheet 39 and the specific reaction detection plate 10 are not limited. .

  In the first embodiment, an example is given in which the specific reaction detection plate is set by placing the specific reaction detection plate 10 where the planar light source sheet 39 is fixed to the stage 31. Instead of this, the following configuration may be adopted. That is, the planar light source sheet 39 may be integrally attached to the specific reaction detection plate 10 and the specific reaction detection plate with the light source device may be placed on the stage 31. By configuring the planar light source sheet 39 to be detachable from the specific reaction detection plate 10, the planar light source sheet 39 can be repeatedly applied. In addition, in recent years, a planar light source sheet can be obtained at a relatively low cost, so that it can also be used as a specific reaction detection plate with a light source device for disposable use.

  The thickness of the planar light source sheet 39 is not particularly limited and can be selected according to the application and purpose, but is, for example, about 0.2 mm to 1.2 mm. As the planar light source sheet 39, an organic EL sheet composed of an organic electroluminescence (EL) element, an inorganic EL sheet composed of an inorganic EL element, and an LED sheet composed of an LED element can be suitably applied.

  The EL sheet has a structure in which a light-emitting layer containing a light-emitting compound (light-emitting material) is sandwiched between a cathode layer and an anode layer, and excitons are injected by recombination by injecting electrons and holes into the light-emitting layer. Is generated. The device emits light by using the emission of light (fluorescence, phosphorescence) when the exciton is generated and deactivated. The layer structure of the EL element is not particularly limited, and any known element can be used without limitation. Examples thereof include those having a layer structure of an anode, a light emitting layer, an electron transport layer, and a cathode, and those having a hole transport layer between the anode and the light emitting layer. The light-emitting layer is a layer that emits light by recombination of electrons and holes injected from the electrode, the electron transport layer, or the hole transport layer, and the light-emitting portion emits light even in the layer of the light-emitting layer. It may be an interface between a layer and an adjacent layer. The inorganic EL sheet can be provided at a particularly low cost, which is advantageous for cost reduction. Therefore, there are uses as a light source for disposable purposes.

  An LED sheet is an element in which light is generated from a bonding surface by bonding a p-type semiconductor and an n-type semiconductor and energizing the p-type semiconductor. An electrode is connected to the element and sealed with resin or the like. The LED sheet is particularly excellent in terms of long life. For example, a luminescence sheet (Lumi Techno Co., Ltd.) (see Non-Patent Document 1) can be applied.

  Next, the specific reaction detection method according to the first embodiment will be described. First, an antibody-magnetic particle complex is prepared. FIG. 4A shows a schematic explanatory diagram of an antibody-magnetic particle complex, and FIG. 4B shows a schematic explanatory diagram of an antigen. The antibody-magnetic particle complex 60 is a particle in which a core is a magnetic particle 61 and a fluorescent molecule 62 and two types of antibodies 63a and 63b are immobilized on the magnetic particle 61. The antigen 70 has epitopes 71a and 71b corresponding to the two types of antibodies 63a and 63b. The antibody 63a and the epitope 71a react specifically, and the antibody 63b reacts specifically with the epitope 71b.

  In this example, the antibody-magnetic particle complex 60 is modified with two types of antibodies 63a and 63b, and an antigen having two epitopes 71a and 71b is taken as an example. One or three or more antibodies may be bound. Similarly, the epitope which an antigen has may be 1 or 3 or more. Further, the antibody-magnetic particle complex 60 to be used is not limited to one type, and a plurality of types of antibody-magnetic particle complexes may be used. When using multiple types of antibody-magnetic particle complexes, for example, fluorescent molecules of different emission bands are labeled on different types of antibody-magnetic particle complexes, and the emission bands are detected when observing the magnetic accumulation pattern. This makes it possible to identify the type of antigen-antibody reaction.

The magnetic particles 61 are not particularly limited as long as they satisfy the conditions of (1) forming a complex with fluorescent molecules and antibodies, and (2) having magnetism that can be accumulated by the magnetic irradiation means 20. Can be applied. For example, magnetite (Fe ₃ O ₄ ), maghemite (Fe ₂ O ₃ ), iron monoxide (FeO), iron nitride, iron (Fe), nickel, cobalt, cobalt platinum chromium alloy, barium ferrite alloy, manganese aluminum alloy, Examples thereof include an iron platinum alloy, an iron palladium alloy, a cobalt platinum alloy, an iron neodymium boron alloy, and a samarium cobalt alloy. The magnetic particles are preferably made of a material having high magnetic anisotropy having high magnetic induction characteristics even if the particle diameter is small. Preferred materials for the magnetic particles 61 include iron nitride, FePt particles, and composites of FePt particles and nanoparticles containing other magnetic metal elements. Alternatively, nanoparticles or microparticles in which magnetic molecules are coated with nonmagnetic molecules may be used. Furthermore, the self-association type magnetic lipid nanoparticles disclosed in Patent Document 6, or lipid-coated magnetic nanoparticles or polymer-coated magnetic nanoparticles may be used.

  The average particle diameter of the magnetic particles 61 is not particularly limited, but is preferably 1 nm or more, and is preferably 500 nm or less in consideration of the flowability in the flow path.

  As a method for forming the antibody-magnetic particle complex 60, a known method can be used without limitation. For example, the antibody-magnetic particle complex 60 can be obtained by binding a fluorescent molecule 62 to a magnetic particle having a plurality of reactive groups via the reactive group and further binding an antibody to a part of the fluorescent molecule. it can. Alternatively, the fluorescent molecule 62 and the antibody 63 may be bonded to magnetic particles having a plurality of reactive groups via the reactive groups. Reactive groups include apidine-biotin bond, epoxy group, tosyl group, ester group, thiol group, amino group, acyl halide group, N-hydroxysuccinimide ester group, aldehyde group, maleimide group, vinyl sulfone group, benzotriazole A carbonate group, a promoacetamide group, etc. are mentioned. Alternatively, the magnetic particles may be coated with a lipid film or a polymer film, and the fluorescent molecules 62 may be introduced onto these surfaces by covalent bonding or the like. Preferable examples of the coating layer include lipids, surfactants, polymers such as polyethylene glycol having an alkoxysilyl group, a chlorosilyl group, an isocyanatosilyl group, a mercapto group at the terminal, and the like.

  The fluorescent molecules 62 need only be introduced at a detectable level and do not need to cover the magnetic particles 61. Further, the number of antibodies in one antibody-magnetic particle complex 60 may be one or more, but it is preferable to introduce a plurality of antibodies so that aggregates are formed by an antigen-antibody reaction.

A well-known thing can be used for a fluorescent molecule without a restriction | limiting. Examples include, for example, 1,5 IAEDANS; 1,8-ANS; 4-methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein; 5-carboxyfluorescein (5-FAM); 5-carboxynaphthofluorescein 5-carboxytetramethylrhodamine (5-TAMRA); 5-hydroxytryptamine (5-HAT); 5-ROX (carboxy-X-rhodamine); 6-carboxyrhodamine 6G; 6-CR 6G; 6-JOE; -Amino-4-methylcoumarin; 7-aminoactinomycin D (7-AAD); 7-hydroxy-4-I methylcoumarin; 9-amino-6-chloro-2-methoxyacridine (ACMA); ABQ; acidic fuchsin Acridine orange; acridine red; acridine yellow Acriflavin; acriflavin fuergen SITSA; aequorin (photoprotein); AFP-autofluorescent protein- (Quantum Biotechnologies) sgGFP; Alexa Fluor (R) 350; Alexa Fluor (R) 430; Alexa Fluor (R) 488 Alexa Fluor (R) 532; Alexa Fluor (R) 546; Alexa Fluor (R) 568; Alexa Fluor (R) 594; Alexa Fluor (R) 633; Alexa Fluor (R) 647; Fluor (R) 660; Alexa Fluor (R) 680; Alizarin Complexone; Alizarin Les Allophycocyanin (APC); AMC, AMCA-S; aminomethylcoumarin (AMCA); AMCA-X; aminoactinomycin D; aminocoumarin; aniline blue; antrocyle stearate; APC-Cy7; APTRA -BTC; APTS; Astrazone Brilliant Red 4G; Astrazon Orange R; Astrazon Red 6B; Astrazon Yellow 7 GLL; Atabulin; Auramin; Aurophosphine G; Aurophosphine; BAO 9 (Bisaminophenyloxadiazole); (High pH); BCECF (low pH); berberine sulfate; β-lactamase; BFP blue-shifted GFP (Y66H); blue fluorescent protein; BFP / GFP FRET; Bisbenzamide; Bisbenzimide (Hoechst); Bis-BTC; Blancophore FFG; Blancophore SV; Body Pe 492/515; 493/503; Body Pe 500/510; Body Pe; 505/515; Body Pe 530/550; Body P 558/568; Body P 564/570; Body P 576/589; Body P 581/591; Body P 630 / 650-X; Body P 650 / 665-X; Body P 665/676; Body P Fl Fl; Body P FL ATP; Body pea R6G SE; body pea TMR; body pea TMR-X conjugate; body pea TMR-X, SE; body pea TR; body pea TR ATP; TR-X SE; brilliant sulfo flavin FF; BTC; BTC-5N; calcein; calcein blue; calcium Crimson -; Calcium Green; Calcium Green -1 ^{Ca 2+} dyes; calcium green -2 ^{Ca 2+;} calcium green -5N ^{Ca 2+;} Calcium Green-C18 Ca ²⁺ ; Calcium Orange; Calcofluor White; Carboxy-X-Rhodamine (5-ROX); Cascade Blue®; Cascade Yellow; Catecholamine; CCF2 (GeneBlazer); CFDA; CFP (Cyan Fluorescent Protein) CFP / YFP FRET; chlorophyll; chromomycin A; chromomycin A; CL-NERF; CMFDA; coelenterazine; Coelenterazine f; coelenterazine fcp; coelenterazine h; coelenterazine hcp; coelenterazine ip; coelenterazine n; coelenterazine O; coumarin phalloidin; C-phycocyanin; CPM I methylcoumarin; CTC; Cy5.1 8; Cy5.5; Cy5; Cy7; Cyan GFP; Cyclic AMP Fluorescence Sensor (FiCRhR); Dabsyl; Dansyl; Dansylamine; Dansylcadaverine; Dansyl chloride; Dansyl DHPE; Dansyl fluoride; Dapoxil 2; dapoxil 3 ′ DCFDA; DCFH (dichlorodihydrofluorescein niacetic acid); DDAO; DHR (dihydrorhodamine 123); di 4-ANEPPS; Di-8-ANEPPS (non-ratio); DiA (4-Di 16-ASP); Dichlorodihydrofluorescein diacetic acid (DCFH); DiD-lipophilic tracer; DiD (DilC18 (5)); DIDS; Dihydrorhodamine 123 (DHR); Dil (DilC18 (3)); I dinitrophenol; DiO (DiOC18 (3)); DiR; DiR (DilC18 (7)); DM-NERF (high pH); DNP; dopamine; Red; DTAF; DY-630-NHS; DY-635-NHS; EBFP; ECFP; EGFP; ELF 97; eosin; erythrosin; erythrosin ITC; ethidium bromide; ethidium homodimer-1 (EthD-1); Europium (111) chloride; EYFP; Fast blue; FDA; Fulgen (pararosaniline); FIF (formaldehyde-induced fluorescence); FITC; Frazo orange; Fluoro-3; Fluoro-4; Fluorescein (FITC); Fluorescein niacetic acid; Fluoro-Gold (hydroxystilbamidine); Fluor-Ruby; Fluor X; FM 1-43; FM 4-46; Fura-2 / BCECF; Genacryl Brilliant Red B; Genacryl Brilliant Yellow 10GF; Genacryl Pink 3G Genacryl Yellow 5GF; Gene Blazer; (CCF2); GFP (S65T); GFP Red Shift (rsGFP); GFP wild-type non-UV excitation (wtGFP); GFP wild-type UV excitation (w GFPuv; glouvate; granular blue; hematoporphyrin; Hoechst 33258; Hoechst 33342; Hoechst 34580; HPTS; hydroxycoumarin; hydroxystilbamidine (fluorogold); hydroxytryptamine; Indo-1, high calcium; Indo-1 Indodicarbocyanine (DiD); Indotricarbocyanine (DiR); Intra White Cf; JC-1; JO JO-1; JO-PRO-1; Laser Pro; Laurodan; LDS 751 ( LDS 751 (RNA); leucophore PAF; leucophore SF; leucophore WS; lysamine rhodamine; risamine rhodamine B; calcein / ethidium homodimer; 1; LO-PRO-1; lucifer yellow; litho tracker blue; litho tracker blue-white; litho tracker green; litho tracker red; litho tracker yellow; litho sensor blue; litho sensor green; Magdara Red (Phloxine B); Mag-Fura Red; Mag-Fura-2; Mag-Fura-5; Mag-India-1; Magnesium Green; Magnesium Orange; Malachite Green; Marina Blue; I Maxilon Brilliant Flavin 10 GFF; Maxilon Brilliant Flavin 8 GFF; Merocyanine; Methoxycoumarin; Mitotracker Green FM; Mitotracker Orange; Mitotracker Red; Mitromycin; Monobromobiman; Mobiman (mBBr-GSH); Monochlorobiman; MPS (Methyl Green Pyronin Stilbene); NBD; NBD Amine; Nile Red; Nitrobenzoxazidole; Noradrenaline; Nuclear Fast Red; Nuclear Yellow; Oregon Green (registered trademark); Oregon Green (registered trademark) 488; Oregon Green (registered trademark) 500; Oregon Green (registered trademark) 514; Pacific Blue; Pararosaniline (Furgen); PBFI; PE-Cy5; PerCP-Cy5.5; PE-Texas Red (Red 613); Phloxine B (Magdara Red); Holwright AR; Holwright BKL; ev; Holwright RPA; Phosphine 3R; Photoresist; Phycoerythrin B [PE]; PKH26 (Sigma); PKH67; PMIA; Pontchrome Blue Black; POPO-1; POPO-3; PO-PRO-1; PRO-3; Primulin; Procion Yellow; Propidium Iodide (P1); PyMPO; Pyrene; Pyronin; Pyronin B; Pyrozar Brilliant Flavin 7GF; QSY7; Quinacrine Mustard; Rhodamine 123; rhodamine 5 GLD; rhodamine 6G; rhodamine B; rhodamine B 200; rhodamine B extra; rhodamine BB; rhodamine BG; rhodamine green; rhodamine faricidin; R: Phycocyanin; R-Phycoerythrin (PE); rsGFP; S65A; S65C; S65L; S65T; Sapphire GFP; SBFI; Serotonin; Sebron Brilliant Red 2B; Red 4G; Sebron I Brilliant Red B; Sebron Orange; Sebron Yellow L; sgBFP® (Super Glow BFP); sgGFP® (Super Glow GFP); SITS (Primulin; Stilbene Isothiosulfonic Acid); SNAFL SNAFL-1; SNAFL-2; SNARF calcein; SNARF1; sodium green; spectrum aqua; spectrum green; spectrum orange; SPTR (6-methoxy-N- (3sulfopropyl) quinolinium); stilbene; sulforhodamine B and C; sulforhodamine extra; SYTO 11; SYTO 12; SYTO 13; SYTO 14; SYTO 15; SYTO 16; SYTO18; SYTO20; SYTO21; SYTO22; SYTO23; SYTO24; SYTO25; SYTO40; SYTO41; SYTO42; SYTO43; SYTO44; SYTO45; SYTO59; SYTO60; SYTO 63; SYTO 64; SYTO 80; SYTO 81; SYTO 82; SYTO 83; SYTO 84; SYTO 85; SYTOX blue; SYTOX green; Tetracycline; tetramethylrhodamine (TRITC); Texas Red®; thiadicarbocyanine (DiSC3); thiazine red R; thiazole orange; thioflavin 5; thioflavin S; thioflavin TON; Fluor White); TIER; TO-PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; Tricolor (PE-Cy5); TRITC tetramethylrhodamine isothiocyanate; True Blue True Red; Ultralight; Uranine B; Ubitex SFC; wt GFP; WW 781; X-Rhodamine; XRITC; Xylene Orange; Y66F; Y66H; Y66W; YO-PRO-1; YO-PRO 3; YOYO-1; YOYO-3; Sybr Green; thiazole orange (interchelating dye); semiconductor nanoparticles such as quantum dots; or caged fluorophores (light or other electromagnetic) Including a combination thereof, or the like. In addition, other light emitting molecules (phosphorescence, etc.) may be used instead of fluorescent molecules.

  The coating layer may be a lipid membrane or a polymer membrane, and the fluorescent molecules 62 may be introduced to these surfaces by covalent bonding or the like. Preferable examples of the coating layer include lipids, surfactants, polymers such as polyethylene glycol having an alkoxysilyl group, a chlorosilyl group, an isocyanatosilyl group, a mercapto group at the terminal, and the like.

  The antibody may be an antibody contained in a body fluid sample obtained from a living body such as plasma, serum, urine, nasal discharge, tears, stool, tissue extract, or a test sample solution such as a culture solution. Alternatively, therapeutic antibodies may be used.

  As the test sample, a body fluid sample obtained from a living body such as plasma, serum, urine, nasal discharge, tears, stool, tissue extract or the like, or a test sample solution such as a culture solution can be used.

  Next, a reagent sample 5 containing the antibody-magnetic particle complex 60 is prepared. The fluid to be prepared is (1) capable of dispersing the antibody-magnetic particle complex 60 and (2) movable within a flow path provided in the specific reaction detection plate 10. Next, a test sample 6 is prepared by collecting an antigen to be examined from a patient or the like, and adjusting viscosity, concentration, etc. as necessary.

  Thereafter, the reagent sample 5 containing the antibody-magnetic particle complex 60 is injected from the first inlet 13 of the specific reaction detection plate 10 and the test sample 6 is injected from the second inlet 14 of the specific reaction detection plate 10. To do. The injection of the reagent sample 5 and the test sample 6 is performed at substantially the same timing. The injection timing may be adjusted according to the viscosity and characteristics of the reagent sample 5 and the test sample 6. In the mixing channel 23, when the reagent sample 5 and the test sample 6 are mixed and the antibody in the reagent sample 5 and a specific antigen are contained in the test sample 6, an antigen-antibody reaction occurs.

  The channel length, channel width, and the like of the mixing channel 23 are appropriately adjusted so that the antigen-antibody reaction occurs in the mixing channel 23. If necessary, physiological saline, phosphate buffer, or the like may be added to the reagent sample 5 and the test sample 6. It is also possible to introduce a gas that does not adversely influence the reaction, such as air, argon, nitrogen, etc., into the specific reaction detection plate 10 to optimize the flow rate. The flow of the fluid is adjusted by the pressure when injecting the sample from the microsyringe. It is also possible to use a micro syringe pump or the like.

  Thereafter, the magnetic field generation flow path 64 in the vicinity of the magnetic irradiation means 20 is observed with the fluorescence microscope 30. Here, when the test sample 6 contains an antigen that specifically reacts with the antibody of the antibody-magnetic particle complex 60 in the reagent sample 5 (when positive), the antigen-antibody-magnetism as shown in FIG. 5A. A particle aggregate 8 is formed. On the other hand, when the antigen specifically reacting with the antibody is negative in the test sample 8, no aggregate is formed as shown in FIG. 5B.

  The flow pressure of the magnetic field generation flow path 24 is set so that the antigen-antibody-magnetic particle aggregate 8 exceeds the adsorption by magnetic force. Thereby, the antigen-antibody-magnetic particle aggregate 8 flows in the magnetic field generation flow path 24 without staying in the vicinity of the magnetic irradiation means 20. On the other hand, in the case of a negative sample, the antigen-antibody-magnetic particle aggregate 8 is not formed, and the magnetic particles having a small size have a higher magnetic force than the fluid pressure and accumulate in the magnetic field generation region by the magnetic irradiation means 20.

  The planar light source sheet 39 provided in the fluorescence microscope 30 irradiates white light and detects the accumulated pattern of magnetic particles. Alternatively, the planar light source sheet 39 is irradiated with excitation light, and the integrated pattern of magnetic particles is detected from the distribution pattern of fluorescence in the magnetic particles. Of course, an accumulation pattern of magnetic particles may be detected by a normal operation using a normal fluorescence microscope. Then, whether the antigen-antibody reaction is positive or negative is determined based on the magnetic particle accumulation pattern in the magnetic field generation region. In other words, if the antibody-magnetic particle complex 60 is observed, it is determined to be negative, and if the antigen-antibody-magnetic particle aggregate 8 is detected, it is determined to be positive.

  As described above, since the antigen-antibody-magnetic particle aggregate 8 forms an aggregate and has a large particle size, the antigen-antibody-magnetic particle aggregate 8 is greatly influenced by the fluid pressure rather than the magnetic force. As a result, the antigen-antibody-magnetic particle aggregate 8 is not accumulated in the vicinity thereof due to the magnetic force of the magnetic irradiation means 20, but flows downstream. On the other hand, the antibody-magnetic particle complex 60 has a smaller particle size than the former, and is more influenced by the magnetic force than the fluid pressure. As a result, it accumulates in the vicinity of the magnetic irradiation means 20. By using this property, it is possible to easily perform positive / negative determination.

  According to the specific reaction detection apparatus 1 according to the first embodiment, negative / positive can be determined by injecting a test sample and a reagent sample into the specific reaction detection plate 10 and observing the magnetic accumulation pattern. Is possible. Therefore, the burden on the inspector can be minimized. Also, the determination operation is easy. From the fluorescence intensity of the aggregation pattern of the antigen-antibody-magnetic particle aggregate, it is possible to carry out a quantitative test of the antigen-antibody reaction. Therefore, it is possible to carry out a highly accurate antigen-antibody reaction test.

  In addition, as in Patent Document 1 and the like, there is an excellent feature that an antibody or antigen immobilization treatment is unnecessary. Therefore, a general-purpose specific reaction detection plate 10 can be provided. In addition, there is an advantage that there is no problem of variation in the immobilization rate due to the immobilization process. If a resin is used for the specific reaction detection plate 10, mass production is possible, and it is also suitable for disposable applications.

  Further, since a planar light source sheet is used as the light source, it is possible to reduce the thickness. In addition, since the planar light source sheet 39 that is a light source is in contact with the specific reaction detection plate 10, the space for the light source can be minimized. Therefore, the miniaturization of the microscope device and the light source device can be realized. Moreover, since the aspect which contact | abuts directly the planar light source sheet | seat 39 to the specific reaction detection plate 10 is employ | adopted, it is excellent also in the point of brightness | luminance and power saving.

[Second Embodiment]
Next, an example of a specific reaction detection apparatus different from the first embodiment will be described. FIG. 6 is a schematic explanatory view showing an example of the specific reaction detection apparatus 1 according to the second embodiment. The second embodiment is different in that the magnetic irradiation means 20a is disposed in the fluorescence microscope 30a. Specifically, the magnetic irradiation means 20a is accommodated in the accommodating part 41 provided in the stage 31a.

  The magnetic irradiation means 20a has a function of irradiating at least a part of the magnetic field generation flow path of the specific reaction detection plate 10a. As shown in FIG. 6, the magnetic irradiation means 20 a is disposed so as to face the specific reaction detection plate 10 a with the planar light source sheet 39 interposed therebetween. FIG. 7 shows an exploded perspective view of the magnetic irradiation means 20a according to the second embodiment. The planar light source sheet 39 and the magnetic irradiation unit 20a may be disposed so as to contact each other as in the second embodiment, or may be disposed at close positions. From the viewpoint of miniaturization of the fluorescence microscope 30a, it is preferable to place the planar light source sheet 39 and the magnetic irradiation means 20a in contact with each other. By arranging the planar light source sheet 39 and the magnetic irradiation means 20a in contact with each other, there is also a merit that reduction in attenuation of magnetic force can be more effectively suppressed.

  The magnetic irradiation means 20a includes a magnet 42, a shield means 43, etc. as shown in FIG. The shield means 43 is composed of a magnetic shielding member. Thereby, directivity can be imparted to the magnetic lines of force of the magnet 42. In addition, as the magnetic irradiation means 20a, what is necessary is just to have the magnet 42, and when it is not necessary to provide directivity to the magnet 42, the shielding means 43 does not need to be provided. In the second embodiment, a cylindrical body is used as shown in FIG.

  In the conventional microscope, when the observation object and the magnetic irradiation means are installed via the light source, there is a problem that the magnetic force becomes weak and the observation under a desired condition cannot be performed. This is because the magnetic force attenuates in proportion to the cube of the distance. That is, when a conventional light source is inserted between the specific reaction detection plate 10a and the magnet, the magnetic force is remarkably attenuated and it is difficult to perform real-time microscope observation.

  On the other hand, according to the microscope apparatus according to the second embodiment, a light source is inserted between the magnetic irradiation means 20a and the specific reaction detection plate 10a by using a very thin planar light source sheet 39 as a light source. However, the magnetic attenuation can be almost ignored. In addition, since the optical path of the fluorescence microscope 30a is not blocked by the magnetic irradiation means 20a, the real-time behavior in the magnetic irradiation region can be observed. In addition, the configuration is simple, and even if the magnetic irradiation means 20a is mounted, the size can be reduced.

  According to the second embodiment, the same effect as that of the first embodiment can be obtained. In addition, since the specific reaction detection plate is not provided with a recess, the specific reaction detection plate can be more versatile. There is a feature that the loss of magnetic force transmitted through the thin planar light source sheet can be minimized. In the case of the arrangement of magnets as in the second embodiment, the planar light source sheet is particularly effective. Furthermore, by using a planar light source sheet, it is possible to avoid the problem that “the optical axis of the transmission light source of the microscope is blocked when a magnet is brought close to the observation region of the plate, and transmission observation is not possible”. .

  If the magnetic irradiation means has a planar structure, it is possible to perform microscopic observation by placing the magnetic irradiation means, the light source, and the specific reaction detection plate 10 on the stage as they are.

[Third Embodiment]
In the specific reaction detection plate according to the third embodiment, the reagent sample channel 21, the test sample channel 22, and the mixing channel 23 are not formed, and a magnetic field generating channel is formed as a channel. The second embodiment is different from the second embodiment in that the reagent sample and the test sample are introduced from the first recess 17b, which is the same recess. The configuration of the other specific reaction detection apparatus is the same as that of the second embodiment.

  FIG. 8 is a schematic top view showing an example of the second substrate 12b of the specific reaction detection plate according to the third embodiment, and FIG. 9 is a cross-sectional view of the specific reaction detection plate of the IX-IX section in FIG. A schematic cross-sectional view is shown. As shown in FIG. 9, the specific reaction detection plate 10b includes a first substrate 11b, a second substrate 12b, two connecting portions 25, and a microsyringe pump 26 that is a circulation means. The second substrate 12b is formed with a first recess 17b, a magnetic field generation flow path 24b, and a third recess 18b (hereinafter also referred to as “internal flow path”). The first substrate 11b has an inlet 13b, an outlet 15b, and a connection portion 25 formed therein. The two connecting portions 25 function as ports for introducing or outflowing fluid. The internal flow path is configured to communicate between the two connecting portions 25. The internal flow path is configured such that the fluid can be moved by the microsyringe pump 26. At least a part of the magnetic field generation flow path 24 b has a region where a magnetic field is generated by the magnetic irradiation means 20.

  The micro syringe pump 26 plays a role of circulating the fluid in the internal flow path. In the example of FIG. 9, a tube connected to the two connecting portions 25 is provided. By using the microsyringe pump 26, it is possible to circulate a larger volume of fluid than the specific reaction detection plate 10b. Further, by using the microsyringe pump 26, it is easy to adjust the relationship between the flow velocity and the magnetic force so that the antigen-antibody reaction can be detected and quantified by the magnetic accumulation pattern. Further, by using the microsyringe pump 26, the configuration of the specific reaction detection plate 10b can be simplified. Note that the microsyringe pump can be applied to both suction and press-fitting.

  The planar light source sheet 39 and the magnetic force generation means 20a are disposed immediately below the specific reaction detection plate 10b as in the second embodiment.

  In addition, the position of the connection part 25 is not limited to the upper surface of the specific reaction detection plate 10b, You may provide in the bottom face and a side surface. Further, the internal flow path is not limited to the configuration of FIG. 8, and may be an internal flow path having a straight line, a branched structure, and a spiral structure.

  According to the antigen-antibody reaction detection method according to the third embodiment, the reagent sample and the test sample are simultaneously injected into the specific reaction detection plate. When a specific antigen-antibody reaction combination exists in the mixed fluid, an antigen-antibody-magnetic particle aggregate 8 is formed by the antigen-antibody reaction. Thereafter, the mixed fluid is injected from the inflow port of the specific reaction detection plate, and by detecting the accumulation pattern of the magnetic particles, the presence or absence of the antigen-antibody reaction is determined, or the antigen-antibody reaction is quantified. In the third embodiment, an example in which the reagent sample and the test sample are simultaneously injected into the specific reaction detection plate is described. However, a mixed fluid in which the reagent sample and the test sample are mixed in advance is prepared, Can also be injected into the specific reaction detection plate. In addition, although the example which uses a micro syringe pump as a circulation means was given, other well-known circulation means can be utilized without a restriction | limiting.

  According to the third embodiment, the same effect as that of the second embodiment can be obtained. Further, the use of the circulation means has an excellent merit that the relation between the magnetic force and the flow velocity for detecting and quantifying the antigen-antibody reaction from the magnetic accumulation pattern can be easily optimized by the circulation means. The specific reaction detection plate can be further shortened and simplified by providing a circulation means.

  The specific reaction detection plate according to the above embodiment is an example, and various modifications can be made. For example, in the specific reaction detection plate 10, an example of a plate in which one inspection lane is formed is shown, but it is also possible to provide a plurality of inspection lanes in one specific reaction detection plate. As a result, the inspection efficiency can be further increased.

  The shape of the flow path can be variously modified. The internal flow path may be arranged two-dimensionally in the plane, or three-dimensionally in the height direction. Further, a valve or the like may be appropriately provided in the internal flow path so that the direction of fluid flow in the internal flow path can be controlled according to the purpose.

  In the above description, an example in which the fluorescence microscope 30, the imaging device 40, and the image processing device 50 are used as the magnetic integrated pattern detection device 3 has been described. However, an imaging device such as a CCD camera or a macro imaging device is used without using the fluorescence microscope. Alternatively, the magnetic accumulation pattern may be directly imaged, and the imaging pattern may be automatically analyzed by a computer to automatically determine whether the antigen-antibody reaction is positive or negative.

  In the above description, the method of fixing fluorescent molecules to magnetic particles has been described. However, detection may be performed by binding other luminescent molecules. Furthermore, without using luminescent molecules such as fluorescent molecules, transmitted light patterns and reflection patterns, magnetic particles combined with various signal substances and sensors corresponding to signals are used to detect magnetic integrated patterns from their outputs, You may make it perform positive / negative determination of an antigen antibody reaction, and a quantitative test | inspection.

  In the above embodiment, an example in which the target for forming a complex with magnetic particles is an antibody. However, an antigen-magnetic particle complex in which an antigen is immobilized on magnetic particles is formed, It is also possible to determine and quantify the presence or absence of antibodies.

  Further, the specific reaction detection apparatus 1 may be provided with a magnetic detection apparatus. Thereby, the behavior of the magnetic particles can be observed in real time. In addition, the amount of magnetic material passing through the flow path can be quantified. In addition, for the purpose of condition (survival, activity, etc.) and integrated monitoring of magnetic materials, laser devices, optical sensors, densitometers, particle analyzers, cell sorters, radioactivity detectors, ultrasonic generators, ultrasonic detectors, etc. May be attached. Moreover, you may attach the microphone etc. which detect the sound which generate | occur | produces by the vibration of a nanoparticle, when an alternating magnetic field is irradiated to a magnetic nanoparticle.

  Moreover, in the said embodiment, although the example which uses a planar light source sheet as a light source was described, it is not limited to this, A well-known light source can be used without a restriction | limiting.

  The specific reaction detection kit according to the present invention can be suitably applied to a ligand-receptor reaction. In this case, the magnetic particles of the reagent sample are modified with either a ligand or a receptor, and whether there is a receptor or ligand that reacts between the ligand or receptor modified with the magnetic particles and the ligand-receptor in the test sample. To detect. Examples of the ligand include epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factor (TGF), insulin-like growth factor (IGF), fibroblast growth factor (FGF), nerve growth factor ( NGF). A receptor is a receptor (PDGF receptor etc.) corresponding to each ligand.

  The specific reaction detection kit according to the present invention can also be suitably applied to complementary internucleic acid reactions. In this case, the magnetic particles of the reagent sample are modified with nucleic acids such as DNA and RNA, and it is detected whether or not there is a nucleic acid that reacts in a complementary manner with the nucleic acid modified with the magnetic particles in the test sample. To do. The complementary nucleic acid reaction can be suitably applied to, for example, detection of a combination of siRNA and target mRNA. For example, siRNA can be modified into magnetic particles to detect whether target mRNA is present in the test sample.

<Example>
Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to the examples.

  Carcinoembryonic Antigen (CEA) —magnetic nanoparticles labeled with near infrared fluorescence (NIRF) forming a complex with an antibody were synthesized. In 1 ml of organic solvent and 1 ml of distilled water, egg yolk phosphatidylcholine, cholesterol, dithiopyridine dipalmitoyl phosphatidylethanolamine (manufactured by Northern Lipid), DiR (fluorescent dye, manufactured by Invitrogen) which is a hydrophobic counterpart of Cy7 cyanine, olein Acid coated iron oxide was mixed in a weight ratio of 16: 8: 1: 1: 22. Then, these were subjected to ultrasonic waves and evaporated to obtain NIRF-labeled magnetic nanoparticles.

  On the surface of the NIRF-labeled magnetic nanoparticles, N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP) and dithiothreitol (DTT) are used for the anti-CEA monoclonal by a conventional treatment method. The antibody was modified. Finally, an antibody-magnetic particle complex that specifically reacts with the target CEA antigen for an antigen-antibody reaction was prepared. It was confirmed that a specific antigen-antibody reaction occurred in the obtained antibody-magnetic particle complex and CEA antigen, and an antigen-antibody-magnetic particle aggregate was formed.

  The specific reaction detection plate was placed on an inverted microscope. Then, as a magnetic irradiation means, a magnet was installed above one end of the magnetic field generation flow path of the specific reaction detection plate, and the specific reaction detection plate was magnetically irradiated. The test sample and the reagent sample were simultaneously injected into the specific reaction detection plate. Then, using a micro syringe pump (NanoJet, manufactured by Chemyx), the test sample and the reagent sample were circulated at 25 nl / min.

  FIG. 10A to FIG. 10C show electron micrographs of magnetic accumulation patterns when a test sample having no CEA antigen that specifically reacts, that is, a negative test sample is used. Since the boundary of the magnetic field generating flow path 24 is difficult to see, the boundary of the flow path is traced in these drawings.

  FIG. 10A shows the result after 45 seconds, FIG. 10B shows the result after 90 seconds, and FIG. 10C shows the result after 135 seconds. After exceeding 90 seconds, washing with water was performed at the same flow pressure. Accordingly, when a negative test sample is used, the antigen-antibody-magnetic particle aggregate 8 is not formed, and the antibody-magnetic particle complex 60 is stepped above the magnetic field generation channel 24 stepwise by a magnetic field. It can be seen that it is attracted.

  FIG. 11A to FIG. 11C show electron micrographs of magnetic accumulation patterns when a positive test sample containing a CEA antigen that reacts specifically is used. Since the boundary of the magnetic field generating flow path 24 is difficult to see, the boundary of the flow path is traced in these drawings.

  FIG. 11A shows the result after 45 seconds, FIG. 11B shows the result after 90 seconds, and FIG. 10C shows the result after 135 seconds. After exceeding 90 seconds, washing with water was performed at the same flow pressure. Accordingly, when a positive test sample is used, the antigen-antibody-magnetic particle aggregate 8 is formed, and the antigen-antibody-magnetic particle aggregate 8 is not attracted by the magnetic field, and the magnetic field generating flow path 24 You can see that it is flowing inside.

  FIG. 12 shows the results of fluorescence distribution measurement for negative and positive samples using a macro imaging system (Lumason FA equipped with 512BE, manufactured by Roper Industries). The excitation light was 750 nm, and fluorescence at 780 nm was measured. As a result, fluorescence at 780 nm was not observed in the positive sample, whereas fluorescence at 780 nm was observed in the negative sample. From this, it can be seen that CEA antigen (100 ng / ml) can be detected by the presence or absence of the disappearance of NIRF-labeled magnetic particles by a magnetic field.

DESCRIPTION OF SYMBOLS 1 Specific reaction detection apparatus 2 Specific reaction detection kit 3 Integrated pattern detection apparatus 5 Reagent sample 6 Test sample 8 Antigen-antibody-magnetic particle aggregate 10 Specific reaction detection plate 11 1st board | substrate 12 2nd board | substrate 13 1st Inlet 14 Second inlet 15 Outlet 16 Recess 17 First recess 18 Second recess 19 Third recess 20 Magnetic irradiation means 21 Reagent sample channel 22 Test sample channel 23 Mixing channel 24 Magnetic field generating channel 25 Connection Unit 26 Micro syringe pump 30 Microscope 31 Stage 32 Lens column 33 Lens barrel 34 Adjustment handle 35 Eyepiece lens 36 Revolver 37 Objective lens 38 Power supply terminal 39 Planar light source sheet 40 Imaging device 50 Image processing device 60 Antibody-magnetic particle complex 61 Magnetic Particle 62 Fluorescent molecule 63 Antibody 70 Antigen

Claims (13)

A specific reaction detection plate having a flow path for determining whether or not a substance in a test sample has a specific reaction with a substance in a reagent sample;
Magnetic irradiation means for generating a magnetic field in at least a part of the flow path of the specific reaction detection plate,
The flow path of the specific reaction detection plate includes a reagent sample containing a magnetic particle complex obtained by surface-modifying one of substances to be subjected to a specific reaction on the magnetic particles, and the other target to be subjected to the specific reaction. Injecting a test sample that forms an aggregate between the magnetic particle complex and the other substance when the specific reaction occurs. Communicated with the inlet of
In the flow path, the specific reaction is detected by making the magnetic accumulation pattern of the magnetic particle complex and the aggregate different depending on the magnetic force by the magnetic irradiation means and the fluid pressure of the fluid in the flow path. Reaction detection kit.   The specific reaction detection kit according to claim 1, further comprising a circulation means for circulating the test sample and the reagent sample at least in the magnetic field generating channel.   The specific reaction detection kit according to claim 1, wherein the magnetic irradiation means includes an electromagnet, a superconducting magnet, or a permanent magnet.   The specific reaction detection kit according to claim 1, wherein the reagent sample and the test sample are injected into one inflow port.   The specific reaction detection kit according to any one of claims 1 to 4, wherein a luminescent molecule is immobilized on the magnetic particles of the reagent sample.   The specific reaction is an antigen-antibody reaction, and the magnetic particles of the reagent sample are modified with either an antigen or an antibody, and the antigen or antibody modified with the magnetic particles in the test sample 6. The specific reaction detection kit according to any one of claims 1 to 5, wherein it is detected whether or not an antibody or an antigen that reacts with an antigen antibody exists.   The specific reaction is a complementary internucleic acid reaction, the nucleic acid of the reagent sample is modified with a nucleic acid, and the reagent reacts with the nucleic acid modified with the magnetic particle in the test sample. The specific reaction detection kit according to any one of claims 1 to 5, wherein it detects whether or not a nucleic acid is present.   The specific reaction is a ligand-receptor reaction, wherein either the ligand or the receptor is modified in the magnetic particle of the reagent sample, and the ligand modified to the magnetic particle in the test sample Alternatively, the specific reaction detection kit according to any one of claims 1 to 5, wherein it detects whether or not a receptor or a ligand that reacts between a receptor and a ligand / receptor exists. A specific reaction detection kit according to claim 1;
A specific reaction detection device comprising: an integrated pattern detection device for detecting a magnetic integrated pattern in the magnetic field generation flow path.   The specific reaction detection device according to claim 9, wherein the detection device includes a light source including a planar light source sheet.   The magnetic irradiation means provided in the specific reaction detection kit is arranged to face at least a part of the specific reaction detection plate via the planar light source sheet. Specific reaction detector. A reagent sample containing a magnetic particle complex in which one of substances to be subjected to a specific reaction is surface-modified to magnetic particles, and a substance to be subjected to the specific reaction modified to the magnetic particles in the reagent sample; A target for determining whether or not a specific reaction occurs, and when the specific reaction occurs, a test sample that forms an aggregate between the magnetic particle complex and the other substance, Injected into the flow path,
The reagent sample and the test sample are mixed, and a magnetic field is generated in at least a part of the flow path by the magnetic irradiation means, and the magnetic force generated by the magnetic irradiation means and the fluid flow in the flow path are flown in the flow path. A specific reaction detection method for detecting the specific reaction by making a magnetic accumulation pattern of the magnetic particle complex and the aggregate different by pressure.   11. The specific reaction detection method according to claim 10, wherein the specific reaction is any one of an antigen-antibody reaction, a complementary nucleic acid reaction, and a ligand-receptor reaction.