JP2003075444A - Chip for measuring substance to be measured, substance measuring apparatus and method therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To achieve a handy making, an efficient and accurate measurement and even higher generality in a chip for measuring substances to be measured and a substance measuring apparatus and method therefor. SOLUTION: The chip 1 for measurement for measuring substances to be measured in a specimen 10 is provided with a chip substrate 2, the first channel- like passage 5A which is provided on the chip substrate 2 to let the specimen 10 pass therethrough, the second channel-like passage 5B which is provided on the chip substrate 2 to let a label substance 12 having a specific bond substance specifically bonded to the substances pass therethrough, the third channel- like passage 5C which is provided on the chip substrate 2 to let a link substance 5C specifically bonded to the matters pass therethrough, a channel-like reaction passage 5D which is formed on the chip substrate 2 with the gathering of the passages 5A, 5B and 5C, and a reaction part 5E which is provided in the reaction passage 5D having an immobilizing substance 13C specifically bonded to the link substance 13 immobilized thereon.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a measuring chip of a measuring object and a measuring object for measuring the amount of the measuring object in a sample by utilizing a specific reaction such as an antigen-antibody reaction. The measuring device, the measuring method of the measuring object, the connecting substance and the labeling substance.

[0002]

2. Description of the Related Art Conventionally, a technique for measuring an object to be measured in a sample has been developed, such as an immunoassay utilizing an antibody-antigen reaction. As such a technique, for example, there is an immunochromatography method. The basic principle of immunochromatography is as follows. That is, a chromatographic medium (for example, a porous membrane such as a nitrocellulose membrane)
In, a first site on which a substance labeled with a marker (labeled substance) is immobilized and a second site on which a specific binding substance that specifically binds to the measurement target is immobilized are formed, Is supplied to the first portion to react the measurement target with the labeling substance. Then, the complex of the measurement target and the labeling substance generated by this reaction is moved to the second site by utilizing the capillary phenomenon, and the specific binding substance-the measurement target-the labeling substance is moved to the second site. The complex is formed, and the amount of the object to be measured is measured based on the color developed by the immobilized labeling substance.

As an example of a technique to which the principle of such an immunochromatography is applied, Japanese Patent Laid-Open No. 5-133956.
Japanese Patent Laid-Open No. 9-1
There is a technique (prior art 2) disclosed in Japanese Patent No. 84840. In the prior art 1, the labeled particles are developed and moved on the chromatographic medium forming the moving layer in the presence of the vinyl-based water-soluble polymer having the oxygen atom-containing polar group. As a result, the labeled particles can be surely and swiftly developed and moved, and further, the effect of the sensitization of the labeled particles by the detection reagent is maintained for a long period of time so that the reaction between the labeled particles and the measurement object is surely caused. Therefore, even if the concentration of the measurement target in the sample is low, the measurement target can be detected reliably within a short time.

Further, in the prior art 2, the labeling substance and the specific binding substance are reacted in the liquid layer of the first portion to form a complex in the liquid layer, and the complex is formed by the capillary phenomenon into the second portion. Then, the amount of the object to be measured is measured based on the degree of color development derived from the labeling substance in the determination unit, and thus the labeling substance and the specific binding substance are separated into the liquid layer. By allowing the reaction to occur inside, the time required for such a reaction can be shortened.

As opposed to the immunochromatography method in which the measurement object is circulated by utilizing the capillary phenomenon as described above, as a measurement method capable of performing measurement while controlling the flow of the measurement object by pressure control or electroosmotic force. There is a technique of circulating a sample in a capillary having a specific binding substance immobilized on the inner wall. Examples of such a technique include the technique disclosed in Japanese Patent Laid-Open No. 60-133368 (Prior Art 3) and Biosensors & Bioelectronics 13 (1998) 825-
830 (A multi-band capillary immunosensor, K. Misiak
os, SE Kakabakos) (Prior Art 4)
There is.

[0006] In the prior art 3, an insoluble carrier on which a labeled substance-capturing substance for capturing a labeling substance is immobilized and an insoluble carrier on which a labeled immunoreaction reagent is held are filled in a capillary, and a sample is inhaled into the capillary for immunization. The reaction is performed, the reaction product or the labeled immunoreaction reagent is bound to the labeled substance-capturing substance to immobilize, and the amount of the measurement target contained in the sample is measured via the immobilized labeling substance. There is.

In the prior art 4, a capillary containing a specific binding substance is filled with a sample containing a fluorescence-labeled measurement target, and the specific binding substance is reacted with the measurement target. The reaction substance is washed away, and the capillary is irradiated with a light beam of a specific wavelength substantially perpendicular to the axial direction of the capillary, and the amount of fluorescence in the sample measured from the end of the capillary is used to measure the amount of the measurement target in the sample. Has become.

[0008]

However, in the immunochromatographic methods such as the above-mentioned prior arts 1 and 2, the flow of the sample is carried out by utilizing the capillary phenomenon. There is a problem that the flow rate cannot be controlled to a predetermined flow rate suitable for the reaction of the measurement object, the labeling substance and the specific binding substance. Furthermore, the chromatographic medium that serves as a place for developing the measurement target is generally composed of a porous film, and the porous film has low transparency.
Further, there is a problem that it is difficult to accurately measure the amount of the reaction product between the specific object and the measurement object because the light scattering in the spectroscopic analysis is large.

Further, in the technique of circulating the sample in the capillary as in the above-mentioned conventional techniques 3 and 4, since the cross-section of the capillary is a closed cross-section, the labeling substance or the labeling substance is limited to a predetermined location in the capillary. It is difficult to immobilize the specific binding substance, and similarly, it is difficult to apply a surface treatment suitable for the analysis system to the inner wall of the capillary, and thus there is a problem that it takes time to manufacture. Further, as a method for immobilizing the specific binding substance in the capillary which is a closed space, there is, for example, one utilizing a photoreaction, and in this method, the specific binding substance is irradiated by irradiating specific light into the capillary. It is designed to be fixed. However, this method has a problem that the chip substrate forming the outer wall of the capillary is limited to the transparent one.

Further, in PCT-WO93 / 24231, a groove portion (barrier portion) for circulating a sample is provided on a chip substrate, and a specific binding substance and a labeling substance are fixed at predetermined positions in the groove portion. There is disclosed a technique in which a lid is mounted on a chip substrate and the groove is configured as a capillary having a closed cross section. In this technology, by setting the shape of the groove (depth, width, path, etc.) in detail, the flow velocity of the sample in the groove can be changed with respect to the reaction between the measurement target, the specific binding substance, and the labeling substance in the sample. I try to optimize. Further, since the specific binding substance and the labeling substance are fixed in the open groove portion before the cap is formed by loading the lid portion on the groove portion, the production can be easily performed.

However, according to this technique, the shape of the groove is set / manufactured so that the flow velocity of the sample is optimal depending on the type of the measurement target in the sample (that is, for each type of the measurement target). Therefore, there is a problem that the versatility is extremely low. In addition to this, as a known technique, there is a measurement chip in which a reaction part in which a specific binding substance is immobilized is formed in one groove provided in a chip substrate. In this technique, first, a sample is flowed into this groove to form a complex of a measurement target substance and a specific binding substance in the sample in the reaction part (first step), and then a labeling substance is added to the groove. It is circulated to generate a complex of the measurement target-specific binding substance-labeling substance in the reaction part (second step),
There is a technique of measuring the amount of the labeling substance bound to the reaction part to measure the amount of the measurement target. However, in this technique, since the sample is first circulated and then the labeling substance is circulated, two steps are required as an operation step, and there is a problem that work efficiency is poor.

Further, generally, the reaction rate between the liquid phase and the solid phase is extremely slow as compared with the reaction rate between the liquid phase and the liquid phase.
In the above technique, in the first step, the specific binding substance (solid phase) immobilized on the reaction part reacts with the sample (liquid layer),
In the second step, the complex (solid phase) of the specific binding substance immobilized on the reaction part and the measurement target in the sample,
Reacts with the labeling substance (liquid layer). That is, since the reaction between the liquid phase and the solid phase is required twice, there is a problem that the reaction time required for the measurement is long and the efficiency is low.

Further, since one groove is also used as the flow path for the sample and the flow path for the labeling substance, it is difficult to make the flow speed of the sample and the flow speed of the labeling substance in the flow path both be optimal. However, the measurement efficiency may be low. Further, in any of the above conventional techniques, a specific binding substance is selected according to the type of the measurement target, and the specific binding substance is
It is necessary to immobilize it on a chromatographic medium or a chip substrate. For this reason, such a measurement medium must be manufactured individually for each type of measurement object, which also poses a problem of low versatility.

The present invention was conceived in view of the above problems, and it is possible to simplify the production of the measuring chip, to perform the measurement efficiently and accurately, and to further expand the versatility of the object to be measured. An object is to provide a chip for measuring an object, a measuring device for an object to be measured, a method for measuring an object to be measured, and a linking substance and a labeling substance.

[0015]

Therefore, a measuring chip of a measuring object of the present invention according to claim 1 is a measuring chip for measuring a measuring object in a sample, and is a chip substrate. A groove-shaped first channel provided on the chip substrate for circulating the sample and a labeling substance having a specific binding substance specifically bound to the measurement object provided on the chip substrate A groove-shaped second flow path, a groove-shaped third flow path provided on the chip substrate and for allowing a connecting substance that specifically binds to the measurement object to flow, the first flow path, and the second flow path. A channel-shaped reaction channel formed by assembling the channel and the third channel on the chip substrate, and an immobilizing substance that is provided in the reaction channel and specifically binds to the connecting substance are fixed. It is characterized in that it is configured with a reaction site.

A measuring chip for measuring an object to be measured according to a second aspect of the present invention is a measuring chip for measuring an object to be measured in a sample, the chip substrate and a covering member for covering the chip substrate. And a first flow path formed between the chip substrate and the covering member for circulating the sample, and formed between the chip substrate and the covering member to specifically bind to the measurement object. A second channel for circulating a labeling substance having a specific binding substance, and a groove-shaped first channel formed between the chip substrate and the covering member for circulating a coupling substance that specifically binds to the measurement target. Three flow paths, a reaction flow path formed between the chip substrate and the covering member, the reaction flow path being formed by assembling the first flow path, the second flow path and the third flow path, and the reaction flow path. Provided on at least one of the chip substrate and the covering member facing the flow path, It is characterized in that immobilization material for different coupling is configured to include a reaction site which is fixed.

According to a third aspect of the present invention, there is provided an apparatus for measuring an object to be measured, wherein the measuring chip according to the first or second aspect and the flow of the sample, the labeling substance and the connecting substance in the measuring chip are controlled. And a measuring means for measuring the labeled substance bound to the reaction site. A measuring device for measuring an object to be measured according to the present invention according to claim 4, comprising a measurement chip through which a sample flows, a flow control means for controlling the flow of the sample through the measurement chip, and a measurement means. An apparatus for measuring an object, wherein the measuring chip is a chip substrate, a groove-shaped reaction channel provided on the chip substrate for allowing the sample to flow therethrough, and specifically bound to the sample and the object to be measured. A labeling substance having a specific binding substance, and a mixing site provided in the reaction channel for mixing three of the linking substance that specifically binds to the measurement target, and the mixing site in the reaction channel. It is provided on the downstream side in the flow direction of the sample with respect to the mixing site, and is configured with a reaction site to which an immobilizing substance that specifically binds to the connecting substance is immobilized, and the measuring means is
It is characterized in that measurement is performed on the labeling substance bound to the reaction site.

The measuring object measuring device of the present invention according to claim 5 comprises a measuring chip through which a sample flows, a flow control means for controlling the flow of the sample in the measuring chip, and a measuring means. A measuring device for measuring an object to be measured, wherein the measuring chip is formed between a chip substrate, a covering member for covering the chip substrate, and the chip substrate and the covering member for circulating the sample. The reaction flow to mix the reaction channel, the analyte, the labeling substance having a specific binding substance that specifically binds to the measurement target, and the linking substance that specifically binds to the measurement target. A mixing portion provided on at least one of the chip substrate and the covering member facing the passage, and the chip substrate and the covering facing the reaction flow channel downstream of the mixing portion in the flow direction of the sample. Installed on at least one of the members And a reaction site on which an immobilizing substance that specifically binds to the linking substance is immobilized, and the measuring means performs a measurement on the labeling substance bound to the reaction site. .

According to a sixth aspect of the present invention, there is provided a method for measuring an object to be measured, which comprises using the measurement chip according to the first or second aspect, wherein the sample flows through the first channel and the second channel. A first step of starting the circulation of the labeling substance through the channel and the circulation of the coupling substance through the third channel to mix the sample, the labeling substance and the coupling substance in the reaction channel; A second step of bringing a mixture of the sample, the labeling substance, and the linking substance into contact with the reaction site of the reaction channel, and a third step of performing a measurement on the labeling substance bound to the reaction site. It is characterized by being configured.

According to a seventh aspect of the present invention, there is provided a method for measuring an object to be measured, which uses the apparatus for measuring an object to be measured according to the fourth or fifth aspect, wherein the flow state of the sample is controlled by the flow control means. While the sample is circulated through the reaction channel, the first step of mixing the sample with the labeling substance and the linking substance at the mixing site, and the flow state controlled by the flow control means The method further comprises a second step of bringing a mixture of the sample, the labeling substance, and the linking substance into contact with the reaction site in the reaction channel, and a third step of measuring the labeling substance bound to the reaction site. It is characterized by being configured.

The connecting substance of the present invention according to claim 8 is a chip substrate, a groove-shaped first channel provided on the chip substrate for allowing a sample to flow therethrough, and an object to be measured provided on the chip substrate. A groove-shaped second flow channel through which a labeling substance having a specific binding substance that specifically binds is circulated, a groove-shaped third flow channel provided on the chip substrate, the first flow channel, and the first flow channel. A groove-shaped reaction channel formed by assembling two channels and the third channel on the chip substrate, and a reaction site provided in the reaction channel and having an immobilizing substance fixed thereon. The third of the measuring chip
It is characterized in that it is a connecting substance that is circulated in the flow channel of (1) and can specifically bind to the measurement target and the immobilized substance at the same time.

According to a ninth aspect of the present invention, there is provided a connecting substance which is formed between a chip substrate, a covering member for covering the chip substrate, and the chip substrate and the covering member, and which allows a sample to flow therethrough. A second flow path, which is formed between the chip substrate and the covering member, for allowing a labeling substance having a specific binding substance that specifically binds to an object to be measured to flow, and the chip substrate and the covering member. A third flow path formed between the first flow path and the third flow path,
A groove-shaped reaction channel formed between the chip substrate and the covering member by assembling the second channel and the third channel, and an immobilizing substance fixed in the reaction channel. Flowed through the third flow path of the measuring chip having the reaction site,
It is a connecting substance, and is characterized in that it can simultaneously and specifically bind to the measurement object and the immobilized substance, respectively.

According to a tenth aspect of the present invention, there is provided a labeling substance according to the present invention, which comprises a chip substrate, a groove-shaped first channel provided on the chip substrate for allowing a sample to flow therethrough, and a groove-shaped channel provided on the chip substrate. A second flow path, a groove-shaped third flow path which is provided on the chip substrate and allows a connecting substance that specifically binds to the measurement object to flow, the first flow path, the second flow path, and A groove-shaped reaction channel provided on the chip substrate, where the third channels are aggregated, and an immobilizing substance which is provided on the chip substrate facing the reaction channel and specifically binds to the connecting substance. A labeling substance, which is circulated in the second channel of a measuring chip having a fixed reaction site, characterized by having a specific binding substance that specifically binds to the measurement target. ..

The labeling substance of the present invention according to claim 11 is a first flow which is formed between a chip substrate, a covering member for covering the chip substrate, and the chip substrate and the covering member, and which allows the sample to flow therethrough. A channel, a second flow path formed between the chip substrate and the covering member, and provided between the chip substrate and the covering member and provided on the chip substrate and specific to the measurement object. A reaction which is formed between the chip substrate and the covering member by assembling the third flow path through which the connecting substance that binds to the substrate flows, the first flow path, the second flow path, and the third flow path. For measurement, which comprises a flow channel and a reaction site which is provided on at least one of the chip substrate and the covering member facing the reaction flow channel and to which an immobilizing substance that specifically binds to the connecting substance is fixed A labeling substance which is circulated in the second channel of the chip, and which is specific to the measurement object Is characterized by having a specific binding substance that binds.

[0025]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described below with reference to the drawings. (A) Description of First Embodiment First, a measurement chip for a measurement target, a measurement device for the measurement target, and a measurement method for the measurement target as the first embodiment of the present invention will be described. FIG. 1 and FIG. 2 are views showing a measuring chip of a measuring object, a measuring device of the measuring object, and a measuring method of the measuring object of the present embodiment. The term "groove" or "groove-shaped flow path" as used below means that the cross section thereof has an open portion.

The measuring chip 1 of this embodiment is shown in FIG.
As shown in (A) and (B), the chip substrate 2, the film-like member 3, and the injection board (cover portion, covering member) 6
And are laminated / polymerized in this order from the bottom. The injection board 6 covers the surface 2A of the chip substrate 2 via the film-shaped member 3, and has a closed cross-sectional flow path (flow paths 5A to 5D) described later between the chip substrate 2, the film-shaped member 3 and the injection board 6. 5) is formed.

The chip substrate 2 here is a plate of polymethylmethacrylate (pMMA) having a thickness of 1 mm, 60 mm ×
It is manufactured by cutting it to 40 mm. In addition, the film-shaped member 3
Is a commercially available double-sided paper tape having a thickness of about 20 μm (= depth of channels 5A to 5D described later) and a width of 40 mm.
It is bonded to the lower chip substrate 2 and the upper injection board 6.

Further, the film-shaped member 3 has a hole 4 formed therethrough.
There is. Branch portions 4A, 4B, 4C and main portion 4 of the hole portion 4
Channel width W of D_A, W_B, W_C, W_DAre both set to 2 mm
Has been done. In addition, the length L of the branched portions 4A, 4B, 4C
_A, L_B, L_CAre set to 14 mm each, the main part 4
Length L of D_DIs set to 30 mm. In addition, each style
Road width W_A~ W_DIs usually 0.1 μm or more and 3 mm or less,
It is preferably 1 μm or more and 1 mm or less. Also, the flow path length
L_A, L_B, L_CIs usually 100 μm or more, 100 mm
The following is preferably 1 mm or more and 50 mm or less. Well
The flow path length L _DRegarding, usually 1mm or more, 100
0 mm or less, preferably 3 mm or more and 500 mm or less
is there.

By stacking the film-shaped member 3 on the chip substrate 2, a groove-shaped flow channel (flow channel having an open portion) is formed on the chip substrate 2. That is, a groove-shaped flow path is formed from the hole 4 of the film-shaped member 3 and the surface 2A of the chip substrate 2. The groove-shaped channel on the chip substrate 2 referred to here is not limited to the one formed on the chip substrate 2 by stacking the film-shaped member 3 on the chip substrate 2 as described above, but also the chip substrate 2 It also includes a groove formed directly in the.

Then, by mounting the injection board 6 on the film-shaped member 3, the groove-shaped flow passage is sealed, and the flow passage 5 having the closed cross-section is formed as described above. To further explain the injection board 6, the injection board 6 is the same as the chip substrate 2 and has a thickness of 1 mm and is made of polymethylmethacrylate (pMM).
It is manufactured by cutting the plate of A) into 60 mm × 40 mm. In addition, the injection board 6 has an injection port 6A for injecting the sample 10 so as to communicate with the upstream end of the channel 5A when being loaded on the measurement chip 1 so as to communicate with the upstream end of the channel 5B. An injection port 6B for injecting the labeling substance 12 is provided in each of the injection ports 6C for injecting the coupling substance 13 so as to communicate with the upstream end of the flow channel 5C. A discharge port 6D for discharging the mixture such as 12 is provided so as to communicate with the downstream end of the reaction channel 5D. Injection board 6
Is used as a lid portion on the measurement chip 1 to form the flow channel 5 in a closed cross-sectional shape. The antibody 13 can be stably distributed.

The inlets 6A, 6B, 6C and the outlet 6D are formed to have a diameter of 2 mm in accordance with the flow path 5 having a width of 2 mm. As the material of the injection board 6, polymethylmethacrylate is used here, but any material that will be described later as a material applicable to the chip substrate 2 can be used. The flow channel 5 includes the respective flow channels 5A to 5D, that is, the first flow channel 5A into which the sample 10 is injected (hereinafter, also simply referred to as flow channel) 5A and the second flow channel into which the labeling substance 12 is injected ( Hereinafter, the flow path 5B, a third flow path 5C into which the connecting substance 13 is injected, and the flow paths 5A, 5B, and 5C are collectively formed. And a reaction site 5 in which an immobilizing substance (here, avidin) 13C that specifically binds to the connecting substance 13 is immobilized in the reaction channel 5D.
E is provided.

Here, the labeling substance 12 and the coupling substance 13 will be described with reference to FIG. The labeling substance 12 is injected into the second flow path 5B, and the first specific binding substance 12A that specifically binds to the measurement target 11 and the labeling unit 12B that is easier to detect than the measurement target 10 are bound to each other. Composed. Also,
The linking substance (biotinylated antibody) 13 is injected into the third channel 5C and is constituted by binding the second specific binding substance 13A and the biotin 13B that specifically bind to the measurement target 11, and the biotin 13B. Is a substance which specifically binds to avidin 13C as an immobilizing substance immobilized on the reaction site 5E.

Therefore, as shown in FIG. 2, the measurement object 11 contained in the sample injected from the first flow path 5A.
And the labeling substance 12 injected from the second channel 5B, and the third
When the connecting substance 13 injected from the flow channel 5C is mixed at the confluence portion (mixing portion) 5F, the first specific binding substance 12A forming the labeling substance 12 and the second specific binding substance forming the connecting substance 13 are mixed. The specific binding substance 13A binds to the measurement target 11, respectively. That is, a complex composed of the labeling substance 12, the measurement target 11 and the connecting substance 13 is formed.

Then, the connecting substance 13 binds to the immobilizing substance 13C fixed to the reaction site 5E. That is, the target substance 11 is measured via the linking substance 13 and the immobilized substance 13C.
Is linked / fixed to the reaction site 5E integrally with the labeling substance 12. As a result, the measurement target 1
Even if it is difficult to directly measure the measurement target 11 because 1 is transparent, the amount of the measurement target 11 can be easily measured via the labeling substance 12.

In order to carry out such a measurement, the measurement object 11 is composed of the first specific binding substance 12A and the coupling substance 13 (specifically, the second specific binding substance 13 in this case).
The condition is that they can be combined with A) at the same time. Also, the first
Specific binding substance 12A and second specific binding substance 13A
May be the same substance as each other, or may be different substances from each other.

Hereinafter, the chip substrate 2, the film-shaped member 3, the measurement object 11, the specific binding substances 12A and 13A, and the connecting substance 1
3 and the immobilizing substance 13C will be further described. First, the chip substrate 2 will be described. As described above, polymethyl methacrylate is used as the material of the chip substrate 2, but the material is not limited to this as long as it is sufficiently solid as a solid phase. . Glass and resin are preferable, but metals, semiconductors, ceramics and the like can also be used. Moreover, since the chip substrate 2 constitutes the flow path 5 together with the film-shaped member 3, the sample 10 and the labeling substance 12 are provided.
It is preferable that the material is stable with respect to the connecting substance 13.

Furthermore, the surface 2A of the chip substrate 2 which constitutes the flow path 5 together with the film member 3 may be subjected to a predetermined surface treatment. Such surface treatment is appropriately performed according to the specimen 10, the labeling substance 12, and the coupling substance 13. For example, the chip substrate 2 is hydrophobic, and the specimen 10, the labeling substance 12, and the coupling substance are If 13 is water-soluble, hydrophilic treatment (surface modification) with acid / alkali can be considered. If the immobilizing substance 13C immobilized on the reaction site 5E is a protein, a surface treatment with, for example, carbodiimide, maleimide, or succinimide is performed to bind the immobilizing substance 13C. Further, in order to suppress non-specific adsorption (adsorption of non-specific substance), for example, surface treatment with alpmine, a surfactant or an artificial polymer may be performed.

Alternatively, instead of performing the surface treatment, a self-assembled film, an LB film, an inorganic thin film or an organic thin film is attached to the surface 2A of the chip substrate 2 so that predetermined surface physical properties can be obtained in the channel 5. You can Next, the film member 3
The film-like member 3 is made of a commercially available paper tape as described above, but if it is a thick film or a thin film made of resin film, paper, metal plate, glass plate, etc. Not limited to. Also here
The film-shaped member 3 is configured by a double-sided tape having adhesive applied on both sides and is attached to the chip substrate 2 or the injection board 6. The method for connecting the film-shaped member 3 is as follows. It is appropriately selected depending on the material of the substrate 2 and the injection board 6 and the like. In addition to bonding with an adhesive, bonding with a solvent / dissolving solvent (for example, resin bonding with a primer), diffusion bonding, anodic bonding,
Eutectic bonding, heat fusion, laser fusion, pressure bonding, etc. Alternatively, an adhesive tape, a pressure-bonding tape, or a self-adsorbing agent may be interposed between each of the film-shaped member 3, the chip substrate 2, and the injection board 6.

Further, the film-like member 3, the chip substrate 2 and the injection board 6 are each provided with irregularities, and these irregularities are fitted and joined, or the film-like member 3, the chip substrate 2 and the injection board 6 are sandwiched by clips. Of course, it does not matter if they are physically connected to each other.
In addition, the thickness of the film-shaped member 3 is the depth of the flow path 5,
The upper limit is generally 400 μm or less, preferably 200 μm or less. Specimen 1 flowing through the flow path 5
0, the labeling substance 12 and the connecting substance 13 contact and bond with the immobilizing substance 13C fixed to the bottom of the flow channel 5, so that the deeper the flow channel 5 is, the less contact with the immobilizing substance 13C at the bottom of the flow channel 5. Since the amount of the specimen 10, the labeling substance 12, and the coupling substance 13 increases, it is preferable to set the thickness of the membrane member 3 (depth of the flow channel 5) to 200 μm or less as described above from the viewpoint of reaction efficiency. is there.

The thickness of the film member 3 (depth of the flow path 5)
Considering the ease of manufacturing the film-shaped member 3 and the thickness of the immobilizing substance 13C fixed to the bottom of the flow path 5, 0.1 μm is obtained.
It is generally m or more. Next, the sample 10 and the measurement target 11 will be described. Specimens 10 include those mainly for medical diagnosis and those for environmental analysis. For medical diagnosis, blood, body fluid, urine, tears, etc. derived from a living body are used for environmental analysis. Is a solution in which water of the sea or river or the atmosphere is dissolved. Further, as the measurement target 11, there is a substance that specifically binds to it (specifically binding substance), and as described above, 2
It is not limited as long as it can bind two or more specific binding substances at the same time, and examples thereof include proteins, organic substances, lipids, sugars,
Peptides, hormones, nucleic acids, etc.

Next, the specific binding substances 12A and 13A will be described. The specific binding substances 12A and 13A are appropriately determined according to the measurement target 11, and include, for example, immunoglobulin and its derivatives F (ab ') 2 and Fa.
b'and Fab, receptors and enzymes and their derivatives, nucleic acids, natural or artificial peptides, artificial polymers, sugar chains, lipids, inorganic substances and organic ligands, viruses, drugs, etc.

Next, the connecting substance 13 and the immobilizing substance 13C will be described. The connecting substance 13 and the immobilizing substance 13C.
May be any combination that specifically binds to each other.
As such a combination, a combination of biotin and avidin as in the present embodiment is preferable because of easy availability. Further, as a method of immobilizing the immobilizing substance 13C on the chip substrate 2 (reaction channel 5D), the immobilizing substance 13C is used.
Is physically adsorbed on the chip substrate 2, and there is a method of chemically bonding the immobilizing substance 13C to the chip substrate 2. As a physical immobilization method, the immobilization substance 13C is used.
Immobilize the immobilized substance 13C on the solid phase through the method of directly immobilizing another substance on the solid phase by physically or chemically immobilizing it on the solid phase (chip substrate 2). There is a way to do it. As the chemical immobilization method, the immobilization substance 13C is directly bound to the solid phase, or the functional group present on the surface of the solid phase is chemically activated before the immobilization substance 13C is bound. There is a method, a method of physically or chemically binding a spacer molecule to a solid phase and binding the immobilizing substance 13C to the solid phase via the spacer molecule.

Further, the immobilizing substance 13C is added to the chip substrate 2
As a method for spotting in (reaction channel 5D),
For example, there are dropping with a dropper, jetting or dropping with a nozzle hole using the principle of an inkjet printer, coating with a tapered pin tip, stamping, and the like. Now, as shown in FIG. 1, the measuring apparatus of the present embodiment is a syringe pump (flow control means) for controlling the flow of the measurement chip 1, the sample 10, the labeling substance 12 and the connecting substance 13 in the flow channel 5. 7 and measuring means (not shown) for measuring the labeling substance 12 bound to the reaction site 5E.

Further, here, as described above, the specimen 10 is used.
A syringe pump is used as a distribution control means 7 for controlling the distribution of the above. Syringe pump 7 has an outer diameter of 6 mm
The silicone tube 7A, the plate 7B made of PDMS (polydimethylsiloxane) material, is connected to the discharge port 6D (downstream end of the reaction channel 5D) of the injection board 6, and the sample 10, the labeling substance 12, and the coupling substance are connected. 13 distribution is controlled. The flow control means is not limited to a syringe pump as long as it can control the flow of the sample 10, the labeling substance 12, and the connecting substance 13, and for example, a positive pressure type pump or a negative pressure type pump may be used as the injection port 6A of the injection board 6, 6B, 6C or outlet 6
You may make it connect to D.

Alternatively, as the flow control means, electrodes are attached to the upstream end and the downstream end of the flow path 5, respectively, and different voltages are applied to these electrodes, so that the sample 10 and the labeling substance 1 can be obtained.
An electroosmotic flow may be generated in 2 and the connecting substance 13. Alternatively, a heating device may be provided in the measurement chip 1 as a flow control means, and a temperature gradient may be generated along the flow path 5 by this heating device. That is, the temperature gradient causes a difference in specific gravity between the specimen 10, the labeling substance 12, and the connecting substance 13 along the flow path 5,
The specimen 10, the labeling substance 12, and the connecting substance 13 are circulated by this difference in specific gravity.

Alternatively, as the flow control means, the inlet 6A,
Electrodes are attached to 6B, 6C and the discharge port 6D, and metal is coated around the injection ports 6A, 6B, 6C and the discharge port 6D, so that a DC electric field is applied to the specimen 10, the labeling substance 12, and the coupling substance 13 in the flow path 5. Alternatively, the ions (the sample 10, the labeling substance 12, and the connecting substance 13) may be electrophoresed to cause electrophoresis. In this case, since the solvent of the sample 10, the solvent of the labeling substance 12, and the solvent of the connecting substance 13 do not move, it is not necessary to seal the flow path 5 with the injection board 6. Therefore, the injection board 6 becomes unnecessary, and the measuring chip is the chip substrate 2 described above.
And the film-shaped member 3, so that the groove formed by the chip substrate 2 and the film-shaped member 3 (groove-shaped channel)
Function as the first flow channel 5A, the second flow channel 5B, the third flow channel 5C, and the reaction flow channel 5D.

Further, the distribution control means may be implemented by combining a plurality of the above methods. Now, the measuring means 8 is not limited as long as it measures the amount of the labeling substance based on the physical properties of the labeling substance, and for example, absorption, fluorescence,
Examples include those that measure evanescent excitation fluorescence, phosphorescence, and chemiluminescence, and those that use surface plasmon resonance, crystal oscillators, and the like. Alternatively, in the case where the labeling substance 12 is a catalyst such as an enzyme, the substrate may be added and reacted, and the product may be detected to perform the measurement. It is also possible to use a photoacoustic measurement method (a method of measuring the amount of a substance by irradiating specific light and measuring a sound wave emitted).

Since the measuring object measuring chip and the measuring object measuring apparatus according to the first embodiment of the present invention are configured as described above, the following procedure (the first embodiment of the present invention will be described. The measuring object (here, TSH) is measured by the measuring method of the measuring object. First, the adjustment of the labeling substance 12 will be described. Labeling substance 1
2 is here composed of anti-human FSH-α subunit antibody-immobilized EuLTX (Ab-EuLTX).

First, 0.05M MES was added to polystyrene particles (EuLTX) containing a Eu complex having a particle diameter of 0.21 μm.
After diluting with (pH 6.0), 2 mL (milliliter) of 1% suspension was prepared, and 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) was removed and washed. , A predetermined amount V (here, 2 mL) of 0.
EuLTX is dispersed in 05M MES (pH 7.0), and then a predetermined amount M (here, 0.8 mg) of anti-human FSH-α subunit antibody (hereinafter, simply referred to as antibody) is added and reacted for 1 hour at room temperature. . Then, this solution was centrifuged to remove unreacted antibody, and BSA-containing Tris buffer (0.3% BSA, 0.1M Tris, pH8.
0) is added to stabilize the particles. At this time, the antibody concentration C1
Is 0.4 mg / mL (C1 = M / V = 0.8 / 2
= 0.4).

Then, the mixture was stirred at room temperature for 30 minutes, centrifuged, washed with purified water, dispersed in 0.05% sodium azide solution, and labeled with (Ab-EuLTX) 1
2 is adjusted. At this point, the unbound antibody concentration C2 was 0.15 mg / mL, and therefore the antibody immobilization rate R was 62.5% [R = (C1-C2) / C
1 × 100 = (0.4−0.15) /0.4×100] Then, in FIG. 1 (A), after the film-shaped member 3 is attached to the chip substrate 2, the chip substrate 2 and the film-shaped member are attached. Predetermined part of the reaction flow channel 5D formed by 3 and 3 (here, a position 10 mm away from the downstream end of the reaction flow channel 5D to the upstream side)
At 1 mg / mL, 1 μL (microliter) of avidin adjusted to 1 mg / mL was dropped as the immobilizing substance 13C, dried at room temperature and atmospheric pressure for 30 minutes, and then vacuum dried at room temperature for 15 minutes to prepare a reaction channel 5D. To form reaction site 5E.

Then, after the injection board 6 was stuck on the membrane member 3, 100 μL of the TSH standard product diluted with pure water and adjusted to 123 μIU / mL was used as the sample 10 from the injection port 6A to the flow path 5A. Drop, Ab
The labeling substance 12 prepared by diluting EuLTX 100-fold with pure water was added dropwise to the channel 5B from the inlet 6B by 100 μL, and further biotinylated 1.1 mg / mL anti-T.
The SH antibody (mouse IgG) was used as the linking substance 13 of 100.
Only μL is dropped from the injection port 6C to the channel 5C.

Next, the syringe pump 7 connected to the discharge port 6D of the injection board 6 is operated to 50 μL / min of the specimen 10, the labeling substance 12 and the connecting substance 13 dropped from the injection ports 6A, 6B and 6C. Is sucked in and circulated toward the reaction channel 5D. Measurement object 1 in sample 10
As shown in FIG. 2, the labeling substance 12 and the linking substance 13 are mixed and bonded to each other at the confluence site 5F (first step), and then move onto the reaction site 5E to reach the reaction site 5E. It further binds to the immobilized specific binding substance 13 (second step). Then, by the same procedure as the above procedure, 100 μL of pure water is sucked from each of the channels 5A, 5B, 5C to clean the channel 5.

Then, when the reaction site 5E was irradiated with ultraviolet rays having a wavelength of 365 nm by a measuring device (not shown),
Red fluorescence resulting from the labeling substance 12 was visually observed at the reaction site 5E, and it was measured that the sample 10 contained the measurement object 11 (third step). Therefore, the measuring tip of the measuring object, the measuring device of the measuring object, and the measuring method of the measuring object according to the present embodiment have the following advantages.

That is, when the measurement object 11 changes, the specific binding substances 12A and 12B also become different substances according to the change, but in the present invention, the substance containing the specific binding substances 12A and 12B is the labeling substance 12. And the connecting substance 13. That is, the immobilized substance 13C fixed to the reaction site 5E can be made constant regardless of the measurement object 11, and the type of the measurement object 11 can be freely changed in one measurement chip (for the same measurement). Various objects to be measured with chips 1
1 can be measured).

As a specific example, if avidin is used as the immobilizing substance 13C to be immobilized on the reaction site 5E, when TSH is measured as the measuring object 11 as described above, an anti-labeling substance 12 is used. TSH can be measured by using a human FSH-α subunit antibody and a biotinylated anti-TSH antibody as the linking substance 13, respectively.
When measuring hCG as the measurement target 11, hCG can be measured by using an anti-human FSH-α subunit antibody as the labeling substance 12 and a biotinylated anti-hCG antibody as the linking substance 13.

Further, this can make the material of the immobilizing substance 13C fixed to the reaction site 5E constant regardless of the kind of the measuring object 11, and thus also simplifies the manufacture of the measuring chip. Further, in the present invention, the reaction of the measurement object 11, the labeling substance 12 and the connecting substance 13 in the sample 10 (first step, reaction between liquid phase and liquid phase), and the measurement object 11, labeling substance 12 The complex of the linking substance 13 and the immobilizing substance 13C immobilized on the reaction site 5E are reacted (second step, reaction between liquid phase and solid phase).

On the other hand, the linking substance is used in the same manner as in the present invention by the above-mentioned conventional technique in which the analyte and the labeling substance are sequentially circulated in this order in one groove having the reaction site to which the specific binding substance is immobilized. When trying to perform measurement by immobilizing the immobilized substance on the reaction site, the reaction site (solid phase)
The linking substance 13, the measurement target 11, and the labeling substance 12 are sequentially flowed in and reacted.

That is, in the prior art, the reaction between the liquid phase and the solid phase must be carried out three times, whereas in the present invention, the reaction between the liquid phase and the solid phase need only be carried out once. . The reaction between the liquid phase and the liquid phase has a higher reaction rate than the reaction between the liquid phase and the solid phase. Therefore, according to the present invention, the time required for the measurement can be shortened and the measurement can be performed efficiently as compared with the prior art. The advantage is that

The syringe pump 7 is used to
Since the flow rates of the labeling substance 12 and the coupling substance 13 can be controlled to a predetermined flow velocity, the sample 10, the labeling substance 12, and the coupling substance 13 can be controlled.
Flow rate of the target substance 11 and the labeling substance 12 in the sample 10.
Also, there is an advantage that the reaction time can be shortened by making the flow rate optimum for the reaction between the linking substance 13 and the measurement can be performed efficiently from this point as well.

Furthermore, by appropriately adjusting the flow velocity by the syringe pump 7 according to the type of the measurement target 11, the measurement targets 11 of various types are measured under the optimum flow velocity by the measuring tip of one specification. There is an advantage that it is possible to do. Further, by the syringe pump 7, for example, before the mixture of the sample 10, the labeling substance 12 and the connecting substance 13 reaches the reaction site 5E, the flow is temporarily stopped and before the mixture with the immobilizing substance 13C of the reaction site 5E is reached. Sample 1
0, the labeling substance 12 and the linking substance 13 are sufficiently reacted, or when the mixture of the sample 10, the labeling substance 12 and the linking substance 13 reaches the reaction site 5E, the flow is temporarily stopped,
It is possible to improve the reaction efficiency between the solid phase (immobilization substance 13C) having a slow reaction rate and the liquid layer (mixture of the specimen 10, the labeling substance 12, and the coupling substance 13). Furthermore, if there is a possibility that the measurement target 11, the labeling substance 12, and the linking substance 13 that would otherwise bind to the immobilized substance 13C at the reaction site 5E may pass through the reaction site 5E without being reacted, a syringe is used. It is also possible to cause the object to be measured 11, the labeling substance 12, and the connecting substance 13 that have passed through the reaction site 5E to flow backward by the pump and to contact the reaction site 5E again.

As described above, the immobilizing substance 13C
Of the measurement target 11, the labeling substance 12 and the coupling substance 13 can be controlled in various ways, and thus the measurement target 11, the labeling substance 12 and the coupling substance 13 can be easily immobilized.
The reaction system can be highly designed and can be set in various ways. Further, since the flow path 5 has a closed cross-section structure and functions as a capillary, it has been widely used from the past.
There is also an advantage that various techniques such as analysis technique and flow channel control in the developed measuring method using a capillary can be used as they are.

As described above as a problem of the prior art, in the immunochromatograph, the material of the flow channel (the place where the measurement object is developed) is limited in principle, and in the technique using the capillary, a specific substance (for example, a fixed substance) is fixed. The material of the flow channel is limited due to the immobilization of the chemical substance 13C) in the capillary. However, in the measurement chip 1, the material of the chip substrate 2 and the injection board 6 forming the flow channel 5 is widely selected. it can.

As a result, not only the spectroscopic measurement can be optimized in terms of the transmission wavelength and background noise, but also surface film treatment is required for the chip substrate 2 such as surface plasmon resonance. It is possible to use a detection system and incorporate a detection element such as a crystal oscillator in the chip substrate 2. Furthermore, since the flow path 5 is formed between the chip substrate 2 and the injection board 6,
Before the chip substrate 2 and the injection board 6 are assembled, the reaction channel 5D (channel 5) is still in an open state, so the solid-phase wall surface forming the reaction channel 5D (here, a predetermined portion of the chip substrate 2). There is an advantage that the immobilizing substance 13C can be easily fixed to the reaction site 5E.

In the above embodiment, the chip substrate 2
The film-like member 3 having the hole portion 4 penetrating therethrough is attached to provide the flow path 5 on the chip substrate 2.
It is also possible to directly form the groove (groove-shaped channel) in the chip substrate 2 without attaching the film-shaped member 3. As a method of directly forming the groove on the chip substrate 2 as described above, for example,
After mechanical processing such as cutting and polishing, or morphology control using lithography, dry etching (eg electron beam,
X-ray irradiation, DRIE), wet etching, electric discharge machining, laser ablation, etc., or a method of forming a groove portion by photolithography, and then the groove portion shape is drawn on a mask. There are a method of removing a predetermined portion of the chip substrate 2 by wet etching, electric discharge machining, laser ablation to form a groove, and a transfer technique using a stamper, compression molding, injection molding or the like.

Alternatively, the groove may be formed at the same time when the chip substrate 2 is formed. As a method of forming such a chip substrate 2, there is, for example, a mold forming. Further, the chip substrate 2 and the groove can be molded at the same time by stereolithography using a photo-curable resin.
In such a case, it is possible to simultaneously design the groove and manufacture the chip substrate 2 and the groove by computer control.

Further, the grooves may be formed in the chip substrate 2 by combining the above methods. Further, since the material of the chip substrate 2 can be widely selected as described above, other known fine processing techniques can be used for such groove processing. Even when the groove is directly formed in the chip substrate 2 as described above, the upper limit of the groove depth is the upper limit of the reaction efficiency, as in the case where the film-like member 3 is attached to the chip substrate 2 to form the flow path. From the point of, 400 μm or less, preferably 200 μm
m or less, and the lower limit is 0.1 μm considering the ease of processing and the thickness of the immobilizing substance 13C fixed to the bottom.
It is general to do the above.

Also in this case, the width of each flow path W _A -W _D is usually 0.1 μm or more and 3 mm or less, preferably 1 μm.
It is m or more and 1 mm or less. Also, the flow path lengths L _A , L _B ,
L _C is usually 100 μm or more and 100 mm or less, preferably 1 mm or more and 50 mm or less. Regarding the flow path length L _D , usually 1 mm or more and 1000 mm or less,
It is preferably 3 mm or more and 500 mm or less.

Further, as shown by the chain double-dashed line in FIG. 1 (B), a channel 5G for a buffer solution for injecting the buffer solution into the sample 10 is provided in order to perform the measurement under certain special conditions. Further, it may be provided. In addition, as shown by the chain double-dashed line in FIG. 1B, in the reaction channel 5D, the reaction site 5E
The flow paths 5H and 5J may be provided on the downstream side of. This channel 5
By properly providing H and 5J (specifically, by appropriately setting the width and depth of the flow channel, the inclination angle with respect to the reaction flow channel 5D, etc.), the flow channel 5D and the flow channels 5H and 5J are separated. Unreacted sample 10, labeling substance 12 and connecting substance 1
It is also possible to separate and collect 3 respectively.

In the above embodiment, the labeling substance 12
Is measured at the reaction site 5E, the labeling substance 12 is separated from the reaction site 5E and recovered after the completion of the circulation of the analyte 10, the labeling substance 12 and the linking substance 13, and the recovered labeling substance Twelve quantities may be measured. In order to separate the labeling substance 12 from the reaction site 5E, for example, a substance similar to the labeling substance 12 is flown,
This substance may be replaced with the labeling substance 12.

When such a mode is preferable,
This is a case where the labeling substance 12 needs to be separated from the chip substrate 2 because the material of the chip substrate 2 and the film-shaped member 3 is not suitable for spectroscopic measurement. Further, in the above-described embodiment,
The width W _{A of the} flow path 5A through which the sample 10 flows and the labeling substance 1
The width W _B of the flow channel 5B through which 2 flows and the width W _C of the flow channel 5C through which the connecting substance 13 flows are set to the same length, but the widths W _A , W _B , and W _C are mutually It is also possible to set different lengths so that the flow passages 5A, 5B, and 5C have different flow passage cross-sectional areas. Specimen 10 flowing in flow channel 5A, marker substance 12 flowing in flow channel 5B, and connecting substance 13 flowing in flow channel 5C
When there is a large difference in each flow rate of
It is effective to set different flow passage cross-sectional areas for A, 5B, and 5C.

That is, the flow channels 5A and 5B will be described. For example, when 100 μL of the sample 10 and 1 μL of the labeling substance 12 are circulated in the flow channels 5A and 5B for measurement, the sample 10 and the label are labeled. It is important for the measurement to be performed with high accuracy by uniformly mixing the substance 12 and reacting the specimen 10 with the labeling substance 12. Then, in order to uniformly mix the sample 10 and the labeling substance 12 at a predetermined ratio, in this case, the sample 10 and the labeling substance 12 flow into the mixing portion 5F at a ratio of 100: 1 per unit time. That is, the ratio of the flow rate FA of the sample 10 per unit time in the channel 5A (hereinafter referred to as flow rate) FA to the flow rate FB of the labeling substance 12 in the channel 5B is 100:
It should be set to 1 (FA / FB = 100/1).

As in this embodiment, the sample 10 in the channel 5A is
In the case where the flow path 5B and the labeling substance 12 in the flow path 5B are sucked from the merging portion 5F side by one flow control means (syringe pump) 7, the flow path cross-sectional areas of the flow path 5A and the flow path 5B are the same. Therefore, it is technically difficult to set a large difference between the flow rate FA of the sample 10 and the flow rate FB of the labeling substance 12, but the width W _A of the flow channel 5A and the width W _{B of the} flow channel 5B are different. By setting the length so that the flow passages 5A and 5B have different flow passage cross-sectional areas, the ratio of the flow velocity FA of the sample 10 to the flow velocity FB of the labeling substance 12 is 100: 1. You can Therefore, even if there is a large difference in the flow rate between the sample 10 and the labeling substance 12, the sample 10 and the labeling substance 12
It is possible to accurately measure by mixing and with a predetermined ratio uniformly.

On the other hand, as described above as the prior art, in the known technique in which the sample and the labeling substance are sequentially passed through the one groove having the reaction site to which the specific binding substance is immobilized, in the sample, In order to efficiently react the measurement target with the labeling substance and perform the measurement with high accuracy, the time during which the analyte and the labeling substance can contact and react with the reaction site (= It is important to set the appropriate transit time).

However, in this conventional technique, in particular, for example, 100 μL of sample and 1 μL are used as in the above case.
When there is a difference in the amount of the sample and the amount of the labeling substance as in the case of performing measurement using the L labeling substance, and the sample and the labeling substance require the same reaction time at the reaction site. For this reason, it is necessary to make the flow rate FA 'of the sample different from the flow rate FB' of the labeling substance (in this case, FA ': FB').
= 100: 1).

By providing a flow control means such as a syringe pump, the flow rate of the sample and the flow rate of the labeling substance are individually controlled by the flow control means, and the flow rate F of the sample is controlled.
It is possible to set A ′ and the flow rate FB ′ of the labeling substance to different values, but if such a difference in flow rate is large as in this example, one flow control means can be used in such a wide range. Distribution control is technically difficult. It is generally practiced to control the flow velocities of the respective flow paths to greatly different speeds by the same flow control means by making the flow path cross-sectional areas of the different flow paths different from each other between the different flow paths. However, in this known technique, since the channels for the sample and the labeling substance are shared, it is naturally impossible to change the channel cross-sectional area for each of the sample and the labeling substance.
Of course, it is possible to control the sample and the labeling substance at greatly different flow rates by using the flow control means having different specifications for controlling the flow of the sample and controlling the flow of the labeling substance. Two types of distribution control means are required, resulting in an increase in cost, which is not realistic.

Therefore, since the measurement chip has the branched channels 5A, B, and 5C, even if there is a large difference in the flow rate between the sample 10, the labeling substance 12, and the coupling substance 13 used for measurement, The measurement can be performed with high accuracy. Here, since the depths of the flow paths 5A, 5B, and 5C are all determined by the thickness of the film-shaped member 3,
Although the flow passage widths W _A , W _B , and W _C are set to different lengths so that the flow passages 5A, 5B, and 5C have different flow passage cross-sectional areas, the flow passages may be provided directly on the chip substrate 2. In such a case, in the flow paths 5A, 5B, 5C, the flow path cross-sectional areas may be made different by changing the flow path depth.
Of course, both the channel depth and the channel width may be set to different values, or only the channel width may be set to different values.

Alternatively, the channel 5A for the reaction channel 5D,
By appropriately setting the respective inclination angles of 5B and 5C, the resistances that the sample 10, the labeling substance 12, and the connecting substance 13 receive when flowing into the reaction channel 5D are different from each other,
It is also possible that the sample 10, the labeling substance 12, and the connecting substance 13 have different flow rates. Also, the flow paths 5A, 5
It is only necessary that B and 5C merge to form a reaction channel 5D,
Flow paths 5A, 5B, 5C as shown in FIGS.
It is not necessary that all of them merge at the same time,
For example, as shown in FIG. 3, the flow channels 5A and 5B may be merged and then the flow channel 5C may be merged.

(B) Description of Second Embodiment Next, a measuring chip of the measuring object, a measuring device of the measuring object, and a measuring method of the measuring object as the second embodiment of the present invention will be described. FIG. 4 and FIG. 5 are views showing a measuring object measuring tip, a measuring object measuring device and a measuring object measuring method according to the present embodiment. The same members as those in the first embodiment described above are designated by the same reference numerals and the description thereof will be omitted.

The measuring chip 21 of this embodiment is shown in FIG.
As shown in (A) and (B), the chip substrate 2, the film-like member 3, and the injection board (covering member) 6 are laminated / polymerized in this order from the bottom. The injection board 6 includes the chip substrate 2 via the film member 3.
Of the reaction flow path 15 having a closed cross-sectional shape, which covers the surface 2A of the
Are formed.

The chip substrate 2 here is a plate of polymethylmethacrylate (pMMA) having a thickness of 1 mm, 60 mm ×
It is manufactured by cutting it to 20 mm. In addition, the film-shaped member 3
Has a thickness of 20 μm (= the depth of the flow path 15) and a width of 15 mm.
A commercially available double-sided tape made of paper is used, the chip substrate 2 is adhered to the lower part, and the injection board 6 is adhered to the upper part. Further, here, a rectangular hole portion 14 having a width (= flow channel width) 2 mm × length (= flow channel length) 30 mm is penetratingly provided in the film-shaped member 3, and the film-shaped member 3 is chipped. By loading on the substrate 2, a groove is formed from the hole 14 of the film member 3 and the surface 2A of the chip substrate 2, and further, the injection board 6 allows the surface 2A of the chip substrate 2 via the film member 3. By coating
A reaction flow channel 15 having a closed cross-sectional shape is formed from the injection board 6 and the groove.

The upper limit of the thickness of the film member 3 (depth of the flow path 15) is 400 μm or less, preferably 200 μm, from the viewpoint of reaction efficiency, as in the first embodiment described above.
The lower limit is 0.1 μm, considering the ease of production and the thickness of the specific binding substance 13 fixed to the bottom.
It is general to do the above. The width of the flow path 15 is
It is usually 0.1 μm or more and 3 mm or less, preferably 1 μm or more and 1 mm or less. The flow path length is usually 1 mm
Or more, 1000 mm or less, preferably 3 mm or more, 50
It is 0 mm or less.

Like the chip substrate 2, the injection board 6 has a thickness of 1 mm and is made of polymethylmethacrylate (p
It is manufactured by cutting a plate (MMA) into 60 mm × 20 mm. The injection board 6 is provided with an injection port 6E for injecting the sample 10 so as to communicate with the upstream end of the reaction channel 15 when the chip 10 for measurement is loaded. A discharge port 6F is provided so as to communicate with the downstream end of the reaction flow path 15 in order to discharge the mixture with 12. The inlet 6E and the outlet 6F
Is formed to have a diameter of 2 mm in accordance with the reaction channel 15 having a width of 2 mm.

In the reaction channel 15, from the upstream side, a labeling site 15A in which the labeling substance 12 is disposed, a mixing site 15B in which the linking substance 13 is disposed, and a reaction site 15C in which the immobilizing substance 13C is immobilized are in this order. Has been formed. As a result, as shown in FIG. 5, the sample 1 flowing through the reaction channel 15
0 is mixed with the labeling substance 12 at the labeling site 15A,
A complex of the measurement target 11 and the labeling substance 12 in the sample 10 is generated, and thereafter, this complex is mixed with the linking substance 13 at the mixing site 15B, and the measurement target 11, the labeling substance 12,
A complex of the linking substance 13 is generated, and the complex is bound to the immobilizing substance 13C at the reaction site 15C.

Immobilization substance 1 to form reaction site 15C
The method for spotting 3C on the chip substrate 2 and the method for fixing it on the chip substrate 2 are the reaction site 5 of the first embodiment.
This is the same as the spotting method / fixing method of the immobilizing substance 13C on the chip substrate 2 in E. The spotting of the labeling substance 12 and the coupling substance 13 into the reaction channel 15 is similar to the spotting method of the immobilization substance 13C, and for example, dropping with a dropper, jetting or dropping with a nozzle hole using the principle of an inkjet printer, It is performed by coating with a tapered pin tip or stamping. In addition, the labeling substance 12 and the linking substance 13 are
Must be mixed with the sample 10 and flow to the reaction site 15C on the downstream side when flowing through the labeling site 15A and the mixing site 15B. Therefore, unlike the immobilization material 13C, the chip substrate 2 is relatively low. It is fixed by the degree of coupling. As the immobilization method, the labeling substance 12 or the connecting substance 1 is used.
There are a method of directly adsorbing 3 to the chip substrate 2, a method of coating the chip substrate 2 with another substance and adsorbing it to the coating film, and a method of admixing 3 with another substance.

The measuring apparatus of this embodiment is shown in FIG.
As shown in FIG.
Specimen 10, labeling substance 12, connecting substance 1 in channel 15
Syringe pump 7 for controlling the flow of 3 and the reaction site 15
It comprises a measuring means (not shown) for measuring the labeling substance 12 bound to C. The measuring chip of the measuring object and the measuring device of the measuring object as the second embodiment of the present invention are configured as described above, and therefore, the procedure shown below (the measurement as the second embodiment of the present invention The measuring object is measured by the measuring method).

First, the Ab-EuLTX is adjusted in the same manner as in the first embodiment described above. Then, in FIG. 4 (A), after the film-shaped member 3 is bonded to the chip substrate 2, the reaction channel 15 formed by the chip substrate 2 and the film-shaped member 3 is formed.
In addition, 1 μL of avidin adjusted to 1 mg / mL as the immobilizing substance 13C, and 1.1 mg / mL of biotinylated anti-TSH antibody (mouse IgG) as the linking substance 13, A
A solution prepared by mixing b-EuLTX and a 10% sucrose solution at a ratio of 9: 1 is spotted as the labeling substance 12 by 2 μL. Then, after drying at room temperature and atmospheric pressure for 30 minutes, vacuum drying is further performed at room temperature for 15 minutes to form the labeling site 15A, the mixing site 15B and the reaction site 15C in the reaction channel 15.

Then, after the injection board 6 was pasted on the film-like member 3, 20 μL of TSH standard product diluted with pure water and adjusted to 1230 μIU / mL was used as the sample 10 from the injection port 6E to the flow path 15. Drop it. Next, the syringe pump 7 connected to the discharge port 6F of the injection board 6 is operated to operate the sample 1 at the injection port 6E.
Aspirate 0 at 10 μL / min.

The specimen 10 circulates through the flow path 15 while the circulation is controlled by the syringe pump 7, and as shown in FIG.
The measurement target 11 in 0 and the labeling substance 12 bind to each other, and further bind to the linking substance 13 at the mixing site 15B (first step). Then, when this mixture moves onto the reaction site 15C, it further binds to the immobilizing substance 13C fixed on the reaction site 15C (second step). And
After the circulation of the sample 10 is completed, 20 μL of pure water is circulated in the channel 15 to wash the channel 5.

Then, when the reaction site 15C was irradiated with ultraviolet rays having a wavelength of 365 nm by a measuring device (not shown), red fluorescence due to the labeling substance 12 was visually observed at the reaction site 15C, and the object to be measured in the sample 10 was observed. It was determined that 11 were included (third step). When a solution containing no TSH (measurement target) was used as the sample 10 and measurement was performed in the same procedure as above, no fluorescence was observed at the reaction site 15C, and the sample 10 contained TSH. I was able to accurately detect that there was no.

Therefore, the measuring tip of the measuring object, the measuring device for the measuring object, and the measuring method of the measuring object according to the present embodiment have the following advantages. That is, since the reaction channel 15 is still open before the chip substrate 2 and the injection board 6 are assembled, the labeling substance is formed on the solid-phase wall surface (here, a predetermined portion of the chip substrate 2) forming the reaction channel 15. 12, there is an advantage that the connecting substance 13 and the immobilizing substance 13C can be easily fixed.

Further, with the syringe pump 7, the sample 1
0, the labeling substance 12 and the connecting substance 13 can be controlled to have a predetermined flow velocity, so that the flow velocity of the sample 10 or the like can be optimized for measurement, and there is an advantage that the reaction time can be shortened and the measurement can be performed efficiently. . Further, since the flow of the sample 10 can be stopped or reversed, the flow state (flow rate, flow direction, etc.) can be appropriately controlled, and there is an advantage that the measurement mode is wide. Furthermore, since the flow velocity can be adjusted appropriately, the measurement target 1
By appropriately adjusting the flow rate by the syringe pump 7 according to one type, there is an advantage that various types of measurement objects 11 can be measured under the optimum flow rate by the measurement tip of one specification.

As described above, the labeling substance 12, the linking substance 13, and the immobilizing substance 13C can be easily immobilized in the reaction channel 15, and the flow of the sample 10 can be controlled in various ways. The reaction system of the labeling substance 12, the coupling substance 13, and the immobilization substance 13C can be refined and can be set in various ways. Further, since the flow path 15 has a closed cross-section structure and functions as a capillary, various techniques such as analysis technology and flow path control in the measuring method using a capillary that has been widely used and developed conventionally can be used as it is. There is also an advantage.

Further, as described above, in the technique using the immunochromatography or the capillary, the material of the flow channel is limited. However, in the measurement chip 21, the chip substrate 2 and the film shape forming the flow channel 15 are formed. Since a wide range of materials can be selected for the member 3 and the injection board 6, not only optimization in spectroscopic measurement is possible, but also a detection system requiring surface film treatment for the chip substrate 2 such as surface plasmon resonance. Can be used, and a detection element such as a crystal oscillator can be incorporated in the chip substrate 2.

In the above embodiment, the chip substrate 2
Although the reaction channel 15 is provided on the chip substrate 2 by adhering the film-shaped member 3 having the hole portion 14 formed therethrough to the chip substrate 2, the groove portion ( The flow path) may be formed directly. The method of forming such a groove portion is the same as that of the chip substrate 2 in the first embodiment.
The method is the same as the method of directly forming the groove portion on. The depth of the groove is also the same as in the first embodiment, the upper limit is 400 μm or less, preferably 200 μm or less from the viewpoint of reaction efficiency, and the lower limit is the ease of processing and the immobilization substance fixed to the bottom. Considering the thickness of 13C, it is generally 0.1 μm or more.

Also in this case, the width of the flow path 15 is usually 0.1 μm or more and 3 mm or less, preferably 1 μm or more,
It is 1 mm or less. The length of the flow path 15 is usually 1 m.
m or more and 1000 mm or less, preferably 3 mm or more, 5
It is 00 mm or less. The flow control means 7 is not limited to the syringe pump as in the first embodiment described above,
For example, a positive pressure type pump or a negative pressure type pump may be used. Alternatively, electrodes may be attached to the upstream end and the downstream end of the flow channel 15, respectively, and different voltages may be applied to these electrodes to generate an electroosmotic flow in the specimen 10.

Alternatively, as the flow control means, electrodes are attached to the inlet 6E and the outlet 6F and a metal is coated around the inlet 6E and the outlet 6F, so that the specimen 10 and the labeling substance 12 in the channel 15 are Connected substance 1
A DC electric field may be applied to 3 to cause the ions (measurement target 11, labeling substance 12, connecting substance 13) to electrophorese. In this case, since the solvent of the sample 10, the solvent of the labeling substance 12, and the solvent of the connecting substance 13 do not move, it is not necessary to seal the flow path 15 with the injection board 6. Therefore, since the injection board 6 is not necessary, the measurement chip is composed of the chip substrate 2 and the film-shaped member 3, and the chip substrate 2 and the film-shaped member 3 are provided.
The groove (groove-shaped channel) formed by the above functions as the reaction channel 15.

Alternatively, as a flow control means, a heating device is provided on the measuring chip 21, and the temperature gradient generated on the measuring chip 21 by this heating device is used to flow the sample 10, the labeling substance 12 and the connecting substance 13. You may do it.
Alternatively, a plurality of these methods may be combined. (3) Others The measuring chip of the measuring object, the measuring device of the measuring object, and the measuring method of the measuring object of the present invention are not limited to the above-described embodiments, and various modifications are made without departing from the spirit of the invention. It is possible to

For example, in each of the above-described embodiments, the reaction sites 5E and 15C are provided on the chip substrate 2 as shown in FIGS. 1A and 4A, respectively.
E and 15C may be provided on the solid phase wall surface forming the reaction channels 5D and 15, and as shown in the measurement chips 1'and 21 'of FIGS. 6A and 6B, the reaction sites 5E and 15C may be provided on the injection board 6. When it is preferable to provide the reaction sites 5E and 15C on the injection board 6 in this manner, for example, the flow paths 5 and 15 are provided.
Is a case of directly forming on the chip substrate 2 by machining. In other words, in this case, as the material of the chip substrate 2, a material (for example, pMMA material) that can be easily machined without being restricted by the immobilization efficiency of the composite material 13C can be selected, while the material of the injection board 6 has the above immobilization efficiency. It is possible to select good polystyrene.

Further, in order to perform electrochemical measurement or the like, an injection board 6 is provided with an element (for example, an electrode).
In the case of embedding in the substrate 2, the reaction sites 5E and 15 are preferably provided on the chip substrate 2 so that the structure of the injection board 6 does not become more complicated and the degree of integration of elements is increased. Similarly, in the above-described second embodiment, the labeling portion 15A and the mixing portion 15B are the same as those in FIG.
Although it is provided on the chip substrate 2 as shown in FIG.
The labeling part 15A and the mixing part 15B may be provided on the solid phase wall surface forming the reaction channel 15, and may be provided on the injection board 6 as shown in FIG. 6 (B).

Further, in each of the above-described embodiments, when forming the flow path 5 between the chip substrate 2 and the injection board 6, the film-like member 3 is used (or directly) on the chip substrate 2 to form the groove. Although an example in which No. 5 is formed is shown in FIG.
As shown in (A), the groove 6a may be provided in the injection board 6 instead of the chip substrate 2. Thus groove 6
A case where it is preferable to form a on the injection board 6 is, for example, a case where quartz having high transparency is used for the chip substrate 2 in order to perform optical observation from the chip substrate 2 side. That is, since it is difficult to form a groove in the quartz (chip substrate 2) by etching, excavation, or the like, if a resin material that is easily etched or excavated is used as the material of the injection board 6, the groove 6a is formed in the injection board 6. Can be easily formed.

In any case, which of the chip substrate 2 and the injection board 6 is provided with the groove is appropriately selected depending on the materials of the chip substrate 2 and the injection board 6, the measurement system, and the like. Of course, FIG.
As shown in (B), the chip substrate 2 and the injection board 6 may be provided with the grooves 2a and 6a, respectively.

Further, the measuring object measuring device may further include a measuring result outputting means such as a printing machine or a monitor for outputting the output result of the measuring means. Further, in the above-described first embodiment, the reaction channel 5D is provided with only one reaction site 5E, but a plurality of reaction sites may be provided in the reaction channel 5D. In this case, the plurality of reaction sites may be provided on only one of the chip substrate 2 and the injection board 6, or may be provided on both the chip substrate 2 and the injection board 6.

Similarly, in the second embodiment described above, the reaction channel 15 is provided with only one reaction site 15C, but a plurality of reaction sites may be provided in the reaction channel 15. Similarly, a plurality of labeling sites or a plurality of mixing sites may be formed in the reaction channel 15. Multiple reaction sites, multiple labeling sites and multiple mixing sites
When provided on one measurement chip, these may be provided on only one of the chip substrate 2 and the injection board 6, or may be provided on both the chip substrate 2 and the injection board 6.

Further, the measuring chips 1 and 21 of each of the above-described embodiments are configured to have one flow channel 5 and 15, respectively, but one measurement chip is provided with a plurality of flow channels. May be. In this case, the flow paths 5 of the first embodiment and the flow path 15 of the second embodiment may be mixed with each other. Further, in the above-described embodiment, whether or not the sample 10 contains the measurement object 11 is detected on / off.
The amount of the measurement object 11 contained in the sample 10 may be quantitatively measured from the fluorescence amount of the labeling substance or the like.

In each of the above embodiments, the connecting substance 1
3 and the example using the biotinylated antibody and the avidin as the immobilizing substance 13C has been described, a tertiary antibody such as a rabbit anti-TSH antibody and a goat anti-rabbit IgG antibody may be used. Specifically, in this case, as shown in FIG. 8, the measurement target 11 and the labeling substance 12 are the rabbit anti-TSH antibody (linking substance) 13 ′ and the goat anti-rabbit IgG antibody 13
The rabbit anti-TSH antibody 13 ′ is linked to the reaction sites 5E and 15C via C ′, and the rabbit anti-TSH antibody 13 ′ is the second specific binding substance 13A and biotin 13B in each of the above embodiments.
It will be a configuration that also serves as.

The method using a tertiary antibody is not as versatile as biotin and avidin, but if a tertiary antibody is used, the antibody itself is used as the linking substance 1.
Since it can be used as 3 ', the step of binding biotin to the antibody can be omitted, the adjustment of the measurement system can be simplified, and the reduction in activity due to modification of the antibody can be avoided. is there.

Further, the chip for measuring an object to be measured, the apparatus for measuring the object to be measured, and the method for measuring the object to be measured according to the present invention measure the object to be measured contained in a biological sample (eg, blood or body fluid). It is widely applicable to medical diagnostics to be detected, environmental diagnostics to measure / detect environmental pollutants contained in the sea, rivers, atmosphere, etc., and measurements used in various researches.

[0108]

As described above in detail, the measuring tip of the measuring object of the present invention according to claim 1, the measuring tip of the measuring object of the present invention according to claim 2, the book according to claim 6 According to the measuring method of the measuring object of the invention, the linking substance according to claims 8 and 9 and the labeling substance of the present invention according to claims 10 and 11, the reaction site is mediated by the measuring substance, the linking substance and the immobilizing substance. It is possible to easily perform the measurement of the measurement target by measuring the labeled substance immobilized on the label. Among the labeled substance, the linking substance and the immobilized substance,
It is the labeling substance and the linking substance that specifically bind to the measurement target. Therefore, by changing the labeling substance and the linking substance according to the measurement target, the immobilization substance immobilized on the reaction site is changed. There is an advantage that it is possible to measure various kinds of measurement objects without using one measurement chip.

In addition, the sample, the labeling substance and the linking substance are
The analyte, the labeling substance, and the coupling substance are allowed to flow through the flow channel, the second flow channel, and the third flow channel, respectively, and the analyte, the labeling substance, and the coupling substance are combined and combined in the reaction channel. The above reaction is a reaction between liquid phases. Since the reaction between liquid phases has a high reaction rate and a high reaction rate, there is an advantage that the reaction time can be shortened and the measurement can be efficiently performed.

Further, the sample, the labeling substance and the connecting substance are simultaneously passed through the first flow channel, the second flow channel and the third flow channel, respectively, so that the sample, the labeling substance and the connecting substance are sequentially passed through one flow channel. Compared with the above, the time required for measurement can be shortened, and from this point, there is an advantage that the measurement can be performed efficiently. In addition, the immobilizing substance is fixed to the reaction channel to provide a reaction site, but since the reaction channel is open to the outside at least during manufacturing, the immobilizing substance is introduced from the opening to the reaction channel. There is an advantage that it is easy to fix and the production of the measuring chip can be simplified. Further, since the material of the chip substrate can be widely selected, there is an advantage that the measurement accuracy can be improved by optimizing the spectroscopic measurement, and further various detection elements can be incorporated into the chip substrate.

According to the measuring device of the measuring object of the present invention as set forth in claim 3, since the flow of the sample, the labeling substance and the linking substance is controlled by the flow control means, the flow rates of the sample, the labeling substance and the linking substance are controlled. There is an advantage that the flow rate can be optimized for the measurement and the measurement can be performed efficiently. Further, there is an advantage that the flow state (flow velocity, flow direction, etc.) can be controlled appropriately, and the measurement can be performed in a wide range. Furthermore, by appropriately adjusting the flow velocity by the flow control means according to the type of the measurement object, various types of measurement objects can be measured in the optimum distribution state with the measurement tip of one specification, and versatility is improved. It has the advantage that it can be expanded.

Further, it is easy to manufacture a measuring chip,
In addition, since the flow of the sample, the labeling substance, and the linking substance can be controlled in various ways, the reaction system of the measurement target, the labeling substance, and the linking substance can be sophisticated, and there is an advantage that various settings can be made. is there. According to the measurement object measuring device of the present invention as set forth in claims 4 and 5, since the flow of the sample is controlled by the flow control means, the flow rate of the sample is determined by measuring the flow rate of the sample in the sample. In addition, the flow rate can be optimized for the reaction of the immobilized substance and the measurement can be performed efficiently, and the flow state can be controlled appropriately, so that the measurement can be performed in a wide range, and moreover, various types of measurement objects can be measured with one specification. The measuring tip of (1) has the advantage that measurement can be performed in an optimal distribution state and versatility can be expanded.

Further, similar to the measuring device for measuring an object to be measured according to claim 3, the production of the measuring chip is easy, and the flow of the sample, the labeling substance and the coupling substance can be controlled in various ways. There is an advantage that the reaction system can be sophisticated and can be set in various ways. According to the measuring method of the measuring object of the present invention as set forth in claim 7,
The flow of the sample can be controlled by the flow control means, and the flow rate of the sample can be optimized for the reaction of the measurement object, the labeling substance, and the linking substance in the sample, so that the measurement can be performed efficiently. Since the distribution state can be appropriately controlled from the outside, measurement can be performed in a wide range, and various types of measurement objects can be measured in an optimum distribution state with a single measurement tip, and versatility can be expanded. There is an advantage.

[Brief description of drawings]

FIG. 1 is a diagram showing a measuring chip of a measuring object and a measuring device of the measuring object according to a first embodiment of the present invention, and FIG. 1A is an enlarged view showing the configurations of the measuring chip and the measuring device. FIG. 3B is a schematic perspective exploded view, and FIG. 3B is a schematic plan view showing an enlarged configuration of the measurement chip with the injection board (covering member) removed.

FIG. 2 is a schematic diagram for explaining a measurement principle in a measurement object measurement chip, a measurement object measurement apparatus, and a measurement object measurement method according to the first embodiment of the present invention.

FIG. 3 is a schematic plan view showing a configuration of a modified example of the measuring chip of the measuring object as the first embodiment of the present invention.

FIG. 4 is a diagram showing a measuring chip of a measuring object and a measuring device of the measuring object according to a second embodiment of the present invention, and FIG. FIG. 3B is a schematic perspective exploded view, and FIG. 3B is a schematic plan view showing an enlarged configuration of the measurement chip with the injection board (covering member) removed.

FIG. 5 is a schematic diagram for explaining a measurement principle in a measurement object measurement chip, a measurement object measurement device, and a measurement object measurement method according to a second embodiment of the present invention.

6 (A) and 6 (B) are schematic perspective exploded views showing an enlarged configuration of a measuring chip as another embodiment of the present invention.

7 (A) and 7 (B) are schematic transverse cross-sectional views of a flow path of a measurement chip as another embodiment of the present invention [XX cross section and FIG. 1 (B)]. 4 (B) is a view corresponding to the YY cross section of FIG.

FIG. 8 is a schematic diagram for explaining a structure of a connecting substance and an immobilizing substance as another embodiment of the present invention.

[Explanation of symbols]

1,1 ', 21,21' measuring tip 2 chip substrates 2a, 6a groove 2A Chip substrate surface 3 Membrane member 4,14 holes 4A, 4B branch part 4C main part 5 channels 5A First channel 5B second flow path 5C Third channel 5D, 15 reaction flow path 5E, 15C Reaction site 5F Confluence part (mixing part) 5G, 5H channel 6 Injection board (cover member) 6A, 6B, 6C, 6E inlet 6D, 6F outlet 7 Syringe pump (flow control means) 7A Silicon tube 7B plate 10 specimens 11 Object to be measured 12 Labeled substances 12A First specific binding substance 12B sign part 13 Connected substances 13A Second specific binding substance 13B Linked substance 13C immobilization material 15A labeled site 15B Mixing part

Claims (11)

[Claims] 1. A measurement chip for measuring an object to be measured in a sample, comprising a chip substrate, a groove-shaped first channel provided on the chip substrate for allowing the sample to flow, and the chip. A groove-shaped second flow channel that is provided on the substrate and allows a labeling substance having a specific binding substance that specifically binds to the measurement target to flow, and that is provided on the chip substrate and is specific to the measurement target A groove-shaped third flow path for circulating a connecting substance that binds to the groove, and a groove-shaped reaction formed on the chip substrate by assembling the first flow path, the second flow path, and the third flow path. A chip for measuring an object to be measured, comprising a flow channel and a reaction site provided in the reaction flow channel and having an immobilizing substance that specifically binds to the connecting substance immobilized thereon. . 2. A measurement chip for measuring an object to be measured in a sample, which is formed between a chip substrate, a covering member for covering the chip substrate, and the chip substrate and the covering member. A first flow path through which the sample flows, and a second flow path through which a labeling substance formed between the chip substrate and the covering member and having a specific binding substance that specifically binds to the measurement target flows And a groove-shaped third flow path that is formed between the chip substrate and the covering member and allows a connecting substance that specifically binds to the measurement object to flow therethrough, and between the chip substrate and the covering member. And a reaction channel formed by assembling the first channel, the second channel and the third channel, and at least the chip substrate and the covering member facing the reaction channel. A reaction part provided on one side and on which an immobilizing substance that specifically binds to the connecting substance is fixed Characterized in that it is configured to include bets, measurement chip of the measuring object. 3. The measurement chip according to claim 1 or 2, a flow control means for controlling the flow of the sample, the labeling substance and the linking substance in the measurement chip, and the label bound to the reaction site. An apparatus for measuring an object to be measured, comprising a measuring means for measuring a substance. 4. A measuring device for measuring a measurement object in which a sample circulates, a flow control means for controlling the flow of the sample in the measurement chip, and a measuring means. Chip for use, a chip substrate, a groove-shaped reaction channel provided on the chip substrate for circulating the sample, the sample, a labeling substance having a specific binding substance that specifically binds to the measurement target, and A mixing portion provided in the reaction channel for mixing three of the linking substances that specifically bind to the measurement target, and a downstream side of the mixing portion in the reaction channel in the flow direction of the sample. And a reaction site on which an immobilizing substance that specifically binds to the linking substance is immobilized, and the measuring means performs a measurement on the labeling substance bound to the reaction site. The measurement of the object Stationary device. 5. A measuring device for measuring an object to be measured, which comprises a measuring chip through which a sample flows, a flow control means for controlling the flow of the sample in the measuring chip, and a measuring means. A chip for chip, a covering member for covering the chip substrate, a reaction channel formed between the chip substrate and the covering member for circulating the sample, and specific to the sample and the object to be measured. At least the chip substrate and the covering member facing the reaction channel so as to mix three of a labeling substance having a specific binding substance that specifically binds and a linking substance that specifically binds to the measurement target. A mixing part provided on one side and provided on at least one of the chip substrate and the covering member facing the reaction flow channel downstream of the mixing part in the flow direction of the sample, and specific to the connecting substance. Immobilization to bind to An apparatus for measuring an object to be measured, which comprises a reaction site on which a substance is immobilized, and wherein the measuring means measures the labeled substance bound to the reaction site. 6. The measurement chip according to claim 1 or 2, wherein the flow of the sample in the first flow channel, the flow of the labeling substance in the second flow channel, and the third flow are performed. A first step of starting the flow of the linking substance through the channel to mix the sample, the labeling substance and the linking substance in the reaction channel; and a mixture of the sample, the labeling substance and the linking substance. An object to be measured, comprising a second step of bringing the reaction channel into contact with the reaction site, and a third step of measuring the labeling substance bound to the reaction site. How to measure things. 7. The apparatus for measuring an object to be measured according to claim 4 or 5, wherein the sample is circulated in the reaction channel while controlling the flow state of the sample by the flow control means. A first step of mixing the sample with the labeling substance and the linking substance at the mixing site; and a mixture of the sample, the labeling substance and the linking substance whose flow state is controlled by the flow control means, An object to be measured, which comprises a second step of contacting the reaction site in the reaction channel and a third step of measuring the labeling substance bound to the reaction site. Measuring method. 8. A chip substrate, a groove-shaped first channel provided on the chip substrate for allowing a sample to flow therethrough, and a specific binding substance provided on the chip substrate for specifically binding to an object to be measured. The groove-shaped second flow channel for circulating the labeling substance, the groove-shaped third flow channel provided on the chip substrate, the first flow channel, the second flow channel, and the third flow channel The third channel of the measuring chip, which is provided with a groove-shaped reaction channel that is assembled and formed on the chip substrate, and a reaction site provided in the reaction channel and to which an immobilizing substance is immobilized. A linked substance which is circulated in the form of a linked substance, which is capable of specifically binding to the measurement target and the immobilized substance simultaneously, respectively. 9. A chip substrate, a covering member for covering the chip substrate, a first channel formed between the chip substrate and the covering member for circulating a sample, the chip substrate and the covering member. A second flow path through which a labeling substance having a specific binding substance that specifically binds to an object to be measured is formed, and a third flow passage formed between the chip substrate and the covering member.
A channel, a groove-shaped reaction channel formed by assembling the first channel, the second channel, and the third channel between the chip substrate and the coating member; A third measuring chip provided with a reaction site provided in the channel and having an immobilizing substance immobilized thereon;
The connecting substance, which is circulated in the flow path, is capable of specifically and simultaneously binding to the measurement target and the immobilized substance, respectively. 10. A chip substrate, a groove-shaped first channel provided on the chip substrate for circulating a sample, a groove-shaped second channel provided on the chip substrate, and on the chip substrate. A groove-shaped third flow path for circulating a connecting substance that specifically binds to the measurement target, the first flow path, the second flow path, and the third flow path, and the chip It is provided with a groove-shaped reaction channel provided on the substrate and a reaction site facing the reaction channel and having an immobilizing substance that is provided on the chip substrate and specifically binds to the connecting substance and fixed. A labeling substance which is a labeling substance which is circulated through the second channel of a measurement chip and which has a specific binding substance which specifically binds to the measurement target. 11. A chip substrate, a covering member for covering the chip substrate, a first channel formed between the chip substrate and the covering member for circulating the sample, the chip substrate and the covering member. A second flow path formed between the chip substrate and the covering member, and a third connecting member which is provided on the chip substrate and which is provided on the chip substrate and which specifically binds to the measurement object. A flow channel, a reaction flow channel formed by assembling the first flow channel, the second flow channel and the third flow channel between the chip substrate and the coating member, and a surface of the reaction flow channel And flow through the second flow path of the measuring chip, which is provided on at least one of the chip substrate and the covering member and has a reaction site to which an immobilizing substance that specifically binds to the connecting substance is fixed A labeling substance that has a specific binding substance that specifically binds to the measurement target It characterized the door, labeling substance.