JP2003098175A - Chip, apparatus, and method for measuring object to be measured - Google Patents

Abstract

PROBLEM TO BE SOLVED: To simplify the manufacturing of chips for measurement, efficiently and accurately perform measurements, and enlarge general versatility in the chip, an apparatus, and a method for measuring an object to be measured. SOLUTION: The chip 1 for measurement for measuring the object to be measured in a specimen 10 is provided with a chip substrate 2, a first groove- shaped channel 5A provided on the chip substrate 2 for passing the specimen 10, a second groove-shaped channel 5B provided on the chip substrate 2 for passing a labeling substance 12 combined with a competitive substance with the object to be measured, a groove-shaped reaction channel 5C formed on the chip substrate 2 by combining the first channel 5A and the second channel 5B, and a reaction section 5D provided for the reaction channel 5C in which the object to be measured and the competitive substance is competitively combined with a specific combining substance and the specific combining substance is fixed.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a chip for measuring an object to be measured for measuring the amount of the object to be measured in a sample by utilizing a specific reaction such as an antigen-antibody reaction. Regarding the measuring device and the method for measuring an object to be measured, specifically, a specific binding substance that specifically binds to the object to be measured, or a competitive substance that competes with the object to be measured and competes with the specific binding substance, The present invention relates to a measurement chip for measuring an object to be measured, a measuring device for the object to be measured, and a method for measuring the object to be measured, which is immobilized in a reaction flow path through which the liquid is circulated.

[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 caused to flow through the groove to form a complex of the measurement object and the specific binding substance in the sample in the reaction part (step 1), and then the labeling substance is circulated through the groove. Then, in the reaction part, the measurement target-specific binding substance-
There is a technique of forming a complex of a labeling substance (step 2) and 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 step 1, the specific binding substance (solid phase) fixed in the reaction part reacts with the sample (liquid layer), and in step 2, the specific binding substance fixed in the reaction part and The complex (solid phase) of the measurement object 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, the reaction time required for measurement is long,
There is a problem of inefficiency.

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 each of the above-mentioned conventional techniques, as shown in FIG.
Of the specific binding substance 53, and the labeling substance 54 is fixed to the reaction part 50. Therefore, it is a necessary condition that the measurement object 52 can simultaneously bind to two or more specific binding substances. In other words, the measurement target is limited to those that can bind to two or more specific binding substances, and thus there is a problem that versatility is low.

The present invention was devised 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 expand the versatility of the object to be measured. An object is to provide a chip for measuring an object, a measuring device for the object to be measured, and a method for measuring the object to be measured.

[0015]

Therefore, a measuring chip of the measuring object of the present invention according to claim 1 is a measuring chip for measuring the measuring object in a sample, and is a chip substrate. A groove-shaped first flow channel provided on the chip substrate for circulating the sample, and a groove-shaped second flow channel provided on the chip substrate for circulating a labeling substance bound to a competitive substance of the measurement object. Channel, a groove-shaped reaction channel formed on the chip substrate by assembling the first channel and the second channel, and the measurement object and the competitor substance provided in the reaction channel. Is characterized in that it is configured with a reaction site to which a specific binding substance that competitively and specifically binds is immobilized.

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 allowing the sample to flow therethrough, and formed between the chip substrate and the covering member to bind to a competitive substance of the measurement target. A second flow path through which the labeled substance is circulated, a reaction flow path formed between the chip substrate and the covering member, and the first flow path and the second flow path are collectively formed, A reaction site provided on at least one of the chip substrate and the covering member facing the reaction flow channel, and having a specific binding substance immobilized to which the measurement target and the competitive substance compete specifically with each other. It is characterized by being configured with.

The measuring chip of the measuring object of the present invention according to claim 3 is a measuring chip for measuring the measuring object in a sample, which is provided on the chip substrate and the chip substrate. A groove-shaped first flow path through which a sample flows, and a label attached to a specific binding substance that is provided on the chip substrate, and the measurement target and the competition substance of the measurement target compete with each other and specifically bind to each other. A groove-shaped second flow channel through which a substance flows, a groove-shaped reaction flow channel formed by assembling the first flow channel and the second flow channel on the chip substrate, and provided in the reaction flow channel It is characterized in that it is provided with a reaction site to which the competitive substance is fixed.

According to a fourth aspect of the present invention, there is provided a chip for measuring an object to be measured, which 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, the measurement object and the measurement object A second flow path through which a labeling substance bound to a specific binding substance that competitively and specifically binds with the competitive substance
A reaction channel formed between the chip substrate and the covering member, the reaction channel being formed by collecting the first channel and the second channel, and the chip substrate facing the reaction channel and the It is characterized in that it is provided on at least one of the covering members and has a reaction site to which the competing substance is fixed. An apparatus for measuring an object to be measured of the present invention according to claim 5 controls the measurement chip according to any one of claims 1 to 4, and the flow of the sample and the labeling substance in the measurement chip. And a measuring means for measuring the labeled substance bound to the reaction site.

An apparatus for measuring an object of measurement according to a sixth aspect of the present invention is a measuring chip in which a sample flows, and a flow control means for controlling the flow of the sample in the measuring chip.
A measuring device for measuring an object to be measured, comprising: a chip substrate; a groove-shaped reaction channel provided on the chip substrate for flowing the sample; and the object to be measured. And a mixing portion provided in the reaction channel for mixing the labeling substance bound to the competing substance and the sample, and provided in the reaction channel downstream of the mixing portion in the flow direction of the sample. The labeling means bound to the reaction site, wherein the measurement target and the competitive substance compete with each other and specifically bind to the reaction site to which a specific binding substance is immobilized. It is characterized by making measurements.

An apparatus for measuring an object to be measured according to a seventh aspect of the present invention comprises a measurement chip through which a sample flows, and a flow control means for controlling the flow of the sample through the measurement chip.
A measuring device for measuring an object to be measured, comprising a measuring means, 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. Provided on at least one of the chip substrate and the covering member facing the reaction channel for mixing the sample with the reaction channel for circulating the sample and the labeling substance bound to the competitive substance of the measurement object. And a mixing site provided on at least one of the chip substrate and the coating member facing the reaction channel downstream of the mixing site in the flow direction of the sample. Is provided with a reaction site to which a specific binding substance that competitively and specifically binds is immobilized, and the measuring means measures the labeling substance bound to the reaction site.

An apparatus for measuring an object to be measured according to claim 8 of the present invention comprises a measurement chip through which the sample flows, and a flow control means for controlling the flow of the sample through the measurement chip.
A measuring device for measuring an object to be measured, comprising: a chip substrate; a groove-shaped reaction channel provided on the chip substrate for flowing the sample; and the object to be measured. A mixing site provided in the reaction channel for mixing the labeling substance bound with the specific binding substance that specifically binds with the competitive substance of the measurement object and the binding substance, and the reaction. The labeling substance bound to the reaction site, wherein the measuring means is provided downstream of the mixing site in the flow direction of the sample in the flow path, and is provided with a reaction site to which the competitive substance is fixed. It is characterized in that the measurement is performed.

An apparatus for measuring an object to be measured of the present invention according to claim 9 comprises a measurement chip through which a sample flows, and a flow control means for controlling the flow of the sample through the measurement chip.
A measuring device for measuring an object, comprising a measuring means, 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. In order to mix the reaction channel through which the sample flows, the labeling substance bound with the specific binding substance that the measurement object and the competitive substance of the measurement object compete with each other and specifically bind, and the sample. A mixing part provided on at least one of the chip substrate and the covering member facing the reaction flow path, and the chip substrate facing the reaction flow path on the downstream side of the mixing position in the flow direction of the sample. It is characterized in that it is provided on at least one of the covering members and is provided with a reaction site to which the competitive substance is fixed, and the measuring means carries out a measurement on the labeling substance bound to the reaction site.

According to a tenth aspect of the present invention, there is provided a method for measuring an object to be measured, wherein the object to be measured is measured using the measuring chip according to any one of the first to fourth aspects. A method of measuring the flow rate of the sample, wherein the labeling substance is circulated in the second flow channel at substantially the same time when the sample is circulated in the first flow channel, and the sample and the labeling substance are mixed in the reaction flow channel. First
A second step of bringing a mixture of the sample and the labeling 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 in that it is configured with the steps of.

According to a method of measuring an object to be measured of the present invention as set forth in claim 11, the device for measuring an object to be measured according to any one of claims 6 to 9 is used to measure the object to be measured. ,
A method for measuring an object to be measured, wherein the sample is circulated through the reaction channel while controlling the flow state of the sample by the flow control means, and the labeling substance disposed in the reaction channel and the A first step of mixing a sample, and a second step of bringing a mixture of the sample and the labeling substance whose distribution state is controlled by the distribution control means into contact with the reaction site in the reaction channel, The third step is a step of measuring the labeling substance bound to the reaction site.

[0025]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described below with reference to the drawings. The term "groove" or "groove-shaped flow path" as used below means that the cross section thereof has an open portion. (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. 1 to 3 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 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 between the chip substrate 2, the film-shaped member 3 and the injection board 6, a flow path 5 (flow path 5A to 5C).

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
Has a thickness of about 20 μm (= channels 5A, 5B, 5 described later).
A commercially available double-sided paper tape having a depth of C) and a width of 40 mm is used and bonded to the lower chip substrate 2 and the upper injection board 6.

The film-shaped member 3 is provided with a Y-shaped hole 4 here. Branch portion 4A of the hole 4,
The flow passage widths of 4B and the main portion 4C are W _A , W _B , and W _C all set to 2 mm. In addition, branch portions 4A, 4B
The lengths L _A and L _B are set to 14 mm, and the length L _C of the main portion 4C is set to 30 mm. The widths of the flow paths W _A , W _B , and W _C are usually 0.1 μm or more and 3
mm or less, preferably 1 μm or more and 1 mm or less. Of the flow path lengths L _A , L _B , and L _C , L _A and L _B are usually 100 μm or more and 100 mm or less, preferably 1 mm or more and 50 mm or less. The flow path length L _C is usually 1 mm or more and 1000
mm or less, preferably 3 mm or more and 500 mm or less.

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. In other words, a groove-shaped flow path is formed from the hole portion 4 of the film-shaped member 3 and the surface 2A of the chip substrate 2 so as to circulate the sample or the labeling substance that labels the measurement target in the sample. 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. Inlet ports 6B for injecting the labeling substance 12 are respectively provided therethrough, and similarly, an outlet port 6C for discharging the mixture of the specimen 10 and the labeling substance 12 communicates with the downstream end of the reaction channel 5C. It is pierced like this.
Measuring chip 1 with injection board 6 as lid
Since the flow path 5 has a closed cross-sectional shape by being loaded in the flow path 5, the sample 10 and the labeling substance 12 can be stably circulated in the flow path 5 by the syringe pump 7 described later.

The inlets 6A and 6B and the outlet 6C are provided.
Is 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 path 5 is such each flow path 5
A to 5C, that is, a first channel (hereinafter, also simply referred to as a channel) 5A into which a sample is injected, a second channel (hereinafter, also simply referred to as a channel) 5B at which a labeling substance is injected, and these Channel 5
A reaction channel 5C formed by assembling A and 5B. A specific binding substance that specifically binds to the measurement target in the sample of the measurement target is immobilized in the reaction flow channel 5C. A reaction site 5D is provided.

Further, here, the inclination angle θ _{A of the} channel 5A with respect to the reaction channel 5C and the inclination angle θ _{B of the} channel 5B with respect to the reaction channel 5C are set to the same angle [θ _A = θ _B. ，
See FIG. 1B]. The labeling substance 12 injected from the second channel 5B is, as shown in FIG.
It is bound to 2A. The term "competitive substance" as used herein refers to a substance that can specifically bind to the specific binding substance of the measurement target, and the specific binding substance means a substance that can specifically bind to the measurement target. That is, the competitive substance means a substance that competes with the measurement target and specifically binds to the specific binding substance.

In general, the competitive substance may be a derivative or an analogue of the object to be measured, but is not limited to this as long as it specifically binds to the specific competitive substance of the object to be measured. It may be a substance having a structure that is significantly different from that of the target object or the measurement target object itself. Then, as shown in FIG. 2, the labeling substance 12 injected from the second channel 5B and the first
The measurement target 11 shown in the sample contained in the sample injected from the flow channel 5A is a merging site (mixing site) of the flow channels 5A and 5B.
After mixing at 5F, the specific binding substance 13 flows into the reaction site 5D of the reaction channel 5C in which the specific binding substance 13 is fixed. Since the specific binding substance 13 can bind to both the substance to be measured 11 and the competitive substance 12A, at this time, the substance to be measured 11 and the competitive substance 12A compete at the reaction site 5D, and thus the substance to be measured. 11 and the labeling substance 12 compete with each other and bind to the specific binding substance 13.

Therefore, the higher the concentration of the measuring object 11 in the sample, the more the measuring object 1 immobilized on the reaction site 5D.
The amount of 1 increases, and the amount of the labeling substance 12 immobilized on the reaction site 5D decreases. Therefore, the concentration of the measurement target 11 can be measured via the labeling substance 12, and even if the measurement target 11 is difficult to be directly measured because the measurement target 11 is transparent, for example, the labeling substance 12
The amount of the measuring object 11 can be easily measured via the.

When the concentration of the measurement object 11 is measured via the labeling substance 12 as described above, the concentration C of the measurement object 11 in the sample and the detection signal (the labeling substance immobilized on the reaction site 5D) The correlation with the level L of the signal representing the quantity) is as shown in FIG. That is, the higher the concentration C of the measurement target, the lower the level of the detection signal detected.

As described above, the measurement of the measurement target 11 by binding the labeling substance 12 (specifically, the competitive substance 12A) and the measurement target 11 to bind to the specific binding substance 13 is called a competitive method. . On the other hand, in the measurement method described with reference to FIG. 10 as the conventional technique, the measurement is performed without causing the labeling substance and the measurement target to compete with each other.

In the competitive method, the object to be measured,
Depending on the order of mixing and reaction of the specific binding substance and the competitive substance, it may be generally called the inhibition method, but here, the inhibition method is also called the competitive method. Hereinafter, the chip substrate 2, the film-shaped member 3, the measurement target 11, the specific binding substance 13
Will be further described.

First, the chip substrate 2 will be described.
As described above, polymethylmethacrylate 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. Also, the chip substrate 2
Forms the flow path 5 together with the film-shaped member 3, so that the sample 1
It is preferable that the material is stable against 0, the labeling substance 12, and the competitive substance 12A.

Further, 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 competitive substance 12A, and, for example, the chip substrate 2 is hydrophobic,
If the sample 10, the labeling substance 12 and the competing substance 12A are water-soluble, hydrophilic treatment (surface modification) with an acid / alkali can be considered. Further, the immobilization product (specific binding substance 13 in the present embodiment and the second embodiment described later, the competitor 12A in the third embodiment and the fourth embodiment described later) immobilized on the reaction site 5D may be a protein. For example, a surface treatment with, for example, carbodiimide, maleimide or succinimide is performed to bind the protein. 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-shaped 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-shaped 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 and the labeling substance 12 are immobilization products fixed to the bottom of the flow path 5 (specific binding substance 13 in the present embodiment and the second embodiment described later, competing substance in the third embodiment and the fourth embodiment described later). 12A), the deeper the channel 5 is, the more the analyte 10 or the labeling substance 12 that does not come into contact with the immobilized substance at the bottom of the channel 5 increases. Therefore, the thickness of the membrane member 3 (the channel 5 From the viewpoint of reaction efficiency, it is preferable to set the depth) to 200 μm or less as described above.

The thickness of the film member 3 (depth of the flow path 5)
Is generally 0.1 μm or more in consideration of the ease of manufacturing the membrane member 3 and the thickness of the immobilization product fixed to the bottom of the channel 5. Next, the sample 10 and the measurement target 11 will be described. Samples 10 are mainly for medical diagnosis and those for environmental analysis. For medical diagnosis, blood, body fluid, urine,
Tears and the like, for environmental analysis, water of the sea or river, a solution in which the atmosphere is dissolved, or the like. Further, the measurement object 11 is not limited as long as a substance (specifically binding substance) that specifically binds to it exists, and examples thereof include proteins, organic substances, lipids, sugars, peptides, hormones, Nucleic acids and the like.

Next, the specific binding substance 13 will be described. The specific binding substance 13 is appropriately determined according to the measurement target 11, and includes, for example, immunoglobulin,
Derivatives such as F (ab ') 2, Fab' 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.

Further, the immobilized substrate (specific binding substance 13 in the present embodiment and the second embodiment described later, competitive substance 12A in the third embodiment and the fourth embodiment described later) chip substrate 2 (reaction channel 5C) The method of immobilizing the immobilization product on the chip substrate 2 may be a method of physically adsorbing the immobilization product on the chip substrate 2 or a method of chemically bonding the immobilization product to the chip substrate 2.
As the physical immobilization method, a method of directly immobilizing an immobilization product on a solid phase (chip substrate 2) and first immobilizing another substance physically or chemically on the solid phase and then immobilizing this substance There is a method in which the immobilized product is adsorbed to the solid phase via Further, as a chemical immobilization method, a method in which an immobilization product is directly bound to a solid phase, a method in which an immobilization group present on the surface of the solid phase is chemically activated and then the immobilization product is bound, a spacer molecule Is physically or chemically bound to the solid phase and the immobilization product is bound to the solid phase via the spacer molecule.

As a method for spotting the immobilized substance on the chip substrate 2 (reaction channel 5C), for example, dropping with a dropper, jetting or dropping with a nozzle hole utilizing the principle of an ink jet printer, or a tapered pin tip is used. There are coating and stamping. Now, as shown in FIG. 1, the measuring apparatus of the present embodiment includes the measuring chip 1 and
A syringe pump (flow control means) 7 for controlling the flow of the sample 10 and the labeling substance 12 in the flow path 5, and a reaction site 5D.
And a measuring means (not shown) for measuring the labeled substance 12 bound to the.

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 and the plate 7B made of PDMS (polydimethylsiloxane) material are connected to the discharge port 6C (downstream end of the reaction channel 5C) of the injection board 6 so that the specimen 10 and the labeling substance 12 flow. To control. The distribution control means is the sample 10
The pump is not limited to a syringe pump as long as it can control the flow between the label substance 12 and the labeling substance 12.
6B or the outlet 6C may be connected.

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 to generate an electroosmotic flow in the specimen 10 and the labeling substance 12. May be. 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 and the labeling substance 12 along the flow path 5, and the difference in specific gravity causes the specimen 10 and the labeling substance 12 to flow.

Alternatively, as the flow control means, the inlet 6A,
Electrodes are attached to 6B and the outlet 6C, and a metal is coated around the inlets 6A, 6B and the outlet 6C to apply a DC electric field to the specimen 10 and the labeling substance 12 in the flow path 5 to measure the ions (measurement target). Object 11, labeling substance 1
2) may be electrophoresed. in this case,
Since the solvent of the specimen 10 and the solvent of the labeling substance 12 do not move, it is not necessary to seal the flow path 5 with the injection board 6. Therefore, since the injection board 6 is not required, the measurement chip is composed of the chip substrate 2 and the film-shaped member 3, and the groove formed by the chip substrate 2 and the film-shaped member 3 (groove-shaped channel). ) Functions as the first flow channel 5A, the second flow channel 5B, and the reaction flow channel 5C.

Further, the distribution control means may be implemented by combining a plurality of the above methods. Now, the measuring means is not limited as long as it measures the amount of the labeling substance based on the physical properties of the labeling substance, and examples thereof include those measuring absorption, fluorescence, evanescent excitation fluorescence, phosphorescence, chemiluminescence, surface plasmon resonance, One that uses a crystal oscillator or the like can be given. 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 chip of the measuring object and the measuring device of the measuring object as the first embodiment of the present invention are configured as described above, the procedure shown below (the first embodiment of the present invention The measuring object is measured by the measuring method of the measuring object). First, the preparation of the labeling substance 12 bound to the competitor 12A will be described. As a compound of the labeling substance 12 and the competitor 12A, here, tylosin-immobilized EuLTX (Ag-EuLTX) is used.
Is adjusted.

First, polystyrene particles (EuLTX) containing a Eu complex having a particle size of 0.21 μm were adjusted to 1%, and 1-
After adding ethyl-3 (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and reacting for 1 hour,
Unreacted EDC is removed by centrifugation, a tylosin solution is added, and the mixture is reacted for 1 hour. Then, unreacted tylosin is removed by centrifugation, and BSA is added to stabilize the particles.

Then, after reacting for 30 minutes, it was centrifuged, washed with purified water and dispersed in a 0.05% sodium azide solution to prepare Ag-EuLTX. Next, in FIG. 1 (A), after adhering the film-shaped member 3 to the chip substrate 2, anti-tylosin (mouse 1gG) is added in the reaction channel 5C formed by the chip substrate 2 and the film-shaped member 3. It is dropped as the specific binding substance 13, dried at room temperature and atmospheric pressure for 30 minutes, and then vacuum dried at room temperature for 1 minute.
It is carried out for 5 minutes to be immobilized in the reaction channel 5C to form the reaction site 5D.

Then, after the injection board 6 was stuck on the film-like member 3, 100 μL of the tylosin standard product as the sample 10 was dropped from the injection port 6A to the flow path 5A, and Ag-EuLTX was added to 100 times with pure water. It is diluted twice and 100 μL of this Ag-EuLTX is dropped into the channel 5B from the injection port 6B. Next, the syringe pump 7 connected to the discharge port 6C of the injection board 6 is operated, and the Ag-EuLTX containing the specimen 10 and the labeling substance 12 at the injection ports 6A and 6B is sucked at 50 μL / min to cause a reaction flow path. Circulate toward 5C. The measurement target 11 and the labeling substance 12 (Ag-EuLTX) in the sample 10 are mixed at the confluence site 5F (step 1), and then move to the reaction site 5D to move the measurement target 11 and the labeling substance 12 to each other. And competitively bind to the specific binding substance 13 immobilized on the reaction site 5D (step 2). Then, according to the same procedure as described above, 100 μm from each of the flow paths 5A and 5B.
The flow path 5 is washed by inhaling L of pure water.

Then, the reaction site 5D is irradiated with ultraviolet rays having a wavelength of 365 nm by a measuring device (not shown) to measure the amount of red fluorescence due to the labeling substance 12 bound to the reaction site 5D. Based on this, the concentration of the measurement object 11 in the sample 10 was measured (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.

In other words, the sample 1
0 and the flow rate of the labeling substance 12 can be controlled to a predetermined flow rate, so that the flow rates of the analyte 10 and the labeling substance 12 are the measurement target 11, the labeling substance 12, and the specific binding substance 13 in the analyte 10.
There is an advantage that the reaction time can be shortened by making the flow rate optimal for the reaction between the two and the measurement can be performed efficiently also from this point. Further, depending on the type of the measuring object 11, the syringe pump 7
Therefore, there is an advantage that various kinds of measurement objects 11 can be measured by a single measurement chip by appropriately adjusting the flow velocity.

Further, by the syringe pump 7, for example, the mixture of the specimen 10 and the labeling substance 12 is added to the reaction site 5D.
Flow is stopped before reaching the specific temperature, and the sample 10 and the labeling substance 12 are sufficiently mixed before contacting with the specific binding substance 13 of the reaction site 5D, or the sample 10 and the labeling substance 1 are
When the mixture of 2 reaches the reaction site 5D, the flow is temporarily stopped, and the solid phase having a slow reaction rate (specific binding substance 13)
-It becomes possible to improve the efficiency of the reaction between the liquid layers (the mixture of the specimen 10 and the labeling substance 12). In addition, the measurement target 11 and the labeling substance 12 that normally bind to the specific binding substance 13 at the reaction site 5D remain unreacted
When there is a possibility of passing through D, the syringe pump may cause the measurement target 11 and the labeling substance 12 that have passed through the reaction site 5D to flow back and contact again with the reaction site 5D.

As will be described later, it is easy to immobilize the immobilized substance (specifically binding substance 13) in the flow path 5, and the flow of the measurement object 11 and the labeling substance 12 can be controlled in various ways. There is also an advantage that the reaction system of the target substance 11, the labeling substance 12 and the specific binding substance 13 can be highly designed and variously set. Further, since the flow path 5 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 which has been widely used and developed conventionally can be used as it is. There is also an advantage.

Further, as described above as a problem of the prior art, in the immunochromatograph, the material of the flow path (the place where the measurement object is developed) is limited in principle, and in the technology using the capillary, the specific binding substance is capillarized. Although the material of the flow path is limited in manufacturing because it is fixed inside, the material of the chip substrate 2 and the injection board 6 that form the flow path 5 can be widely selected in the measurement chip 1.

As a result, not only the spectroscopic measurement can be optimized in terms of 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 5C (channel 5) is still in an open state, so the solid-phase wall surface forming the reaction channel 5C (here, a predetermined portion of the chip substrate 2). The immobilized substance (specific binding substance 13 in the first embodiment) is easily immobilized on the reaction site 5D.
Is provided.

Further, the non-competitive method described as the prior art with reference to FIG. 8 can measure only the measurement object which can simultaneously bind with a plurality of specific binding substances as described above. On the other hand, in the present measurement chip, the measurement is performed by the competitive method as described above, and as shown in FIG. 2, the measurement target 11 can be bound only to the specific binding substance 13 immobilized on the reaction site 5D. Good. That is, the measurement can be performed as long as it is an object to be measured that can simultaneously bind with one or more specific binding substances. Therefore, there is an advantage that measurement can be performed on a wider variety of measurement objects.

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 can 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 μ considering the ease of processing and the thickness of the specific binding substance 13 fixed to the bottom.
It is generally m or more.

Also in this case, the widths of the channels W _A , W _B and W _C are usually 0.1 μm or more and 3 mm or less, preferably 1 μm or more and 1 mm or less. Also, the flow path length L
_{Of A} , L _B and L _C , L _A and L _B are usually 10
It is 0 μm or more and 100 mm or less, preferably 1 mm or more and 50 mm or less. The flow path length L _C is usually 1 mm or more and 1000 mm or less, preferably 3 mm or more and 500 mm or less. Also, FIG.
As shown by the chain double-dashed line in (B), a flow path 5G for a buffer solution for injecting the buffer solution into the sample 10 may be further provided in order to perform measurement under a certain special condition. Further, as shown by the chain double-dashed line in FIG. 1B, in the reaction channel 5C, a channel 5H may be provided on the downstream side of the reaction site 5D. By appropriately providing this flow channel 5H (specifically, by appropriately setting the width and depth of the flow channel, the inclination angle with respect to the reaction flow channel 5C, etc.), the flow channel 5C and the flow channel 5H are interposed. Thus, the unreacted sample 10 and the labeling substance 12 can be separated and collected.

In the above embodiment, the labeling substance 12
Is measured at the reaction site 5D, the labeling substance 12 is separated from the reaction site 5D and recovered after the completion of the flow of the specimen 10 and the labeling substance 12, and the amount of the recovered labeling substance 12 is measured. You may make it measure. To separate the labeling substance 12 from the reaction site 5D, for example,
It suffices to flow a substance similar to the labeling substance 12 onto the reaction site 5D so that this substance and the labeling substance 12 are replaced. Alternatively, a substance similar to the competitor substance 12A is caused to flow on the reaction site 5D so that the competitor substance 12A becomes the labeled substance 1A.
It may be possible to replace this substance with 2 together.

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 is set to the same length, but the width W _A and the width W _B are set to different lengths and the flow channel 5A and the flow channel 5B are flow channels. The cross-sectional areas may be different. When there is a large difference between the flow rates of the sample 10 flowing in the flow channel 5A and the labeling substance 12 flowing in the flow channel 5B, the flow channel 5A and the flow channel 5B are set to have different flow channel cross-sectional areas. Is effective.

That is, 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, it is necessary to mix the sample 10 and the labeling substance 12 uniformly with high accuracy. It is important for making measurements. 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, 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 may be set to 100: 1 (FA / FB = 1
00/1).

As in the present 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 one groove having the reaction site to which the specific binding substance is fixed, 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 prior art, in particular, for example, as in the above case, 100 μL of sample and 1 μL of sample are used.
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 control means individually controls the flow rate of the sample and the flow rate of the labeling substance, 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 general practice 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 flow paths for the sample and the labeling substance are shared, it is naturally impossible to change the cross-sectional area of the flow path 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
It is not realistic because it requires different kinds of distribution control means and increases costs.

Therefore, since the measurement chip has the branched channels 5A and 5B, the sample 1 used for measurement is
Even if there is a large difference in the flow rate between 0 and the labeled substance 12,
The measurement can be performed with high accuracy. Note that, here, the depths of the flow paths 5A and 5B are both the film-shaped member 3
Because it is determined by the thickness, the channel width W _A, passage 5A by setting different lengths W _B, but the passage sectional area is made different in 5B, a flow path chip substrate 2 When directly provided, the flow channel cross-sectional areas may be made different by setting the flow channel depths at different values in the flow channels 5A and 5B. Of course, the flow channel depth and the flow channel width may be set. May be set to different values, or only the flow path width may be set to a different value.

Alternatively, by setting the inclination angle θ _A of the flow channel 5A with respect to the reaction flow channel 5C and the inclination angle θ _B of the flow channel 5B with respect to the reaction flow channel 5C to different angles, the sample 10 is removed from the flow channel 5A. The resistance received when flowing into the reaction flow channel 5C and the resistance received when the labeling substance 12 flows into the reaction flow channel 5C from the flow channel 5B are made different so that the flow rates of the specimen 10 and the labeling substance 12 are different. It is also possible to do so. (B) Description of Second Embodiment Next, a measuring chip of a measuring object, a measuring device of the measuring object, and a measuring method of the measuring object as a 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 is a plate of polymethylmethacrylate (pMMA) having a thickness of 1 mm, which is 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 channel 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
Usually 0.1 μm or more and 3 mm or less, preferably 1
It is not less than μm and not more than 1 mm. The length of the flow path 15 is usually 1 mm or more and 1000 mm or less, preferably 3 mm or more and 500 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 6D 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, and similarly, the sample 10 and the labeling substance are injected. A discharge port 6E 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 6D and the outlet 6E
Is formed to have a diameter of 2 mm in accordance with the reaction channel 15 having a width of 2 mm.

A labeling site (mixing site) 15A in which the labeling substance 12 is arranged is formed in the reaction channel 15, and a reaction site 15B in which the specific binding substance 13 is immobilized is provided on the downstream side thereof.
Are formed, and the labeling substance 12 is bound to the competitive substance 12A as shown in FIG. 3 as in the first embodiment. As a result, the sample 10 flowing through the reaction channel 15 is
As shown in FIG. 2, at the labeling site 15A, the labeling substance 12 bound to the competitive substance 12A is mixed. Then, this mixture flows into the reaction site 15B, and the measurement object 11 and the labeling substance 12 (specifically, the competitive substance 12A) in the sample 10 compete with the specific binding substance 13 fixed to the reaction site 15B. And then join.

The method for spotting the specific binding substance 13 on the chip substrate 2 to form the reaction site 15B and the method for fixing the specific binding substance 13 on the chip substrate 2 are the same as the chip substrate of the specific binding substance 13 on the reaction site 5D of the first embodiment. It is the same as the spotting method / fixing method to 2. The spotting of the labeling substance 12 (including the competing substance 12A) into the reaction channel 15 is similar to the spotting method of the specific binding substance 13, for example, dropping with a dropper or a nozzle hole utilizing the principle of an inkjet printer. It is performed by spraying or dropping, coating with a tapered pin tip, stamping, or the like. Further, since the labeling substance 12 must mix with the specimen 10 and flow to the reaction site 15B on the downstream side when the specimen 10 flows through the labeling site 15A, unlike the specific binding substance 13, the chip substrate 2 has a relatively low degree of binding. Examples of the immobilization method include a method of directly adsorbing the labeling substance 12 on the chip substrate 2, a method of coating the chip substrate 2 with another substance and adsorbing the labeling substance 12 on the coating film, and a method of immobilizing the labeling substance 12 on another side. There is a method of adsorbing by mixing with a substance.

The measuring apparatus of this embodiment is shown in FIG.
As shown in FIG.
A syringe pump 7 that controls the flow of the sample 10 and the labeling substance 12 in the flow path 15 and a measuring unit (not shown) that measures the labeling substance 12 bound to the reaction site 15B are configured. 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 Ag-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 2), a specific binding substance 13 (2 μL) was spotted with an anti-tylosin antibody (mouse 1 gG) at a predetermined position (here, a position 10 mm away from the downstream end of the reaction channel 15C to the upstream side), and a predetermined position ( Here, 10 mm from the upstream end of the reaction channel 15C to the downstream side.
2 μL of a solution prepared by mixing Ag-EuLTX and a 10% sucrose solution at a ratio of 9: 1 is spotted at a separated position). 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 and the reaction site 15B in the reaction channel 15.

Then, after the injection board 6 is attached on the film-like member 3, 20 μL of the tylosin standard product as the sample 10 is dropped into the flow path 15 from the injection port 6D. Next, the syringe pump 7 connected to the discharge port 6E of the injection board 6 is operated, and the injection port 6D
The sample 10 is aspirated at 10 μL / min. The sample 10 flows through the flow path 15 while the flow is controlled by the syringe pump 7, and when the sample 10 comes into contact with the labeling substance 12 at the labeling site 15A, the sample 10 and the labeling substance 12 are mixed (step 1).
Then, when the mixture of the specimen 10 and the labeling substance 12 moves onto the reaction site 15B, the measurement object 11 and the labeling substance 1
2 competes with 2 to bind to the specific binding substance 13 immobilized on the reaction site 15B (step 2). Then, the sample 10
After the circulation of the above is completed, 20 μL of pure water is passed through the channel 15 to wash the channel 5.

Then, the reaction site 15B is irradiated with ultraviolet rays having a wavelength of 365 nm by a measuring device (not shown) to measure the amount of red fluorescence caused by the labeling substance 12 bound to the reaction site 15B. Labeled substance 1
The concentration of the measurement object 11 in the sample 10 was measured via the concentration of 2 (third step). In addition, when a solution containing no tylosin (measurement target) was used as the sample 10 and the measurement was performed in the same procedure as above, a stronger fluorescence amount than the above measurement was measured, and the validity of this measurement was confirmed. Was verified.

Therefore, the measuring tip of the measuring object, the measuring device of the measuring object, and the measuring method of the measuring object of this 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 attached to the solid-phase wall surface (here, a predetermined portion of the chip substrate 2) forming the reaction channel 15C. There is an advantage that 12 (including the competitive substance 12A) and the specific binding substance 13 can be easily immobilized.

Further, the sample 10
And the flow rate of the labeling substance 12 can be controlled to a predetermined flow rate,
There is an advantage that the flow rate of the sample 10 can be set to the optimum flow rate for measurement, 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. Further, since the flow rate can be adjusted appropriately, various kinds of measurement objects 11 can be measured by a single measurement tip by appropriately adjusting the flow rate with the syringe pump 7 according to the type of the measurement object 11. There is an advantage.

As described above, since the labeling substance 12 and the specific binding substance 13 can be easily immobilized on the reaction channel 15 and the flow of the sample 10 can be controlled in various ways, the measurement object 11, the labeling substance There is an advantage that the reaction system of 12 and the specific binding substance 13 can be highly designed and variously set. 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 and the capillaries, the material of the flow channel is limited. However, in the measurement chip 21, the chip substrate 2 forming the flow channel 15 and the film shape 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. Further, 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 specific binding fixed to the bottom. Considering the thickness of the substance 13, 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.
It is 1 mm or less. The length of the flow path 15 is usually 1 mm or more and 1000 mm or less, preferably 3 mm.
mm or more and 500 mm or less. The flow control means 7 is not limited to the syringe pump as in the first embodiment described above, and may be a positive pressure type pump or a negative pressure type pump, for example. 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 6D and the outlet 6E, and metal is coated around the inlet 6D and the outlet 6E, whereby the specimen 10, the labeling substance 12, Competitor 1
A DC electric field may be applied to 2A to cause the ions (measurement target 11, labeling substance 12, and competing substance 12A) to electrophorese. In this case, the solvent of the sample 10 and the labeling substance 12
Since the solvent does 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 required, the measurement chip is composed of the chip substrate 2 and the film-shaped member 3, and the groove formed by the chip substrate 2 and the film-shaped member 3 (groove-shaped channel). ) Functions as the reaction channel 15.

Alternatively, as a flow control means, a heating device may be provided on the measuring chip 21, and the specimen 10 and the labeling substance 12 may be circulated by utilizing the temperature gradient generated on the measuring chip 21 by this heating device. good. Alternatively, a plurality of these methods may be combined. (C) Description of Third Embodiment Next, a measurement chip of a measurement object, a measurement device for the measurement object, and a measurement method of the measurement object as a third embodiment of the present invention will be described. FIG. 6 is a view showing a measuring object measuring chip, a measuring object measuring device, and a measuring object measuring method according to the present embodiment. In addition, the same reference numerals are given to those described in each of the above-described embodiments, and the description thereof will be omitted. In addition, description will be made by diverting FIG.

In the measuring chip 1 of the first embodiment, as shown in FIG.
As shown in, the specific binding substance 13 is fixed to the reaction site 5D, and the labeling substance 12 to which the competitive substance 12A is bound is injected from the second channel 5B. On the other hand, as shown in FIG. 6, in the measuring chip 1'of the present embodiment, the competitive substance 12A is immobilized on the reaction site 5D,
The labeling substance 12 to which the specific binding substance 13 is bound is injected from the second channel 5B.

The structure of the other measuring chip and measuring device is the same as that of the first embodiment, as shown in FIG. 1, and the description thereof is omitted. The measuring chip of the measuring object and the measuring device of the measuring object as the third embodiment of the present invention are configured as described above, and therefore, the procedure shown below (the measurement as the third embodiment of the present invention The measuring object is measured by the measuring method).

First, the preparation of the labeling substance 12 bound to the specific binding substance 13 will be described. As a compound of the labeling substance 12 and the specific binding substance 13, here, anti-tylosin antibody-immobilized EuLTX (Ab-EuLTX) is prepared. First, the polystyrene particles (EuLTX) containing a Eu complex having a particle size of 0.21 μm were adjusted to 1%, and 1-
After adding ethyl-3 (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and reacting for 1 hour,
Unreacted EDC is removed by centrifugation, an anti-tylosin antibody solution is added, and the mixture is reacted for 1 hour. Then, unreacted anti-tylosin antibody is removed by centrifugation, and BSA is added to stabilize the particles.

After reacting for 30 minutes, the mixture was centrifuged, washed with purified water and dispersed in a 0.05% sodium azide solution to prepare Ab-EuLTX. Next, in FIG. 1 (A), after bonding the film-shaped member 3 to the chip substrate 2, in the reaction channel 5C formed by the chip substrate 2 and the film-shaped member 3, tylosin previously bonded to BSA is formed. Is dropped as competitor substance 12A, dried at room temperature and pressure for 30 minutes, and then vacuum dried at room temperature for 1 minute.
It is carried out for 5 minutes to be immobilized in the reaction channel 5C to form the reaction site 5D.

Then, after the injection board 6 was stuck on the membrane member 3, 100 μL of the tylosin standard product as the sample 10 was dropped from the injection port 6A to the flow path 5A, and Ab-EuLTX was diluted with 100% pure water. It is diluted twice, and 100 μL of this Ab-EuLTX is dropped into the channel 5B from the injection port 6B. Next, the syringe pump 7 connected to the discharge port 6C of the injection board 6 is operated to operate the specimen 10 and Ab-E at the injection ports 6A and 6B.
uLTX is aspirated at 50 μL / min to flow toward the reaction channel 5C. Measurement target 11 and Ab-E in specimen 10
The labeling substance 12 in uLTX is mixed at the confluence site 5F (step 1), and then moves to the reaction site 5D, and the labeling substance 12 which is not bound to the measurement object 11 is the reaction site. It binds to the competitor substance 12A immobilized on 5D (step 2). In other words, at the confluence portion 5F and the reaction portion 5D, the measurement target substance 11 and the competitive substance 12A compete with and bind to the specific binding substance 13 bound to the labeling substance 12.

Next, by the same procedure as the above procedure, 100 μL of pure water was sucked from each of the flow paths 5A and 5B to wash the flow path 5, and the reaction site 5D was measured by a measuring device (not shown).
The amount of the labeling substance 12 fixed on the is measured. In this measurement, the lower the concentration of the measurement target substance in the sample, the greater the amount of competing substance 12A that passes through the specific binding substance 13 and the labeling substance 12
Will be combined with. That is, the lower the concentration of the measurement object in the sample, the greater the amount of the labeling substance 12 immobilized on the reaction site 5D via the competitive substance 12A. Therefore, as in the first embodiment, as shown in FIG. 3, the higher the concentration C of the measurement target, the lower the level of the detection signal of the labeling substance.

According to this embodiment, the same effects as those of the first embodiment can be obtained, and the following advantages can be obtained. That is, in the detection chip 1 ′ of the present embodiment, the reaction between the measurement object 11 in the sample 10 and the labeled body 12 (first step, reaction between liquid phase and liquid phase), and the measurement object 11 and The reaction between the complex of the labeled body 12 and the competitive substance 12A immobilized on the reaction site 5D (second step, reaction between liquid phase and solid phase) is performed. On the other hand, in the above-described conventional technique in which the analyte and the labeling substance are sequentially passed through one groove having the reaction site to which the specific binding substance is immobilized, the analyte is immobilized on the measurement target and the reaction site contained in the analyte. Reaction with immobilized specific binding substance (step 1, reaction between liquid phase and solid phase), and specific binding substance immobilized at reaction site-
Reaction between the complex of the measurement target and the labeling substance (Step 2,
The reaction between the liquid phase and the solid phase) is performed.

That is, in the conventional technique, the reaction between the liquid phase and the solid phase must be performed twice, whereas in the present invention, the reaction between the liquid phase and the solid phase only needs to be performed 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

(D) Description of Fourth Embodiment Next, a chip for measuring an object to be measured, a measuring device for the object to be measured, and a method for measuring the object will be described as a fourth embodiment of the present invention. FIG. 7 is a diagram showing a measuring tip of the measuring object, a measuring device of the measuring object, and a measuring method of the measuring object of the present embodiment. Further, the description will be made by diverting FIG. 4 used in the description of each of the above embodiments.

In the measuring chip 21 of the second embodiment, as shown in FIG. 5, the labeling substance 12 to which the competing substance 12A is bound is fixed to the mixing site 15A, and the measurement target 11 is fixed to the reaction site 15B. ing. On the other hand, in the measuring chip 21 'of the present embodiment, as shown in FIG.
It is fixed to A and the competitive substance 12A is fixed to the reaction site 15B.

The structure of the other measuring chip and measuring device is the same as that of the second embodiment, as shown in FIG. 4, and the description thereof is omitted. The measuring chip of the measuring object and the measuring device of the measuring object as the fourth embodiment of the present invention are configured as described above, and therefore, the procedure shown below (the measurement as the fourth 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 third 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.
At a predetermined position (here, a position 10 mm away from the downstream end of the reaction flow channel 15C to the upstream side) in advance.
Tylosin bound to the
While spotting only L, a solution in which Ab-EuLTX and a 10% sucrose solution were mixed at a ratio of 9: 1 was placed at a predetermined position (here, a position 10 mm away from the upstream end of the reaction channel 15C on the downstream side). Spotting only 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 and the reaction site 15B in the reaction channel 15.

Then, after the injection board 6 is stuck on the film-like member 3, 20 μL of the tylosin standard product as the sample 10 is dropped into the flow path 15 from the injection port 6D. Next, the syringe pump 7 connected to the discharge port 6E of the injection board 6 is operated, and the injection port 6D
The sample 10 is aspirated at 10 μL / min. Explaining with reference to FIG. 7, the specimen 10 flows through the flow path 15 while the circulation is controlled by the syringe pump 7, and the specimen 10 and the labeling substance 12 are mixed when they come into contact with the labeling substance 12 at the labeling site 15A. (Step 1). Then, when the mixture of the specimen 10 and the labeling substance 12 moves onto the reaction site 15B,
One of the labeling substances 12 that is not bound to the measurement target 11 binds to the competitor 12A immobilized on the reaction site 5D (step 2). That is, in the mixing part 15A and the reaction part 15B, the measurement target 11 and the competitive substance 12A compete with and bind to the specific binding substance 13 bound to the labeling substance 12, as in the third embodiment. .

Then, after the flow of the sample 10 is completed, 20 μL of pure water is circulated in the channel 15 to wash the channel 5,
The reaction site 15B is irradiated with ultraviolet rays having a wavelength of 365 nm by a measuring device (not shown) to measure the amount of red fluorescence due to the labeling substance 12 bound to the reaction site 15B.
Based on this fluorescence amount, the concentration of the measurement object 11 in the sample 10 was measured via the concentration of the labeling substance 12 (third step).

When a solution containing no tylosin (object to be measured) was used as the sample 10, the measurement was carried out in the same procedure as described above. As a result, a stronger fluorescence amount than the above measurement was measured. The legitimacy was verified. In this embodiment, the measurement is performed in this way, and the same effect as that of the second embodiment is obtained. (E) Others The measuring object measuring chip, the measuring object measuring device, and the measuring method of the measuring object of the present invention are not limited to the above-described embodiments, and are variously modified without departing from the spirit of the invention. It is possible to

For example, in each of the above-described embodiments, the reaction sites 5D and 15B are provided on the chip substrate 2 as shown in FIGS. 1A and 4A, respectively.
D and 15B may be provided on the solid wall surface forming the reaction channels 5C and 15, and the reaction sites 5D and 15B may be provided on the injection board 6 as shown in FIGS. 8A and 8B. good. As a case where it is preferable to provide the reaction sites 5D and 15B on the injection board 6 as described above, for example, the channels 5 and 15 are directly formed on the chip substrate 2 by machining. That is, in this case, the material of the chip substrate 2 is an antibody (second specific binding substance).
It is possible to select a material (for example, pMMA material) that can be easily machined regardless of the immobilization efficiency, and, on the other hand, select polystyrene having a high immobilization efficiency as the material for the injection board 6.

Further, in order to carry out electrochemical measurement or the like, some element (for example, an electrode) is used to inject the injection board 6.
In the case of embedding into the substrate, the specific binding substance 13 is immobilized on the chip substrate 2 (reaction so as not to make the structure of the injection board 6 more complicated and to increase the degree of integration of the device). The parts 5D and 15 are provided on the chip substrate 2).

Similarly, in the second and fourth embodiments described above, the labeling site 15A is provided on the chip substrate 2 as shown in FIG. 4A, but the labeling site 15A is provided in the reaction channel 15. It is sufficient if it is provided on the solid-phase wall surface that forms
It may be provided on the injection board 6 as shown in FIG. 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 groove 5 is formed (or directly) using the film-shaped member 3 on the chip substrate 2. An example 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.

When it is preferable to form the groove 6a in the injection board 6 as described above, for example, the chip substrate 2 is provided so that optical observation can be performed from the chip substrate 2 side.
This is the case when using highly transparent quartz. 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 in accordance with the material of the chip substrate 2 and the injection board 6 and the measuring system. 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 measurement object measuring device may be further provided with a measurement 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 and third embodiments, the reaction channel 5C is provided with only one reaction site 5D on which the specific binding substance is immobilized, but different types of specific sites are provided. A plurality of reaction sites having the binding substance immobilized thereon may be provided in the reaction channel 5C. 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 above-described second and fourth embodiments, the reaction channel 15 is provided with only one reaction site 15B on which the specific binding substance is immobilized, but they are different from each other. The reaction channel 15 may be provided with a plurality of reaction sites on which different types of specific binding substances are immobilized. In this case, a labeling substance corresponding to each specific binding substance is immobilized in the reaction channel 15 to form a plurality or a single labeling site. Each labeling site is provided upstream of the reaction site on which the corresponding specific binding substance is immobilized. In this case, the plurality of reaction sites and the plurality of labeling 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. May be.

The measuring chips 1, 1 ′, 21, 21 ′ of each of the above-mentioned embodiments have one flow path 5, 1 respectively.
However, the measuring chip may be provided with a plurality of channels such as channels 5 and 15 having a labeling site, a reaction site and the like. In this case, the flow paths of different types may be mixed, for example, as shown in FIG.
The Y-shaped channel 5 and the linear channel 15 shown in (1) may be mixed in one chip substrate.

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 sample (for example, blood or body fluid) of biological origin. 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.

[0117]

As described in detail above, the measuring chip of the measuring object of the present invention according to claims 1 and 2 and claim 10 are provided.
In the measurement method of the measurement object of the present invention described,
While flowing through the flow channel, the labeling substance bound to the competitive substance of the measurement target is passed through the second flow channel, and the sample and the labeling substance are mixed in the reaction flow channel. The mixture of the sample and the labeling substance flows into the reaction site, and at the reaction site, the labeling substance and the measurement target substance compete with each other via the competing substance and bind to the specific binding substance.

Therefore, the higher the concentration of the analyte in the sample, the smaller the amount of the labeling substance bound to the reaction site. By utilizing this relationship, the measurement of the labeling substance bound to the reaction site is performed. It is possible to measure the object. In the measurement by the non-competitive method, the measurement target had to bind simultaneously with a plurality of specific binding substances, but in the measurement of the present invention, the measurement target only needs to bind to one or more specific binding substances at the same time. There is an advantage over the non-competitive method that it is possible to measure a variety of specific binding substances and to expand the versatility.

Further, by allowing the sample and the labeling substance to simultaneously flow through the first flow channel and the second flow channel, respectively, the time required for the measurement can be compared to the case where the sample and the labeling substance are sequentially flowed through one flow channel. The advantage is that the measurement can be performed efficiently and the measurement can be performed efficiently. In addition, a specific binding 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 specific binding from this opening to the reaction channel. There is an advantage that the substance can be easily fixed and the production can be simplified. Also,
Since the material of the chip substrate can be widely selected, it is possible to optimize the spectroscopic measurement and improve the measurement accuracy.
Further, there is an advantage that various detecting elements can be incorporated into the chip substrate.

In the chip for measuring an object to be measured according to the present invention described in claims 3 and 4, and the method for measuring an object to be measured according to the present invention described in claim 10, the sample is circulated through the first flow path and is The labeling substance bound to the binding substance is circulated through the second channel, and the sample and the labeling substance are reacted in the reaction channel via the specific binding substance. As a result, in the reaction channel, the binding substance of the measurement target substance and the labeling substance in the sample and the labeling substance that was excessively supplied to the measurement target substance and did not bind to the measurement target substance will be mixed. Then, when this mixed solution flows into the reaction site, only the labeling substance that has not bound to the measurement target binds to the competitor at the reaction site via the specific binding substance.

That is, this means that in the reaction channel, the competitive substance and the object to be measured compete with and bind to the specific binding substance bound to the labeling substance. Therefore,
A chip for measuring an object to be measured according to claim 1, and claim 2.
Similar to the measurement chip of the described measurement object, the measurement object only needs to be able to bind to one or more specific binding substances at the same time, and it becomes possible to perform measurement on various kinds of specific binding substances, and it is said that versatility can be expanded. There are advantages.

Further, the reaction between the substance to be measured in the sample and the labeling substance is a reaction between liquid phases, and the reaction between liquid phases has a high reaction rate and a high reaction rate. It has the advantage of being efficient. Furthermore, by allowing the sample and the labeling substance to simultaneously flow through the first channel and the second channel, respectively, the time required for the measurement can be shortened as compared with the case where the sample and the labeling substance are sequentially passed through one channel. ,
From this point as well, there is an advantage that the measurement can be performed efficiently.

Further, the competitive substance is fixed to the reaction channel to provide a reaction site. However, since the reaction channel is open to the outside at least during manufacturing, the competitive substance is introduced into the reaction channel from this opening. There is an advantage that it is easy to fix the device and the manufacturing 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 apparatus for measuring an object to be measured of the fifth aspect of the present invention, the flow control means controls the flow of the sample and the labeling substance. This has the advantage that 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 target, various types of measurement targets can be measured with a single measurement chip,
There is an advantage that versatility can be expanded.

Further, it is easy to manufacture a measuring chip,
Moreover, since the distribution of the sample and the labeling substance can be controlled in various ways,
The reaction system of the measurement target, the labeling substance and the specific binding substance,
There is an advantage that it can be highly designed and various settings can be made. According to the measuring object measuring device of the present invention described in claims 6 to 9, since the measurement is performed by the competitive method similarly to the chip for measuring the measuring object according to claims 1 to 4, the measuring object is It is only necessary to be able to bind at least one specific binding substance at the same time, and it becomes possible to measure various types of specific binding substances, which has the advantage of expanding versatility.

Further, since the flow of the sample is controlled by the flow control means, the flow rate of the sample is set to the optimum flow rate for the reaction between the measurement object, the labeling substance, the specific binding substance and the competitive substance in the sample. Since measurement can be performed efficiently and the distribution state can be controlled appropriately, measurement can be performed in a wide range, and various types of measurement objects can be measured with a single measurement chip, expanding versatility. There is an advantage that you can.

Further, as in the measuring apparatus for measuring an object to be measured according to claim 5, the production of the measuring chip is easy, and the flow of the sample and the labeling substance can be controlled in various ways. The advantages are that it can be highly designed and various settings can be made. According to the method for measuring an object to be measured of the present invention as set forth in claim 11, the flow of the sample is controlled by the flow control means, and the flow velocity of the sample is set to the object to be measured in the sample.
Since it can be optimized for the reaction between the labeling substance, the specific binding substance and the competitive substance, the measurement can be performed efficiently, and the distribution state can be appropriately controlled from the outside.
There is an advantage that the measurement can be performed in a wide range, and various kinds of measurement objects can be measured by the measurement chip having one specification, and the versatility can be expanded.

[Brief description of drawings]

FIG. 1 is a diagram showing a chip for measuring an object to be measured and a measuring device for the object to be measured as a first embodiment and a third embodiment of the present invention, and FIG. FIG. 2B is a schematic perspective exploded view showing an enlarged view of FIG.
FIG. 3 is a schematic plan view showing an enlarged configuration of a measuring chip with an 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 diagram showing a relationship between a measured concentration in a sample and a detection signal of a labeling substance at a reaction site according to the chip for measuring an object to be measured, the measuring device for the object to be measured, and the method for measuring the object to be measured of the present invention. Is.

FIG. 4 is a diagram showing a measuring chip of a measuring object and a measuring device of the measuring object as a second embodiment and a fourth embodiment of the present invention, and (A) is a configuration of the measuring chip and the measuring device. FIG. 2B is a schematic perspective exploded view showing an enlarged view of FIG.
FIG. 3 is a schematic plan view showing an enlarged configuration of a measuring chip with an 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.

FIG. 6 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 third embodiment of the present invention.

FIG. 7 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 a fourth embodiment of the present invention.

8A and 8B are schematic perspective exploded views showing an enlarged configuration of a measuring chip as another embodiment of the present invention.

9 (A) and 9 (B) are schematic cross-sectional views of a flow channel of a measurement chip as another embodiment of the present invention [XX cross-section and view of FIG. 1 (B)]. 4 (B) is a view corresponding to the YY cross section of FIG.

FIG. 10 is a diagram for explaining a measurement principle of a conventional non-competitive method.

[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, 15 reaction channel 5D, 15B Reaction site 5F Confluence part (mixing part) 5G, 5H channel 6 Injection board (cover member) 6A, 6B, 6D inlet 6C, 6E outlet 7 Syringe pump (flow control means) 7A Silicon tube 7B plate 10 specimens 11 Object to be measured 12 Labeled substances 12A Competitor 13 Specific binding substances 15A labeled site (mixed site)

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, which is provided on the substrate and allows the labeling substance bound to the competitive substance of the measurement object to circulate, and the first flow channel and the second flow channel, are formed on the chip substrate. A groove-shaped reaction channel, and a reaction site provided in the reaction channel, on which a specific binding substance that is capable of specifically binding through competitive competition between the measurement target and the competitive substance is immobilized. Is characterized by being
A chip for measuring an object to be measured. 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 channel for circulating the sample; a second channel formed between the chip substrate and the covering member for circulating a labeling substance bound to a competitive substance of the measurement target; and the chip substrate A reaction channel formed between the first flow channel and the second flow channel, and the chip substrate and the coating member facing the reaction flow channel. An object to be measured, which is provided on at least one side, and is provided with a reaction site on which a specific binding substance that competitively and specifically binds to the object to be measured and the competitive substance is fixed. Chip for measuring things. 3. A measurement chip for measuring an object to be measured in a sample, which comprises a chip substrate, a groove-shaped first channel provided on the chip substrate for allowing the sample to circulate, and the chip. A groove-shaped second flow channel which is provided on the substrate, and through which the labeling substance bound to the specific binding substance, in which the measurement target and the competitive substance of the measurement target compete with each other and specifically binds, is flowed; Constituting a groove-shaped reaction channel formed by assembling one channel and the second channel on the chip substrate, and a reaction site provided in the reaction channel and having the competitive substance fixed thereon A measuring tip for measuring an object to be measured. 4. 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 specific flow path formed between the chip substrate and the covering member for allowing the sample to flow therethrough, wherein the measurement target and the competitive substance of the measurement target compete with each other and specifically bind to each other. A second flow path for circulating the labeling substance bound to the binding substance, and a reaction flow formed between the chip substrate and the covering member and formed by collecting the first flow channel and the second flow channel. And a reaction site provided on at least one of the chip substrate and the covering member facing the reaction channel and having a reaction site on which the competing substance is fixed, Chip for measuring things. 5. The measurement chip according to claim 1, a flow control means for controlling the flow of the sample and the labeling substance in the measurement chip, and the measurement chip bound to the reaction site. A measuring device for measuring an object to be measured, comprising a measuring means for measuring the labeled substance. 6. A measurement device for measuring an object to be measured, comprising a measurement chip through which a sample flows, a flow control means for controlling the flow of the sample in the measurement chip, and a measuring means. The chip for use is a chip substrate, a groove-shaped reaction channel provided on the chip substrate for allowing the sample to flow therethrough, and the reaction flow for mixing the analyte with the labeling substance bound to the competitive substance of the measurement target. A mixing site provided in the channel and a specific site provided downstream of the mixing site in the reaction channel in the flow direction of the sample so that the measurement target and the competitive substance compete with each other and specifically bind to each other. Device for measuring an object to be measured, characterized in that it comprises a reaction site on which a specific binding substance is fixed, and the measuring means measures the labeling substance bound to the reaction site. 7. 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 use in bonding, a chip substrate, 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 bound to a competitive substance of the measurement target. A mixing portion provided on at least one of the chip substrate and the covering member so as to face the reaction flow channel so as to mix the labeled substance with the sample, and on the downstream side in the flow direction of the sample with respect to the mixing portion. A reaction site, which is provided on at least one of the chip substrate and the covering member facing the reaction flow channel, and on which a specific binding substance that binds specifically to the measurement target and the competitive substance in a competitive manner is fixed Configured with The measuring device for measuring an object to be measured, wherein the measuring means measures the labeled substance bound to the reaction site. 8. A measurement device for measuring an object to be measured, comprising: a measurement chip through which a sample circulates; a flow control means for controlling the flow of the sample in the measurement chip; and a measuring means. Is a chip substrate, a groove-shaped reaction channel provided on the chip substrate for allowing the sample to flow therethrough, and the measurement target and a competitive substance of the measurement target compete with each other for specific binding. The labeling substance bound to the selective binding substance,
A mixing part provided in the reaction channel for mixing the sample, and a reaction provided in the reaction channel downstream of the mixing part in the flow direction of the sample and fixed with the competitive substance A measuring device for measuring an object to be measured, characterized in that the measuring means measures the labeled substance bound to the reaction site. 9. An apparatus for measuring an object to be measured, comprising: a measurement chip through which a sample circulates; a flow control means for controlling the flow of the sample in the measurement chip; and a measuring means. Chip, a chip substrate, a covering member for covering the chip substrate, a reaction channel formed between the chip substrate and the covering member for circulating the sample, the measurement object and the measurement object A labeling substance bound to a specific binding substance that competitively and specifically binds with a competitor of
A mixing part provided on at least one of the chip substrate and the covering member so as to face the reaction flow path to mix the sample, and the reaction flow path downstream of the mixing part in the flow direction of the sample. Facing at the same time as at least one of the chip substrate and the covering member, and comprising a reaction site to which the competitive substance is fixed, and the measuring means measures the labeling substance bound to the reaction site. A measuring device for measuring an object to be measured. 10. A method for measuring an object to be measured, which comprises measuring the object to be measured using the measuring chip according to claim 1. The first step of causing the labeling substance to flow through the second flow channel and mixing the sample and the labeling substance in the reaction flow channel at substantially the same time when the sample and the labeling substance are passed through the channel, A second step of bringing the mixture into contact with the reaction site of the reaction channel, and a third step of measuring the labeling substance bound to the reaction site. How to measure the object. 11. A method of measuring an object to be measured using the apparatus for measuring an object to be measured according to any one of claims 6 to 9, which comprises: A sample is circulated through the reaction flow channel while controlling the flow state by the flow control means to mix the labeling substance arranged in the reaction flow channel with the sample.
And a second step of bringing the mixture of the sample and the labeling substance whose flow state is controlled by the flow control means into contact with the reaction site in the reaction channel, and the step of binding to the reaction site. A method for measuring an object to be measured, comprising a third step of measuring a labeling substance.