JP2003114229A - Microchannel chip, measuring device and measuring method using microchannel chip - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a microchannel chip, and a measuring device and a measuring method using the microchannel chip capable of simplifying manufacture thereof, measuring efficiently and accurately, and enlarging versatility. SOLUTION: This microchannel chip for measuring a measuring object in a specimen 10 comprises a chip body, a first fine passage 5A provided in the chip body, for passing the specimen, a second fine passage 5B provided in the chip body, for passing a labeling material 12 having a first specifically bonding material to be bonded specifically with the measuring object, a fine reaction passage 5D provided in the chip body and formed by assembling the first passage 5A and the second passage 5B together, and a reaction site 5E provided in the reaction passage 5D having a second specifically bonding material immobilized thereon to be bonded specifically with the measuring object. In the chip, the first passage 5A, the second passage 5B and the reaction passage 5D are arranged stereoscopically.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a microchannel chip and a measurement using a microchannel chip for measuring the amount of an object to be measured in a sample by utilizing a specific reaction such as an antigen-antibody reaction. The present invention relates to a measuring method using a device and a microchannel chip.

[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 & Bioselect.
tronics 13 (1998) 825-830
(A multi-band capillary i
mmunosensor, K .; Misiakos,
S. E. There is a technique (conventional technique 4) disclosed in Kakabakos).

[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 microchannel 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, the sample is first poured into this groove,
In the reaction part, a complex of the measurement object in the sample and the specific binding substance is formed (first step), and then the labeling substance is circulated in the groove, and in the reaction part, the measurement target-specific binding substance. There is a technique of forming a complex of a labeling substance (second step) 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 the first step, the specific binding substance (solid phase) immobilized on the reaction part reacts with the sample (liquid layer),
In the second step, the complex (solid phase) of the specific binding substance immobilized on the reaction part and the measurement target in the sample,
Reacts with the labeling substance (liquid layer). That is, since the reaction between the liquid phase and the solid phase is required twice, there is a problem that the reaction time required for the measurement is long and the efficiency is low.

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

The present invention was devised in view of the above problems, and facilitates the manufacture of a microchannel chip, enables efficient and accurate measurement, and further expands the versatility of the microchannel. An object is to provide a measuring device and a measuring method using a chip and a microchannel chip.

[0015]

Therefore, the microchannel chip of the present invention according to claim 1 is a microchannel chip for measuring an object to be measured in a sample, which comprises a chip body and the chip body. A minute first channel provided in the chip for circulating the sample, and a minute second channel provided in the chip body for circulating a labeling substance having a first specific binding substance that specifically binds to the measurement target. A flow channel, a minute reaction flow channel provided in the chip body and formed by collecting the first flow channel and the second flow channel, and a micro reaction flow channel provided in the reaction flow channel and specific to the measurement object The second specific binding substance to be bound is configured to have a fixed reaction site, and the first flow path, the second flow path and the reaction flow path are three-dimensionally arranged. .

The microchannel chip of the present invention according to claim 2 is a microchannel chip for measuring an object to be measured in a sample, which comprises a chip body and a micro-particle provided in the chip body for circulating the sample. A first flow path, a minute second flow path provided in the chip body for flowing a labeling substance having a competitive substance of the measurement object, and the first flow path and the second flow path provided in the chip body. A reaction in which a minute reaction channel formed by aggregating channels is fixed, and a specific binding substance that is provided in the reaction channel and that specifically binds to the measurement target and the competitive substance in a competitive manner is immobilized. It is configured with parts and
The first flow channel, the second flow channel, and the reaction flow channel are three-dimensionally arranged.

The microchannel chip of the present invention as set forth in claim 3 is a microchannel chip for measuring an object to be measured in a sample, comprising a chip body and a micro-particle provided in the chip body for circulating the sample. And a minute second channel that is provided in the chip body and allows a labeling substance having a specific binding substance that is provided in the chip main body and that specifically binds to the measurement target and the competition substance of the measurement target to specifically bind. A channel, a minute reaction channel provided in the chip body and formed by collecting the first channel and the second channel, and a reaction provided in the reaction channel and to which the competitive substance is fixed It is configured with parts and
The first flow channel, the second flow channel, and the reaction flow channel are three-dimensionally arranged.

A microchannel chip according to a fourth aspect of the present invention is the microchannel chip according to any one of the first to third aspects, in which the first flow path and the second flow path are substantially vertical. It is characterized in that it is formed so as to relatively approach / assemble along with to form the reaction channel.
A microchannel chip according to a fifth aspect of the present invention is the microchannel chip according to any one of the first to fourth aspects, wherein the chip body is formed by laminating a plurality of laminated members. It has a feature.

The microchannel chip of the present invention according to claim 6 is a microchannel chip for measuring an object to be measured in a sample, which comprises a chip body and a micro-particle provided in the chip body for circulating the sample. A first flow path, a minute second flow path provided in the chip body for flowing a labeling substance having a specific binding substance that specifically binds to the measurement target, and the measurement provided in the chip body A minute third flow path for circulating a connecting substance that specifically binds to an object,
A minute reaction channel provided in the chip body and formed by the first channel, the second channel, and the third channel assembled together, and a specific substance that is provided in the reaction channel and is specific to the connecting substance. A reaction site to which an immobilizing substance that binds physically is fixed, and the first flow path, the second flow path, the third flow path, and the reaction flow path are three-dimensionally arranged. Is characterized by.

A microchannel chip according to a seventh aspect of the present invention is the microchannel chip according to the sixth aspect, wherein the first flow path and the second flow path are relatively close / assembled in a substantially vertical direction. It is characterized in that it is formed so as to form the reaction channel. An eighth aspect of the present invention provides the microchannel chip according to the sixth or seventh aspect, wherein the chip body is formed by laminating a plurality of laminated members.

[0021] A measuring device of the present invention according to claim 9 is the microchannel chip according to any one of claims 1 to 5, and a flow controlling the flow of the sample and the labeling substance in the microchannel chip. It is characterized by comprising a control means and a measurement means for measuring the labeled substance bound to the reaction site. The measuring device of the present invention according to claim 10 is a microchannel chip according to any one of claims 6 to 8, and a flow for controlling the flow of the sample, the labeling substance and the connecting substance in the microchannel chip. It is characterized by comprising a control means and a measurement means for measuring the labeled substance bound to the reaction site.

The measurement method of the present invention according to claim 11 is a measurement method using the microchannel chip according to any one of claims 1 to 5, wherein the sample is circulated in the first flow path. , And starting circulation of the labeling substance in the second flow path,
A first step of mixing the sample and the labeling substance in the reaction channel, and a second step of bringing the 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.

The measurement method of the present invention according to claim 12 is the measurement method using the microchannel chip according to any one of claims 6 to 8, wherein the sample is circulated in the first flow path. Starting the circulation of the labeling substance in the second flow channel and the circulation of the coupling substance in the third flow channel to mix the sample, the labeling substance and the coupling substance in the reaction flow channel A first step, a second step of bringing a mixture of the sample, the labeling substance, and the linking substance into contact with the reaction site of the reaction channel; and a measurement of the labeling substance bound to the reaction site. It is characterized by comprising three steps.

[0024]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described below with reference to the drawings. It is to be noted that the three-dimensionally arranged flow passages below mean that the first flow passage, the second flow passage, and the reaction flow passage (the third flow passage in the fourth and fifth embodiments) are in the same horizontal plane. That is not fully placed in
In other words, in at least one of the first flow path, the second flow path, and the reaction flow path (the third flow path in the fourth and fifth embodiments), at least a part of the flow path is It means that it is out of the horizontal plane. (A) Description of First Embodiment First, a microchannel chip, a measuring apparatus and a measuring method using the microchannel chip as a first embodiment of the present invention will be described. 1 to 3 are views showing a microchannel chip of the present embodiment, a measuring apparatus using the microchannel chip, and a measuring method.

Microchannel chip 1 of this embodiment
Is a chip substrate 2 as shown in FIGS. 1 (A) and 1 (B).
1, a film-shaped member 32, an intermediate substrate 22, a film-shaped member 31, and an injection board (lid part, covering member) 6 are laminated / polymerized in this order from the bottom. ) Laminated members 21, 32, 22, 31,
A chip body is formed by 6, and a minute channel (comprising channels 5A to 5D) 5 having a closed cross-sectional shape described later is three-dimensionally arranged in this chip body.

The substrates 21 and 22 and the injection board 6 are each manufactured by cutting a plate of polymethylmethacrylate (pMMA) having a thickness of 1 mm into 60 mm × 40 mm. In addition, the film members 31 and 32 have a thickness of about 20 μm (= the depth of the flow paths 5A to 5D) and a width of 4
Commercially available 0 mm double-sided paper tapes are used. As a result, the substrates 21 and 22 are inserted through the film member 32.
Is bonded, and the intermediate substrate 22 and the injection board 6 are bonded via the film-shaped member 31. As a result, the laminated members 21, 32, 22, 31, and 6 are integrally assembled.

Further, the film member 31 has a cutout portion 31A.
And a cut-out portion 32A is formed in the film member 32. The intermediate substrate 22 is provided with holes 22A and 22B. These holes 22A and 22B are cut out 32A of the film member 32 below when the intermediate substrate 22 is attached to the film member 32. Both ends 32Aa, 32A
Positioned to connect to b.

Further, the hole portion 22B has the film-like member 3 on the upper side.
When 1 is attached, it is connected to a predetermined position (30 mm from the right end in FIG. 1) of the cutout portion 31A of the film member 31. The hole 22A is connected to a hole 31B provided in the film member 31, and the hole 31B is connected to the hole 6A of the injection board 6 when the injection board 6 is attached to the upper portion. It has become so.

In addition, both ends of the cutout portion 31A are adapted to be connected to the holes 6D and 6B of the injection board 6 when the injection board 6 is attached to the upper portion. The hole 6A is an injection part for injecting the sample 10, the hole 6B is an injection part for injecting the labeling substance 12, and the hole 6D is for discharging the mixture of the sample 10 and the labeling substance 12. Therefore, the discharge portion 6D is provided.

With this structure, the injection section 6A, the hole section 31B, the hole section 22A, the cutout section 32A, and the flow path (first flow path) 5A for circulating the sample 10 through the hole section 22B are formed, and the injection section 6B is formed. , And a channel (second channel) 5B for circulating the labeling substance 12 from the upstream side [the right side in FIGS. 1 (a) and 1 (b)] of the connection portion 5F of the cutout portion 31A with the hole portion 22B. And the connecting portion 5 of the cutout portion 31A is formed.
A reaction channel 5D formed by merging the channels 5A and 5B is formed on the downstream side of F (the left side in FIGS. 1A and 1B). Further, the second specific binding substance 13 that specifically binds to the measurement target in the sample 10 is immobilized on the injection board 6 facing the reaction channel 5D. That is, the reaction channel 5D is provided with the reaction site 5E on which the second specific binding substance 13 is immobilized.

Thus, the main parts of the flow paths 5B and 5D are formed in the film-shaped member 31, and the main part of the flow path 5A is formed in the film-shaped member 3.
2 is formed. That is, the flow paths 5B and 5D and the flow path 5
It is a three-dimensional arrangement formed on a horizontal plane different from A. By the way, when the measurement object can simultaneously bind two or more specific binding substances of the same type, or simultaneously can bind two or more specific binding substances of different types, it is immobilized on the reaction site 5E. Since the specific binding substance and the labeling substance having the specific binding substance can be bound at the same time, the measurement target can be measured through the labeling substance having physical properties advantageous for the measurement.

That is, as shown in FIG. 2, the labeling substance 12 injected into the flow channel 5B is a specific binding substance (first specific binding substance) 1 that specifically binds to the measurement target substance in the sample.
The measurement target 11 having 2A and contained in the sample injected from the first flow channel 5A is bonded to the measurement target 11 at the merging site (mixing site) 5F of the flow channels 5A and 5B via the specific binding substance 12A. Further, the binding substance between the measurement target 11 and the labeling substance 12 binds to the specific binding substance (second specific binding substance) 13 via the measurement target 11 in the reaction site 5E of the reaction channel 5D. .

Therefore, since the complex composed of the labeling substance 12 (specific binding substance 12A), the measurement target substance 11 and the specific binding substance 13 is fixed to the reaction site 5E, the measurement target substance 11 is transparent, for example. Therefore, even if it is difficult to directly measure the measurement target 11, the amount of the measurement target 11 can be easily measured via the labeling substance 12.

The injection board 6 is mounted on the microchannel chip 1 as a lid to form the channel 5 in a closed cross-section, so that the syringe pumps 71 and 72, which will be described later, are used in the channel 5 to detect the specimen 10 and the label. The substance 12 can be stably distributed. Further, the flow passage widths WA, W of the respective flow passages 5A, 5B, 5D
B and WD are set to 2 mm here, but usually 0.1 μm or more and 3 mm or less, preferably 1 μm or more,
It is 1 mm or less. The flow path lengths LA and LB are set to 14 mm here, but normally 100
μm or more and 100 mm or less, preferably 1 mm or more and 5
It is 0 mm or less. Although the flow path length LD is set to 30 mm here, it is usually 1 mm or more,
1000 mm or less, preferably 3 mm or more, 500 mm
It is the following.

Further, the hole portion 31B of the film member 31 and the hole portions 22A and 22B of the intermediate substrate 22 are formed to have a diameter of 2 mm in accordance with the flow path 5 having a width of 2 mm. Hereinafter, the substrates 21, 22, the film members 31, 32, the injection board 6, the measurement target 11, and the specific binding substance 13 will be further described. First, the substrates 21 and 22 and the injection board 6 (hereinafter referred to as the substrates 21, 22 and 6) will be described. As the material of the substrates 21, 22 and 6, polymethyl methacrylate is used here as described above. However, it 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. In addition, the substrates 21, 22, and 6 are the film-shaped members 3
Since the flow path 5 is configured with 1, 32, it is preferable that the material is stable with respect to the specimen 10 and the labeling substance 12.

Furthermore, a predetermined surface treatment may be applied to the surfaces of the constituent substrates 21, 22, 6 that form the flow path 5. Such surface treatment is performed by the specimen 10 and the labeling substance 12.
(Furthermore, in the fourth and fifth embodiments to be described later, the connecting substance 15 is further applied)
For example, if the substrates 21, 22, 6 are hydrophobic,
If the labeling substance 12 (and the linking substance 15 in the fourth and fifth embodiments described later) is water-soluble, hydrophilic treatment (surface modification) with an acid / alkali can be considered. If the specific binding substance 13 immobilized on the reaction site 5E is a protein, surface treatment with, for example, carbodiimide, maleimide, or succinimide is performed to bind the specific binding substance 13. Furthermore, in order to suppress non-specific adsorption (adsorption of non-specific substances),
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 used as the substrate 21,
22 and 6 may be attached to the surface forming the flow path 5 so that predetermined surface physical properties can be obtained in the flow path 5.
Next, the film-shaped members 31 and 32 will be described. Although the film-shaped members 31 and 32 are composed of commercially available paper tapes as described above here, resin films, paper, metal plates, glass plates, etc. If it is a thick film or a thin film made of Further, here, the film-shaped members 31, 32
Is composed of double-sided tape with adhesive applied on both sides and is attached to the substrates 21, 22, and 6.
The method for connecting the film members 31, 32 is as follows.
It is appropriately selected depending on the material of the substrate, the substrates 21, 22, 6 and the like. In addition to bonding with an adhesive, bonding with a solvent / solvent (eg resin bonding with a primer), diffusion bonding, anodic bonding, Crystal bonding, heat fusion, laser fusion, pressure bonding, etc. Alternatively, the film-shaped members 31, 32, the substrates 21, 2
An adhesive tape, a pressure-bonding tape, or a self-adsorbing agent may be interposed between each of Nos. 2 and 6.

Further, the film members 31, 32, the substrates 21, 2
Protrusions and depressions are provided on each of 2, 6, and the protrusions and depressions are fitted into each other to be joined, or the film-shaped members 31, 32, the substrates 21, 22,
Of course, it does not matter if they are physically connected, such as by sandwiching 6 with clips and connecting them. Further, the thickness of the film members 31, 32 is the depth of the flow path 5, and the upper limit is generally 400 μm or less, preferably 200 μm or less. Specimen 10 and labeling substance 12 flowing through the flow path 5
(In the fourth and fifth embodiments to be described later, the connecting substance 15 is further contacted with the specific binding substance 13 fixed to the bottom of the flow channel 5, so that the deeper the flow channel 5 is, the more the flow channel 5 becomes. Since the amount of the analyte 10 or the labeling substance 12 that does not come into contact with the specific binding substance 13 at the bottom of the membrane increases, the film-shaped member 31,
The thickness of 32 (depth of the flow path 5) is 200 μm as described above.
The following setting is preferable from the viewpoint of reaction efficiency.

The thickness of the film members 31 and 32 (the flow path 5
In general, the depth is 0.1 μm or more in consideration of the ease of manufacturing the film members 31 and 32 and the thickness of the specific binding substance 13 fixed to the bottom of the channel 5. .. Next, the sample 10 and the measurement target 11 will be described. As the sample 10, there are a sample mainly for medical diagnosis and a sample for environmental analysis.
It is blood, body fluid, urine, tears, etc. derived from a living body, and for environmental analysis, it is 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 there is a substance that specifically binds to it (specifically binding substance), and examples thereof include proteins, organic substances, lipids,
Sugars, peptides, hormones, nucleic acids, etc.

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 of F (ab ') 2, Fab', Fab,
Receptors, enzymes and their derivatives, nucleic acids, natural or artificial peptides, artificial polymers, sugar chains, lipids, inorganic substances and organic ligands, viruses, drugs, etc.

As a method of immobilizing the specific binding substance 13 on the injection board 6, a method of physically adsorbing the specific binding substance 13 on the chip substrate 2 or a method of chemically chipping the specific binding substance 13 is used. There is a method of bonding to the substrate 2. As a physical immobilization method, a specific binding substance 13 is directly contacted with a solid phase (injection board 6) to immobilize it, and first, another substance is physically or chemically immobilized on the solid phase, There is a method of adsorbing the specific binding substance 13 to the solid phase via this substance. As the chemical immobilization method, a method of directly binding the specific binding substance 13 to the solid phase, or a method of chemically activating the functional group present on the surface of the solid phase before the specific binding substance 13 There are a method of binding and a method of physically or chemically binding a spacer molecule to a solid phase and binding the specific binding substance 13 to the solid phase via the spacer molecule.

As a method for spotting the specific binding substance 13 on the injection board 6 (reaction channel 5D), for example, dropping with a dropper, jetting or dropping with a nozzle hole utilizing the principle of an ink jet printer, and a tapered shape. There are coating and stamping with a pin tip. As shown in FIG. 1, the measuring device of the present embodiment includes the microchannel chip, syringe pumps (flow control means) 71 and 72 for controlling the flow of the sample 10 and the labeling substance 12 in the flow channel 5, It comprises a measurement means (not shown) for measuring the labeling substance 12 bound to the reaction site 5E.

The syringe pumps 71 and 72 have an outer diameter of 6 mm.
71 made of silicon tubes 71A, 72A, PDMS (polydimethylsiloxane) material plate 71
B and 72B are connected to the injection ports 6A and 6B of the injection board 6 to control the flow of the sample 10 and the labeling substance 12. The flow control means is not limited to a syringe pump as long as it can control the flow of the sample 10 and the labeling substance 12, and for example, a positive pressure type pump or a negative pressure type pump can be used as the injection ports 6A and 6B of the injection board 6 or the discharge port. You may make it connect to the exit 6D.

Alternatively, as a flow control means, electrodes are attached to the upstream end and the downstream end of the flow path 5, respectively, and different voltages are applied to these electrodes so that an electroosmotic flow is generated in the specimen 10 and the labeling substance 12. May be. Alternatively, a heating device may be provided in the microchannel 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 6D, and a metal is coated around the inlets 6A, 6B and the outlet 6D to apply a DC electric field to the sample 10 and the labeling substance 12 in the flow path 5 to measure the ions (measurement target). 11, labeling substance 1
2) may be electrophoresed. In this case, since the solvent of the sample 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. When the injection board 6 is not provided, the specific binding substance 13 is fixed to the intermediate substrate 22 and the reaction flow path 5 is formed.
The reaction site 5E may be provided on D.

Further, the distribution control means may be implemented by combining a plurality of the above methods. 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, and crystal vibration. Those that use children and the like can be mentioned. 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 microchannel chip and the measuring apparatus using the microchannel chip as the first embodiment of the present invention are configured as described above, the procedure shown below (the first embodiment of the present invention will be described. The measurement is performed by a measuring method using a microchannel chip). First, the adjustment of the labeling substance 12 will be described. The labeling substance 12 here is composed of anti-human FSH-α subunit antibody-immobilized EuLTX (Ab-EuLTX).

First, a polystyrene particle (EuLTX) containing a Eu complex having a particle diameter of 0.21 μm was added to 0.05 M MES.
The mixture was diluted with (pH 6.0), 2 mL (ml) of 1% suspension was prepared, 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) was added, and the mixture was stirred at room temperature for 1 hour. After the reaction, this solution was centrifuged to remove unreacted EDC, and EuLTX was dispersed in a predetermined amount V (here, 2 mL) of 0.05 M MES (pH 7.0), and then the anti-human FSH-α sub A predetermined amount of a unit antibody (hereinafter, also simply referred to as an antibody) M (here, 0.8 m
g) Add and react at room temperature for 1 hour. Then, this solution was centrifuged to remove unreacted antibody, and BSA-containing Tris buffer (0.3% BSA, 0.1M Tris, p
H8.0) is added to stabilize the particles. At this time, the antibody concentration C1 is 0.4 mg / mL (C1 = M / V = 0.
8/2 = 0.4).

Then, the mixture was stirred at room temperature for 30 minutes, centrifuged, washed with purified water, dispersed in a 0.05% sodium azide solution, and labeled with (Ab-EuLTX) 1
2 is adjusted. At this point, the unbound antibody concentration C2 was 0.15 mg / mL, and therefore the antibody immobilization rate R was 62.5% [R = (C1-C2) /
C1 × 100 = (0.4−0.15) /0.4×10
0] Then, after bonding the film-shaped member 31 to the injection board 6, the reaction channel 5D formed by the cut-out portion 31A of the injection board 6 and the film-shaped member 31.
5.0 mg that specifically binds to the measurement target 11 [here, hCG (human chorionic gonadotropin)] at a predetermined site (here, a position 10 mm away from the downstream end of the reaction channel 5D to the upstream side). / ML of anti-hCG antibody as a specific binding substance 13 (1 μL (microliter)) was dropped and dried at room temperature and atmospheric pressure for 30 minutes, and then vacuum dried at room temperature for 15 minutes to be immobilized on the reaction channel 5D. , Reaction site 5E was formed.

After the intermediate substrate 22, the film-shaped member 32, and the substrate 21 are assembled around the film-shaped member (double-sided tape) 32, this is film-shaped on the injection board 6 on which the reaction site 5 is formed. The microchannel chip was assembled by assembling via the member 31 (double-sided tape).
After that, as the sample 10, 10 μL of 1 IU / mL hCG standard product prepared in 6% bovine serum albumin was dropped from the injection port 6A into the channel 5A, and Ab-
EuLTX is diluted 100 times with pure water to prepare the labeling substance 12, and 10 μL of the labeling substance 12 is dropped from the injection port 6B into the flow channel 5B. Next, for each inlet 6A, 6B,
Syringe pump 71 via plates 71B and 72B,
After connecting 72 respectively, syringe pumps 71, 72
Are operated to cause the specimen 10 and the labeling substance 12 at the injection ports 6A and 6B to flow toward the reaction channel 5D at 10 μL / min. Target substance 11 and labeling substance 1 in specimen 10
2 and 2 are mixed and combined at the confluence site 5F (first step), and then moved to the reaction site 5E to react with the reaction site 5E.
It further binds to the specific binding substance 13 immobilized on (2nd step). Then, by the same procedure as above,
50 μL of 100 μL of pure water is supplied from each of the flow paths 5A and 5B.
The flow path 5 is washed by circulating at a flow rate of μL / min.

When the reaction site 5E was irradiated with ultraviolet rays having a wavelength of 365 nm by a measuring device (not shown),
Red fluorescence resulting from the labeling substance 12 was visually observed at the reaction site 5E, and it was determined that the measurement target 11 in the sample 10 was included (third step). Therefore, the microchannel chip, the measuring apparatus and the measuring method using the microchannel chip of the present embodiment have the following advantages.

That is, in the present invention, the reaction between the measurement object 11 and the labeling substance 12 in the sample 10 (first step, reaction between liquid phase and liquid phase), and the measurement object 11 and the labeling substance 12 And the specific binding substance 13 immobilized on the reaction site 5E (second step, reaction between liquid phase and solid phase). On the other hand, when attempting to perform the measurement by the above-mentioned conventional technique in which the analyte and the labeling substance are sequentially circulated in this order in one groove having the reaction site to which the specific binding substance is immobilized, the immobilization substance is immobilized on the reaction site. Immobilize,
The measurement object 11 and the labeling substance 12 are allowed to sequentially flow into the reaction site (solid phase) for reaction.

That is, in the prior art, 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 need only 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

Further, since the flow rates of the sample 10 and the labeling substance 12 can be controlled to the predetermined flow rates by the syringe pumps 71 and 72, the flow rates of the sample 10 and the labeling substance 12 can be changed to those of the measurement object 11 and the labeling substance 12 in the sample 10. There is an advantage in that the reaction time can be shortened by making the flow rate optimum for the reaction, and the measurement can be performed efficiently from this point as well. Furthermore, the measurement object 11
By appropriately adjusting the flow rate with the syringe pumps 71 and 72 according to the type of
There is an advantage that 1 can be measured under the optimum flow rate by the microchannel chip of one specification.

Further, with the syringe pumps 71 and 72, for example, before the mixture of the specimen 10 and the labeling substance 12 reaches the reaction site 5E, the flow is temporarily stopped and before the contact with the specific binding substance of the reaction site 5E. The sample 10 and the labeling substance are sufficiently reacted with each other, or when the mixture of the sample 10 and the labeling substance 12 reaches the reaction site 5E, 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). Furthermore, when there is a possibility that the measurement object 11 and the labeling substance 12 that originally bind to the specific binding substance 13 at the reaction site 5E may pass through the reaction site 5E without being reacted, a syringe pump It is also possible to cause the measurement target 11 and the labeling substance 12 that have passed through the reaction site 5E to flow backward and to come into contact with the reaction site 5E again.

By the way, in general, the labeling substance 12 is the sample 1
Since the molecular weight is larger than 0, it is difficult to mix the specimen 10 and the labeling substance 12 with each other. Therefore, in the present microchannel chip 1, the channel 5 is three-dimensionally formed, and the channels 5A and 5A
As a configuration in which B is approached / aggregated along a substantially vertical direction (with respect to the vertical direction, that is, the vertical direction), that is, along the flow channel depth direction,
The sample 10 and the labeling substance 12 are mixed sufficiently.

That is, since the microchannel chip has a fine flow path, the sample 10 and the labeling substance 12 which have flowed into the reaction flow path 5D from the flow paths 5A and 5B are separated into two layers in the reaction flow path 5D. They flow and are mixed by mutual molecular diffusion at the interface between the specimen 10 and the labeling substance 12. Therefore, as shown in the plan view of FIG.
When 5B and 5C are formed on the same horizontal plane, the sample 10 and the labeling substance 12 are mixed with each other when the labeling substance 1 is mixed.
The distance (mixing distance) D1 at which 2 diffusely moves to the specimen 10 side
And the shorter the distance (mixing distance) D2 by which the sample 10 diffuses and moves to the side of the labeling substance 12, that is, the narrower the channel width WD of the reaction channel 5D, the more effectively. On the other hand, the higher the flow rate of the sample 10 flowing through the reaction channel 5D, the higher the analysis sensitivity can be improved. Therefore, it is preferable to increase the cross-sectional area of the reaction channel 5D.

Therefore, in order to secure both the mixing performance and the analytical sensitivity, the depth of the reaction channel 5D must be increased. However, it is extremely difficult to form a thin and deep flow path in a substrate, expensive equipment is required to manufacture such a flow path, and the material of the substrate is also limited. Therefore, it is practically difficult to configure the reaction channel 5D with the above-mentioned shape so as to perform highly accurate measurement.

On the other hand, the present microchannel chip 1
Then, as shown in the side view of FIG. 3B, the flow path 5 is formed three-dimensionally and the flow paths 5A and 5B are approached / assembled along the substantially vertical direction (that is, the depth direction of the flow path). In addition, the mixing distances D1 and D2 are along the flow channel depth direction. Therefore, in order to secure both the mixing performance by reducing the mixing distances D1 and D2 and the analytical sensitivity by securing the flow rate, the reaction channel 5D (specifically, the mixing portion 5F) is made shallow and its width is narrow. The shape can be easily manufactured with inexpensive equipment, and the material of the chip body can be selected in a wide range. That is, there is an advantage that the specimen 10 and the labeling substance 12 are sufficiently mixed and highly accurate measurement can be performed.

Further, the microchannel chip 1 is formed by laminating the two-dimensionally processed laminated members 21, 32, 22, 31, and 6 to form the flow channel 5 as a whole. Because it is configured in three dimensions,
There is an advantage that the flow path 5 having a complicated three-dimensional shape can be manufactured more easily. Further, as described above, it is easy to immobilize the specific binding substance 13 in the channel 5, and the measurement target 11
Since the distribution of the labeling substance 12 and the distribution of the labeling substance 12 can be variously controlled, there is an advantage that the reaction system of the measurement target 11 and the labeling substance 12 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 a measuring method using a capillary which has been widely used and developed conventionally can be used. There is also an advantage that it can be used as it is. Further, as described above as a problem of the conventional technique, in the immunochromatograph, the material of the flow channel (field of development of the measurement target) is limited in principle, and in the technique using the capillary, a specific substance (for example, a specific binding substance) 13) is immobilized in the capillary, the material of the flow channel is limited in manufacturing, but the microchannel chip 1 of the present embodiment
Then, a wide range of materials can be selected for the chip substrates 21 and 22 and the injection board 6 that form the flow path 5.

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. Further, the laminated members 21, 32, 2
2, 31, and 6 form a chip body, and the laminated members 21, 32, 22, 31, and 6 are formed by the cutouts and holes formed in the laminated members 21, 32, 22, 31, and 6.
Since the flow path 5 is formed between them, the reaction flow path 5D (flow path 5) is still in an open state before the laminated members 21, 32, 22, 31, and 6 are assembled, so that the reaction There is an advantage that the reaction site 5E can be provided by easily immobilizing the specific binding substance 13 on the solid wall surface (here, a predetermined portion of the injection board 6) forming the flow path 5E.

Further, in order to perform the measurement under a certain special condition, for example, a buffer solution flow path for injecting the buffer solution into the specimen 10 may be further provided. Also, FIG.
As shown by the chain double-dashed line in (B), in the reaction channel 5D, a channel 5H may be provided on the downstream side of the reaction site 5E.
By properly providing this flow path 5H (specifically,
By appropriately setting the width and depth of the flow channel, the inclination angle with respect to the reaction flow channel 5D, etc.), the unreacted sample 10 and the labeling substance 12 are separated and collected via the flow channel 5D and the flow channel 5H. It is also possible to do.

Further, in the above embodiment, the labeling substance 12
Is measured at the reaction site 5E, the labeling substance 12 is separated from the reaction site 5E and recovered after completion of the circulation of the specimen 10 and the labeling substance 12, You may make it measure. To separate the labeling substance 12 from the reaction site 5E, for example,
A substance similar to the labeling substance 12 may be flowed so that this substance and the labeling substance 12 are replaced.

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 materials of the chip substrate 2 and the film-shaped members 31 and 32 are not suitable for spectroscopic measurement. Further, in the above-described embodiment, the width WA of the flow channel 5A for circulating the sample 10 and the width WB of the flow channel 5B for circulating the labeling substance 12 are set to the same length. The lengths may be set to be mutually different so that the flow passages 5A and 5B have different flow passage cross-sectional areas. Specimen 10 and channel 5B flowing through channel 5A
If there is a large difference with the labeling substance 12 that distributes
In this way, it is effective to set different flow passage cross-sectional areas in the flow passages 5A and 5B.

That is, for example, when 100 μL of the sample 10 and 1 μL of the labeling substance 12 are circulated in the channels 5A and 5B for measurement, the sample 10 and the labeling substance 12 are mixed uniformly to form the sample 10. Reacting with the labeling substance 12 is important for accurate measurement. 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 flow rate of the sample 10 in the channel 5A per unit time (hereinafter referred to as flow velocity) FA
And the flow rate FB of the labeling substance 12 in the flow channel 5B is set to 100: 1 (FA / FB = 100/1).

Thus, in order to set the ratio of the flow rate FA of the sample 10 to the flow rate FB of the labeling substance 12 to 100: 1, the flow path 5
The width WA of A and the width WB of the flow channel 5B may be set to different lengths so that the flow channel 5A and the flow channel 5B have different flow channel cross-sectional areas. Thereby, 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 can be mixed uniformly at a predetermined ratio and accurate measurement can be performed.

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 where the specific binding substance is immobilized, in the sample, In order to efficiently react the measurement target with the labeling substance and perform the measurement with high accuracy, the time during which the analyte and the labeling substance can contact and react with the reaction site (= It is important to set the appropriate transit time).

However, in this conventional technique, in particular, for example, as in the above case, 100 μL of sample and 1 μL of sample are used.
When there is a difference between 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 reaction time of the sample and the labeling substance at the reaction site is about the same. 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 ′: F
B '= 100: 1).

By providing a flow control means such as a syringe pump, the flow rate of the sample and the flow rate of the labeling substance are individually controlled by the flow control means, and the flow rate F of the sample is F.
It is possible to set A ′ and the flow rate FB ′ of the labeling substance to different values, but if the flow rate difference is extremely large as in this example, such a flow control means may be used. It is technically difficult to control distribution over a wide area.

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 of course impossible to change the cross-sectional area of the flow path for each of the sample and the labeling substance.

On the other hand, since the present microchannel chip has the branched channels 5A and 5B, even if there is a large difference in the flow rate between the sample 10 and the labeling substance 12 used for the measurement, the measurement can be performed accurately. It is possible to do.
Here, since the depths of the flow channels 5A and 5B are both determined by the thickness of the film-shaped members 31 and 32, the flow channel width W
By setting A and WB with different lengths, the flow paths 5A,
5B have different flow passage cross-sectional areas, but when the flow passage is directly provided on the chip substrate 2, the flow passages 5A, 5
In B, the flow channel cross-sectional area may be made different by changing the flow channel depth, and, of course, both the flow channel depth and the flow channel width may be set to different values. (B) Description of Second Embodiment Next, a microchannel chip according to a second embodiment of the present invention,
A measuring device and a measuring method using a microchannel chip will be described. 4 and 5 are views showing a microchannel chip of the present embodiment, a measuring device using the microchannel chip, and a measuring method. In addition,
The same reference numerals are given to those already described in the first embodiment, and the description thereof will be omitted. In addition, FIG. 1 used in the description of the first embodiment will also be used for description.

The microchannel chip of this embodiment is
The microchannel chip of the first embodiment is configured as shown in FIG. 1 similarly to the microchannel chip of the first embodiment, but the configuration of the labeling substance injected into the second flow path is different. That is, in the microchannel chip of the first embodiment, as shown in FIG. 2, the labeling substance 12 has the first specific substance 12A and the reaction site is composed of the second specific binding substance. On the other hand, as shown in FIG. 4, in the microchannel chip of the present embodiment, the labeling substance 12 ′ has the competitive substance 14 of the measurement object 11.

The term "competitive substance" as used herein means a substance capable of specifically binding to a specific binding substance of an object to be measured, and
The specific binding substance refers to 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 analogue of the measurement target, but is not limited to this as long as it specifically binds to the specific competitor of the measurement target, and the measurement target It may be a substance having a greatly different structure or the measurement object itself.

Other than this, the configuration is the same as that of the first embodiment, and the description thereof is omitted. Since the microchannel chip and the measuring device according to the second embodiment of the present invention are configured as described above, the measurement is performed by the following method (the measuring method according to the second embodiment of the present invention). First, the adjustment of the labeling substance 12 'will be described. Here, the labeling substance 12 ′ is the tylosin-immobilized EuLTX (A
g-EuLTX).

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, the mixture was centrifuged, washed with purified water, dispersed in 0.05% sodium azide solution, and labeled with (Ag-EuLTX) 12 '.
Is adjusted. Then, in FIG. 1A, after the film-shaped member 31 is attached to the injection board 6, a predetermined portion of the reaction channel 5D (here, the cut-out portion 31A of the injection board 6 and the film-shaped member 31 (here, Anti-tylosin (mouse 1 gG) was dropped as a specific binding substance 13 at a position 10 mm away from the downstream end of the reaction channel 5D to the upstream side, and dried at room temperature and atmospheric pressure for 30 minutes, and then vacuumed at room temperature. It is dried for 15 minutes to be immobilized in the reaction channel 5D to form the reaction site 5E.

Then, after the intermediate substrate 22, the film-shaped member 32, and the substrate 21 are assembled around the film-shaped member (double-sided tape) 32, this is film-shaped on the injection board 6 in which the reaction site 5E is formed. The microchannel chip was assembled by assembling via the member 31 (double-sided tape). Then, as the sample 10, the tylosin standard product
Only 0 μL is dropped from the injection port 6A into the channel 5A, and the standard substance 12 ′ is adjusted by diluting Ag-EuLTX 100 times with pure water, and 100 μL of this standard substance 12 ′ is prepared.
Only from the inlet 6B is dropped into the flow path 5B.

Next, the plate 7 is attached to each of the inlets 6A and 6B.
After connecting the syringe pumps 71 and 72 via 1B and 72B, respectively, the syringe pumps 71 and 72 are operated, respectively, and the reaction channel 5D at 10 μL / min of the specimen 10 and the labeling substance 12 ′ at the injection ports 6A and 6B. To distribute to. As shown in FIG. 4, the measurement object 11 and the labeling substance 12 ′ in the sample 10 are mixed at the confluence site 5F (first step), and then moved to the reaction site 5E to be measured. 11 and the labeling substance 12 'competitively react with the reaction site 5E.
It binds to the specific binding substance 13 immobilized on (2nd step).

At this time, the lower the concentration of the substance to be measured in the sample, the more the competitive substance 14 binds to the labeling substance 12 ′ via the specific binding substance 13. That is, the lower the concentration of the measurement target substance in the sample, the greater the amount of the labeling substance 12 ′ fixed to the reaction site 5E via the competitive substance 14. Then, following the same procedure as above, 100 μL of pure water was injected into each of the channels 5A and 5B to wash the channel 5, and then the reaction site 5E was irradiated with ultraviolet rays having a wavelength of 365 nm by a measuring device (not shown). Then, the amount of red fluorescence caused by the labeling substance 12 'bound to the reaction site 5E was measured, and the concentration of the measurement target 11 in the sample 10 was measured based on this amount of fluorescence (third step).

Therefore, the concentration of the measuring object 11 can be measured through the labeling substance 12 ', and the measuring object 1
Even if it is difficult to directly measure the measurement target 11 because 1 is transparent, the amount of the measurement target 11 can be easily measured via the labeling substance 12 ′. In addition, like this,
When measuring the concentration of the measurement target 11 through the labeling substance 12 ', the concentration C of the measurement target 11 in the sample and the level L of the detection signal (the signal indicating the amount of the labeling substance immobilized on the reaction site 5E) The correlation with is as shown in FIG. That is, the higher the concentration C of the measurement target, the lower the detection signal S is detected.

As described above, the competitive method is to perform the measurement on the measurement target 11 by causing the labeling substance 12 '(specifically, the competitive substance 14) and the measurement target 11 to compete with each other and bind to the specific binding substance 13. Say. On the other hand, in the measurement method of the first embodiment described with reference to FIG. 2, the measurement is performed without causing the labeling substance and the measurement target to compete with each other, and thus this measurement method is referred to as a non-competitive method in comparison with the competitive method. .

In the above 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. The microchannel chip of this embodiment has the following advantages in addition to the advantages of the microchannel chip of the first embodiment.

In other words, in the first embodiment (non-competitive method) described with reference to FIG. 2, only the measurement object that can be bound simultaneously with a plurality of specific binding substances can be measured, whereas in the present microchannel chip. The measurement is performed by the competitive method as described above, and the measurement target 11 is measured as shown in FIG.
Need only bind to the specific binding substance 13 immobilized on the reaction site 5E. 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. (C) Description of Third Embodiment Next, a third embodiment of the present invention will be described. FIG. 6 is a diagram showing a microchannel chip of the present embodiment, a measuring device using the microchannel chip, and a measuring method. In addition, the same reference numerals are given to those already described in each of the above-described embodiments, and the description thereof will be omitted. Further, FIG. 1 used in the description of each embodiment will also be used for description.

In the above-described microchannel chip of the second embodiment, as shown in FIG. 4, the specific binding substance 13 is immobilized on the reaction site 5E, and the labeling substance having the competitive substance 14 is introduced from the second channel 5B. 12 are to be injected. On the other hand, as shown in FIG. 6, in the microchannel chip of the present embodiment, the competitive substance 1 is present in the reaction site 5E.
4 is fixed, and the specific binding substance 1 from the second channel 5B.
The labeling substance 12 having 2A is injected.

The structure of the other micro-channel chip and the measuring device is the same as that of the first embodiment, as shown in FIG. 1, and the description thereof is omitted. Since the microchannel chip according to the third embodiment of the present invention is configured as described above, the measurement is performed according to the procedure described below (the measuring method according to the third embodiment of the present invention).

First, the adjustment of the labeling substance 12 will be described. Here, the labeling substance 12 is composed of anti-tylosin antibody-immobilized EuLTX (Ab-EuLTX). First, polystyrene particles (EuLTX) containing a Eu complex having a particle size of 0.21 μm were adjusted to 1%, and 1-ethyl-3 (3
-Dimethylaminopropyl) -carbodiimide hydrochloride (EDC) was added and reacted for 1 hour, then unreacted ED
Remove C by centrifugation and add anti-tylosin antibody solution to 1
React for hours. Then, unreacted anti-tylosin antibody is removed by centrifugation, and BSA is added to stabilize the particles.

Then, after reacting for 30 minutes, the mixture was centrifuged, washed with purified water, and dispersed in a 0.05% sodium azide solution to prepare a labeling substance (Ab-EuLTX) 12. Next, in FIG. 1A, after the film-shaped member 31 is attached to the injection board 6, the injection board 6 and the cutout portion 31 of the film-shaped member 31.
Tyrosine preliminarily bound to BSA is dropped as a competitor substance 14 at a predetermined portion of the reaction flow channel 5D constituted by A (here, at a position 10 mm away from the downstream end of the reaction flow channel 5D to the upstream side), at room temperature. After being dried at normal pressure for 30 minutes, vacuum drying is further performed at room temperature for 15 minutes to immobilize in the reaction channel 5D to form the reaction site 5E.

After the intermediate substrate 22, the film-shaped member 32, and the substrate 21 are assembled around the film-shaped member (double-sided tape) 32, this is film-shaped on the injection board 6 in which the reaction site 5E is formed. The microchannel chip was assembled by assembling via the member 31 (double-sided tape). Then, as the sample 10, the tylosin standard product
Only 0 μL is dropped from the inlet 6A into the channel 5A, and Ab-EuLTX is diluted 100 times with pure water to adjust the standard substance 12, and 100 μL of this standard substance 12 is dropped from the inlet 6B into the channel 5B. To do. Next, each inlet 6
After connecting the syringe pumps 71 and 72 to A and 6B via the plates 71B and 72B, respectively, the syringe pumps 71 and 72 are operated, and the injection ports 6A and 6B are connected.
The specimen 10 and the labeling substance 12 of 10 μL / min are circulated toward the reaction channel 5D. As shown in FIG.
The measuring object 11 and the labeling substance 12 in the inside are merged with each other at a confluence site 5F.
Are mixed in (first step). After that, reaction site 5
After moving to E, the labeling substance 12 that is not bound to the measurement target 11 binds to the competing substance 14 immobilized on the reaction site 5E (second step). That is, at the confluence portion 5F and the reaction portion 5E, the measurement target substance 11 and the competitor substance 14 compete with and bind to the specific binding substance 12A bound to the labeling substance 12.

Next, by the same procedure as above, 100 μL of pure water was injected into each of the channels 5A and 5B to wash the channel 5, and the labeling substance fixed to the reaction site 5E by a measuring device (not shown). Twelve quantities are measured (third step). In this measurement, the lower the concentration of the measurement target substance in the sample, the more the competitive substance 14 will bind to the labeling substance 12 via the specific binding substance 12A. That is, the lower the concentration of the measurement object in the sample, the larger the amount of the labeling substance 12 immobilized on the reaction site 5E via the competitive substance 14. Therefore, as in the second embodiment, as shown in FIG. 5, the higher the concentration C of the measurement target, the lower the level of the detection signal of the labeling substance. (D) Description of Fourth Embodiment Next, a microchannel chip according to a fourth embodiment of the present invention,
A measuring device and a measuring method using a microchannel chip will be described. 7 and 8 are views showing a microchannel chip of the present embodiment, a measuring apparatus using the microchannel chip, and a measuring method. In addition,
The same reference numerals are given to those already described in each of the above embodiments, and the description thereof will be omitted.

The present microchannel chip is constructed as shown in FIG. 7, and the third channel 5C is added to the microchannel chip of each of the embodiments shown in FIG. The reaction channels 5A, 5B, and 5C merge to form a reaction channel 5D. The added third flow path 5C is configured substantially the same as the second flow path 5B. That is, as shown in FIG. 7, the injection board 6
A hole portion (injection port) 6C is formed on the right side of the hole portion 6A in the drawing, a hole portion 31C is formed on the film member 31 on the right side of the hole portion 31B in the drawing, and a hole is formed in the intermediate substrate 22. Parts 22A, 22
Holes 22C and 22D are respectively formed on the right side in the drawing of B, and further, a cutout portion 32B is formed on the film member 32 on the right side of the cutout portion 32A. Then, a third flow path 5C is formed from the injection port 6C, the hole portion 31C, the hole portion 22C, the cutout portion 32B, and the hole portion 22D.
The channel 5C joins the second channel 5B at the hole 22D.

In this microchannel chip, as shown in FIG. 8, the sample 10 is injected from the first channel 5A and
The first that specifically binds to the measurement target 11 from the flow path 5B
The labeled substance 12 having the specific binding substance 12A is injected, and the linking substance (biotinylated antibody) 15 is supplied from the third channel 5C.
Is injected, and an immobilizing substance (avidin in this case) 16 that specifically binds to the connecting substance 15 is immobilized in the reaction site 5E of the reaction channel 5D.

The linking substance 15 is the second specific binding substance 13 that specifically binds to the measurement object 11 and biotin 15A.
And are combined, and biotin 15A has a reactive site 5
It specifically binds to avidin 16 immobilized on E as an immobilizing substance. The coupling substance 15 and the immobilization substance 16 may be any combination that specifically binds to each other. As such a combination, a combination of biotin and avidin as in the present embodiment is preferable because of easy availability.

The hole (inlet) 6C has an outer diameter of 6 m.
A syringe pump (flow control means) is connected through a plate made of m silicon tube and PDMS (polydimethylsiloxane) material (all not shown). The channel width WC of the channel 5C is 2 m here.
m, usually 0.1 μm or more and 3 mm or less, preferably 1 μm
It is m or more and 1 mm or less. The flow path length L _C is 14 mm here, usually 100 μm or more and 100 mm or less, preferably 1 mm or more and 50 mm or less.

The rest of the configuration is similar to that of the above-mentioned embodiment, and therefore its explanation is omitted. Since the microchannel chip and the measuring device as the fourth embodiment of the present invention are configured as described above, the measurement is performed by the following method (the measuring method as the fourth embodiment of the present invention). That is, to explain with reference to FIG. 8, each syringe pump connected to the injection board 6 is operated to operate the injection port 6
The sample 10, the labeling substance 12, and the connecting substance 15 are circulated from A, 6B, and 6C toward the reaction channel 5D. The measurement object 11, the labeling substance 12, and the connecting substance 15 in the sample 10 are
As shown in FIG. 8, they are mixed at the merging site 5F and bonded to each other (first step), and then move to the reaction site 5E and further bind to the immobilizing substance 16 fixed to the reaction site 5E. (Second step). Then, by the same procedure as above, 100 μL of pure water is made to flow into each of the channels 5A, 5B, 5C to clean the channel 5.

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

That is, when the measurement object 11 changes, the specific binding substances 12A and 13 also become different substances according to the change, but in the present invention, the substance containing the specific binding substances 12A and 13 is the labeling substance 12. And the connecting substance 15. That is, the immobilization substance 16 fixed to the reaction site 5E can be fixed regardless of the measurement target 11, and the type of the measurement target 11 can be freely changed in one microchannel chip (same microchannel). It is possible to measure various kinds of measurement objects 11 with a chip).

As a specific example, if avidin is used as the immobilizing substance 16 immobilized on the reaction site 5E, as described above, when TSH is measured as the measuring object 11, the anti-labeling substance 12 is used as the anti-labeling substance 12. TSH can be measured by using a human FSH-α subunit antibody and a biotinylated anti-TSH antibody as the linking substance 15, respectively,
When measuring hCG as the measurement target 11, hCG can be measured by using an anti-human FSH-α subunit antibody as the labeling substance 12 and a biotinylated anti-hCG antibody as the linking substance 15.

In this embodiment, the measuring object 11
However, the first specific binding substance 12A and the linking substance 15 (specifically, the second specific binding substance 13 in this case) can be simultaneously bound. Further, the first specific binding substance 12A and the second specific binding substance 13 may be the same substance or different substances.

In each of the above embodiments, the connecting substance 1
5 and the example using the biotinylated antibody and the avidin as the immobilization substance 16 has been described, a tertiary antibody such as a rabbit anti-TSH antibody and a goat anti-rabbit IgG antibody may be used. Specifically, in this case, as shown in FIG. 9, the measurement object 11 and the labeling substance 12 are mediated by a rabbit anti-TSH antibody (linking substance) 15 ′ and a goat anti-rabbit IgG antibody (immobilizing substance) 16 ′. As a result, the rabbit anti-TSH antibody 15 'serves as the second specific binding substance 13 and biotin 15A in the above embodiment.

Although the method using the tertiary antibody is not as versatile as biotin and avidin, the antibody itself can be used as the linking substance 1 if the tertiary antibody is used.
Since it can be used as 5, the step of binding biotin to the antibody can be omitted, the adjustment of the measurement system can be simplified, and further, the decrease in activity due to the modification of the antibody can be avoided. . (F) Description of Fifth Embodiment Next, a microchannel chip according to a fifth embodiment of the present invention,
A measuring device and a measuring method using a microchannel chip will be described. FIG. 10 is a diagram showing a microchannel chip of the present embodiment, a measuring apparatus and a measuring method using the microchannel chip. In addition, the same reference numerals are given to those already described in each of the above-described embodiments, and the description thereof will be omitted.

The microchannel chip of this embodiment is
Although configured as shown in FIG. 10, the microchannel chip of the fourth embodiment has one measurement system, whereas it has three measurement systems and can simultaneously measure three types of specimens. You can do it. Specifically, three rows of flow channels 5A for circulating the sample 10 are provided in parallel, and each flow channel 5A is joined with a flow channel 5B formed by branching from the injection port 6B for circulating the labeling substance. The flow channel 25 branched from the injection port 26 for flowing the pH adjusting buffer merges, and the flow channel 5C branched from the injection port 6C for flowing the connecting substance 15 merges. ing. In addition, a reaction site 5E is provided in each of the reaction channels 5D formed by merging the channels 5A, 5B, and 5C.

Then, here, the flow paths 5A, 5C, 25
Are formed in the same horizontal plane, and the flow path 5B is formed outside the horizontal plane. In addition, the injection ports 6A, 6B, 6
Syringe pumps (not shown) are connected to C and 26, respectively, and the present embodiment includes these syringe pumps, the microchip, and a measuring device (not shown) that measures the amount of the labeling substance 12 bound to the reaction site 5E. Measuring device is configured.

Since the microchannel chip as the fifth embodiment of the present invention is configured as described above, in addition to the advantages of the microchannel chip of the fourth embodiment described above, measurement for three different analytes 10 can be performed simultaneously. It has the advantage that it can be done. When a configuration functionally equivalent to that of the microchannel chip shown in FIG. 10 is to be configured in a plane (when each flow channel is arranged in the same horizontal plane),
For example, as shown in FIG. 11, crossing of the flow paths occurs at a portion indicated by X in the drawing. That is, when the reagent injected from one injection port is branched and supplied to each of the three or more flow paths provided in parallel as described above, the type of the reagent is limited to two. In the microchannel chip of the present embodiment, three or more types (here, three types) of reagents are provided in each of three or more channels (here, three) without being restricted by such regulation by crossing the channels. It is possible to supply. (H) Others The microchannel chip of the present invention, the measuring device and the measuring method using the microchannel chip are not limited to the above-described embodiments, and can be variously modified without departing from the spirit of the invention. .

For example, in the above-described first to fifth embodiments, the planar laminated members 6, 31, 22, 32, 2 are formed.
Although the micro channel chip is configured by stacking 1 of the above, it is also possible to form the micro channel chip as an integrated body by using a photo curable resin such as a UV curable resin. In the above embodiment, The flow paths 5 are provided on the substrates 21 and 22 by attaching the film members 31 and 32 having the cutouts 31A and 32A penetrating to the substrates 21 and 22, respectively. It is also possible to directly form the grooves (groove-shaped flow paths) in the substrates 21, 22, 6 without using. For example, as shown in FIGS. 12A and 12B, the flow paths 5B and 5D are directly formed on the injection board 6 or the intermediate substrate 22, or the flow channels 5B and 5D are shown in FIGS.
The channels 5A and 5C may be formed directly on the chip substrate 21 or the intermediate substrate 22 as shown in FIG. Alternatively, FIG.
As shown in (E) and (F), the flow paths 5C and 5D are formed directly on both the injection board 6 and the intermediate substrate 22, or the flow paths 5A and 5C are directly formed on both the chip substrate 21 and the intermediate substrate 22. It may be formed.

Which of the injection board 6, the intermediate substrate 22 and the chip substrate 21 is provided with the flow path is appropriately selected according to various conditions. For example, when quartz with high transparency is used for the injection board 6 to optically measure the amount of the standard substance 12 immobilized on the reaction site 5E from the injection board 6 side, the quartz (injection board 6) is etched or excavated. Therefore, it is difficult to form the groove, and therefore, the intermediate substrate 22 is made of a resin material that is easily etched or excavated, and the flow path is formed on the intermediate substrate 22 side.

As described above, as a method for directly forming the grooves on the substrates 21, 22, and 6, for example, mechanical processing such as cutting and polishing, or morphological control using lithography, followed by dry etching (for example, electron beam, X-ray irradiation, DRI
E), a method of forming a groove portion such as wet etching, electric discharge machining, laser ablation, or the like, first, the groove shape is drawn on a mask by photolithography, and then the above-described dry etching, wet etching, electric discharge is performed based on this drawing. There are a method of forming grooves by removing predetermined portions of the substrates 21, 22, 6 by processing and laser ablation, and a transfer technique using a stamper, compression molding, injection molding or the like.

Alternatively, the channels can be formed at the same time when the substrates 21, 22, 6 are formed.
Examples of the molding method of 1, 22, 6 include molding with a mold. It is also possible to mold the substrates 21, 22, 6 and such flow paths simultaneously by stereolithography using a photo-curable resin. In such a case,
It is also possible to simultaneously manufacture the substrates 21, 22, 6 and the flow path by computer control.

Further, the substrate 2 is obtained by combining the above-mentioned methods.
The channels 1, 2, 6 may be molded. Further, since the materials of the substrates 21, 22, and 6 can be widely selected as described above, other known fine processing techniques can be used for such flow path processing. In addition, the substrate 2
Even when the flow channels are directly formed in 1, 2, 6, 6, the film-shaped member 3
In the same manner as in the case where the channels 1 and 32 are attached to the chip substrates 21 and 22 to form the channels, the upper limit of the channel depth is 400 μm or less, preferably 200 μm or less from the viewpoint of reaction efficiency, and the lower limit. Considering the easiness of processing and the thickness of the specific binding substance 13 fixed to the bottom, it is generally 0.1 μm or more.

Also in this case, the width of each of the flow paths WA to WD is usually 0.1 μm or more and 3 mm or less, preferably 1 μm.
It is m or more and 1 mm or less. Also, the flow path lengths LA, L
B and LC 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 LD is usually 1 mm or more and 1000 mm or less, preferably 3 mm or more and 500 mm or less.

In each of the above embodiments, the reaction site 5
E is provided on the injection board 6 as shown in FIGS. 1 (A) and 7 (A), respectively, but the reaction site 5E may be provided on the solid-phase wall surface forming the reaction channel 5D, It may be provided on the chip substrate 22 so as to face the reaction channel 5D. Reaction board 5E, injection board 6
Which of the chip substrate 22 and the chip substrate 22 is provided is appropriately determined according to various conditions. For example, when the channel 5 is directly formed on the chip substrate 22 by machining,
It is preferable to provide the reaction site 5E on the injection board 6. That is, as the material of the chip substrate 22, the first
A material (for example, pMMA material) that is easy to machine regardless of the immobilization efficiency of the specific binding substance, etc., is selected, while polystyrene is selected as the material of the injection board 6 with high immobilization efficiency.

Further, in order to carry out electrochemical measurement or the like, an injection board 6 is provided with an element (for example, an electrode).
In the case of embedding into the substrate, the reaction site 5E is formed on the chip substrate 22 so that the structure of the injection board 6 does not become more complicated and the degree of integration of elements is increased.
It is desirable to be provided in. Further, as the measuring device using the microchannel chip, the measuring result outputting means such as a printing machine or a monitor for outputting the output result of the measuring means may be further provided.

In each of the above embodiments, the reaction flow channel 5
Although only one reaction site 5E is provided in D, a plurality of reaction sites may be provided in the reaction channel 5D. In this case, the plurality of reaction sites may be provided on only one of the chip substrate 22 and the injection board 6, or may be provided on both the chip substrate 22 and the injection board 6.

Further, in the above-described embodiment, whether or not the sample 10 contains the measurement object 11 is detected on / off. However, how much the measurement object 11 is contained in the sample 10 is detected. It may be possible to quantitatively measure the presence or absence of the fluorescent substance from the labeling substance. Further, the microchannel chip, the measuring device and the measuring method using the microchannel chip of the present invention are used for medical diagnosis for measuring / detecting an object to be measured contained in a biological sample (for example, blood or body fluid), sea / river It can be widely applied to environmental diagnosis for measuring / detecting environmental pollutants contained in air, atmosphere, etc., and measurement used in various researches.

[0115]

As described above in detail, according to the measuring method using the microchannel chip of the present invention described in claim 1 and the microchannel chip of the present invention described in claim 11, it is fixed at the reaction site. By performing the measurement with respect to the labeling substance, it is possible to easily perform the measurement with respect to the measurement target. Further, the sample and the labeling substance are circulated in the first flow channel and the second flow channel, respectively, and the sample and the labeling substance are collected. Since they are merged and bonded in the reaction channel, the reaction between the measurement target and the labeling substance in the sample becomes a reaction between liquid phases. Since the reaction between liquid phases has a high reaction rate and a high reaction rate, there is an advantage that the reaction time can be shortened and the measurement can be efficiently performed.

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 reduced as compared with the case where the sample and the labeling substance-linking substance are sequentially passed through one channel. There is an advantage that the measurement can be shortened and the measurement can be performed efficiently also from this point. Further, since the first flow path, the second flow path, and the reaction flow path are three-dimensionally arranged, the mixing distance can be formed along the vertical direction, that is, the depth direction. The shallower the flow path, the higher the mixing performance. Making the reaction channel shallow is a direction that facilitates processing. Therefore, it is possible to form the reaction channel shallow and sufficiently mix the analyte and the labeling substance, which are generally difficult to mix. There are advantages.

In the measuring method using the microchannel chip of the present invention according to claim 2 and claim 3 and the microchannel chip of the present invention according to claim 11, the competitive substance and the object to be measured compete with each other and are specific. The higher the concentration of the analyte that binds to the binding substance, the less the amount of the labeling substance that binds to the reaction site decreases.By utilizing this relationship, the labeling substance bound to the reaction site can be measured. It is possible to measure the measurement target.

In the measurement by the non-competitive method, the substance to be measured had to bind with a plurality of specific binding substances at the same time. Therefore, it has an advantage over the non-competitive method in that it can measure various kinds of specific binding substances and can expand versatility. In the microchannel chip of the present invention according to claim 4 and claim 7, the first channel and the second channel are configured to relatively approach / assemble along a substantially vertical direction to form a reaction channel. Has been done. The shorter the diffusion movement distance (mixing distance) of molecules required for mixing the sample and the labeling substance, the easier the sample and the labeling substance are mixed, but in the present microchannel chip, the mixing distance is the vertical direction, That is, they are formed along the depth direction, so that the shallower the reaction flow channel, the higher the mixing performance. Making the reaction channel shallow is a direction that facilitates processing. Therefore, it is possible to form the reaction channel shallow and sufficiently mix the analyte and the labeling substance, which are generally difficult to mix. There are advantages.

Since the microchannel chip of the present invention according to claim 5 and claim 8 is formed by laminating a plurality of laminated members, the three-dimensional structure is obtained by laminating the laminated members processed two-dimensionally. Since it is possible to dispose the flow path, it is possible to easily manufacture the flow path. In addition, since a wide range of materials can be selected, it is possible to optimize spectroscopic measurement and improve measurement accuracy.
There is an advantage that various detection elements can be incorporated.

Further, although the reaction site is provided in the reaction flow channel, the reaction flow channel is open to the outside at least during manufacturing, so that it is easy to provide the reaction site in the reaction flow channel from this opening. Then, there is an advantage that the manufacture of the microchannel chip can be simplified. Further, since the microchannel chip can be easily manufactured and the distribution of the specimen, the labeling substance and the like can be controlled in various ways, there is an advantage that the reaction system of the measurement object and the labeling substance can be set highly and variously.

According to the measuring method using the microchannel chip of the present invention described in claim 6 and the microchannel chip of the present invention described in claim 12, among the labeling substance, the linking substance and the immobilizing substance, the substance to be measured is Is specifically bound by the labeling substance and the linking substance. Therefore, by changing the labeling substance and the linking substance according to the measurement target, the immobilization substance immobilized at the reaction site can be There is an advantage that it is possible to measure various kinds of measurement objects with one microchannel chip.

According to the measuring device using the microchannel chip of the present invention as defined in claims 9 and 10, the flow control means controls the flow of the sample and the labeling substance, so that the flow rate of the sample and the labeling substance is controlled. However, there is an advantage that the flow rate can be optimized for the measurement and the measurement can be performed efficiently. In addition, the distribution state (flow velocity, distribution direction, etc.) can be controlled appropriately,
There is an advantage that the measurement can be performed in a wide range. further,
By appropriately adjusting the flow velocity by the flow control means according to the type of the measurement object, various types of measurement objects can be measured with the microchannel chip of one specification in the optimum distribution state, and versatility can be expanded. There is an advantage.

[Brief description of drawings]

FIG. 1 is a diagram showing a microchannel chip and a measuring device according to a first embodiment of the present invention, FIG. 1A is a schematic perspective exploded view showing an enlarged configuration of a microchannel chip and a measuring device; B) is a schematic plan view showing an enlarged configuration of the microchannel chip in a state where the injection board (covering member) is removed.

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

FIG. 3 is a diagram for explaining the effect of the microchannel chip, the measuring device, and the measuring method according to the first embodiment of the present invention, in which (A) shows channels 5A, 5B, and 5C formed on the same horizontal plane. A schematic plan view showing the mixing distance in the case,
(B) is a schematic side view showing a mixing distance when the channels 5A, 5B, and 5C are three-dimensionally arranged.

FIG. 4 is a schematic diagram for explaining a measurement principle in a microchannel chip, a measuring device and a measuring method as a second embodiment of the present invention.

FIG. 5 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 a microchannel chip, a measuring device and a measuring method as a second embodiment of the present invention.

FIG. 6 is a schematic diagram for explaining a measurement principle in a microchannel chip, a measuring device and a measuring method as a third embodiment of the present invention.

FIG. 7 is a diagram showing a microchannel chip and a measuring device according to a fourth embodiment of the present invention, FIG. 7A is a schematic perspective exploded view showing an enlarged configuration of the microchannel chip and the measuring device; B) is a schematic plan view showing an enlarged configuration of the microchannel chip in a state where the injection board (covering member) is removed.

FIG. 8 is a schematic diagram for explaining a measurement principle in a microchannel chip, a measuring device and a measuring method as a fourth embodiment of the present invention. It

FIG. 9 is a schematic diagram for explaining the structures of a connecting substance and an immobilizing substance according to another embodiment of the present invention.

FIG. 10 is a schematic view showing a configuration of a microchannel chip and a measuring device as a fifth embodiment of the present invention.

FIG. 11 is a schematic diagram showing the configuration of a comparative example for explaining the effects of the microchannel chip and the measuring apparatus as the fifth embodiment of the present invention.

12 (A) to (F) are schematic cross-sectional views of a flow channel of a microchannel chip as another embodiment of the present invention.

[Explanation of symbols]

5 channels 5A First channel 5B second flow path 5C Third channel 5D reaction channel 5E reaction site 5F Confluence part (mixing part) 5G, 5H channel 6 Injection board (laminated member, covering member) 6A, 6B, 6C, 6E, 26 inlet 6D, 6F outlet 10 specimens 11 Object to be measured 12,12 'labeling substance 12A First specific binding substance 13 Second specific binding substance 14 Competitor 15,15 'linked substances 16,16 'Immobilization material 21 Chip substrate (laminated member) 22 Intermediate substrate (laminated member) 22A, 22B, 22C, 22D Holes 31, 32 Membrane member (laminated member) 31A, 32A, 32B Cutout 32Aa, 32Ab End of cutout 31B, 31C hole 71, 72, 73, 74 Syringe pump 71B, 72B plate 71A, 72A Silicon tube

Claims (12)

[Claims] 1. A microchannel chip for measuring an object to be measured in a sample, comprising: a chip main body; and a minute first microparticle provided in the chip main body for circulating the sample.
A channel, a minute second channel provided in the chip body for flowing a labeling substance having a first specific binding substance that specifically binds to the measurement target, and a second channel provided in the chip body. A minute reaction channel formed by assembling one channel and the second channel, and a second specific binding substance that is provided in the reaction channel and specifically binds to the measurement target are fixed. And a reaction site, wherein the first flow channel, the second flow channel, and the reaction flow channel are three-dimensionally arranged. 2. A microchannel chip for measuring an object to be measured in a sample, comprising: a chip main body;
A flow channel, a minute second flow channel provided in the chip body for allowing a labeling substance having a competitive substance of the measurement object to flow therethrough, and the first flow channel and the second flow channel provided in the chip body. A minute reaction flow channel formed by assembly and a reaction site provided in the reaction flow channel and having a specific binding substance immobilized thereon that specifically binds to the measurement target and the competitive substance by competition are immobilized. A microchannel chip having the above-mentioned structure, wherein the first channel, the second channel, and the reaction channel are three-dimensionally arranged. 3. A microchannel chip for measuring an object to be measured in a sample, comprising: a chip main body;
A flow path, and a minute second flow path provided in the chip main body for flowing a labeling substance having a specific binding substance that the measurement target and a competitive substance of the measurement target compete with each other to specifically bind A minute reaction channel provided in the chip body and formed by collecting the first channel and the second channel, and a reaction site provided in the reaction channel and having the competitive substance immobilized thereon. A microchannel chip having the above-mentioned structure, wherein the first channel, the second channel, and the reaction channel are three-dimensionally arranged. 4. The first flow path and the second flow path are formed so as to relatively approach / assemble along a substantially vertical direction to form the reaction flow path. The microchannel chip according to claim 1. 5. The chip body is formed by stacking a plurality of stacked members.
5. The microchannel chip according to any one of items 4 to 4. 6. A microchannel chip for measuring an object to be measured in a sample, comprising: a chip body, and a micro first microchip provided in the chip body for circulating the sample.
A flow channel, a minute second flow channel provided in the chip main body for circulating a labeling substance having a specific binding substance that specifically binds to the measurement target, and a microscopic second flow channel provided in the chip main body for the measurement target A microscopic third flow path through which a connecting substance that specifically binds is circulated, and a microscopic flow path that is provided in the chip body and is formed by the first flow path, the second flow path, and the third flow path gathering together A reaction channel and a reaction site provided in the reaction channel and having an immobilized substance that specifically binds to the connecting substance immobilized thereon, the first channel, the second channel, A microchannel chip, wherein the third channel and the reaction channel are three-dimensionally arranged. 7. The first flow path and the second flow path are formed so as to relatively approach / assemble along a substantially vertical direction to form the reaction flow path. Item 7. A microchannel chip according to item 6. 8. The chip body is formed by laminating a plurality of laminated members.
Alternatively, the microchannel chip according to the item 7. 9. The microchannel chip according to any one of claims 1 to 5, a flow control means for controlling the flow of the sample and the labeling substance in the microchannel chip, and the microchannel chip bound to the reaction site. A measuring device using a microchannel chip, comprising a measuring means for measuring the labeled substance. 10. The microchannel chip according to any one of claims 6 to 8, a flow control means for controlling the flow of the sample, the labeling substance and the connecting substance in the microchannel chip, and the reaction. A measuring device using a microchannel chip, comprising a measuring means for measuring the labeled substance bound to the site. 11. A measurement method using the microchannel chip according to any one of claims 1 to 5, wherein the sample is circulated in the first channel and in the second channel. The first step of starting the circulation of the labeling substance to mix the sample and the labeling substance in the reaction channel, and bringing the mixture of the sample and the labeling substance into contact with the reaction site of the reaction channel A measuring method using a microchannel chip, comprising a second step and a third step for measuring the labeled substance bound to the reaction site. 12. A measurement method using the microchannel chip according to any one of claims 6 to 8, wherein the sample flows in the first flow path and the sample flows in the second flow path. A first step of starting the circulation of the labeling substance and the circulation of the linking substance through the third flow path to mix the sample, the labeling substance and the linking substance in the reaction flow path; The method comprises a second step of bringing a mixture of the labeling substance and the linking substance into contact with the reaction site of the reaction channel, and a third step of performing a measurement on the labeling substance bound to the reaction site. And a measuring method using a microchannel chip.