JP2007187616A - Analyzing chip and analyzing method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an analyzing chip used in the analysis of the structure/function of protein or the reaction using the protein, and an analyzing method. <P>SOLUTION: The analyzing chip is provided which keeps connected to a reaction chamber through a flow channel on the downstream side of the reaction chamber. A solution containing a reaction product is stored in a storage tank to perform measurement. In this arrangement, the quantitative analysis having reproducibility of the reaction product becomes possible. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention mainly relates to an analysis chip used for analysis of protein structure / function or reaction using protein, and an analysis method thereof. In particular, the present invention relates to an analysis chip used for protein analysis using an antigen-antibody reaction and an analysis method thereof.

  Immunoassay and analysis using a selective binding substance are known to be one of important analytical methods in fields such as medicine and biochemistry. However, the conventional enzyme immunoassay (ELISA) performed using test tubes, microplates, microtubes, microwells, etc. has steps such as introduction of multiple types of reagents, reaction, and washing, and the operation is complicated. Therefore, there is a disadvantage that a long time is required for the analysis, or the amount of reagent to be used is large because the volume of the container is large. Therefore, by using an analysis chip in which microchannels (microchannels) on the order of micrometers are formed on a single substrate, reaction is performed using the characteristics of microvolume, large specific surface area, and short diffusion movement distance. Based on the results and knowledge of improving efficiency and building various chemical systems, as one of them, immunoassay has been applied to analysis chips.

  That is, microbeads are used as an immobilization carrier for immobilizing an antibody or antigen that selectively binds to an antigen or antibody in a sample solution in a microchannel of an analysis chip, and the antibody or antigen is bound to the microbead. As a result, it is possible to continuously introduce and react multiple types of reagents, perform washing, and perform an antigen-antibody reaction efficiently.

  Analysis of proteins using an analysis chip, that is, antigens and antibodies as selective binding substances, is generally performed as follows.

  An antigen or antibody as a selective binding substance for capturing a measurement target component is bound to a solid support such as glass, resin substrate, plastic bead, plastic test tube or paper, and these capture antigen or antibody is immobilized. A liquid containing the antibody or antigen to be identified is added to the carrier. After a certain reaction time, when the capture antigen or antibody reacts with the component to be measured, they are bound to the carrier.

  Examples of the method for detecting the measurement target component include the following examples. That is, a step of previously labeling a measurement target component with a radioactive, fluorescent or enzymatically active labeling substance, a step of reacting these labeled measurement target component with a capture antigen or antibody immobilized on a carrier, and A method including a step of measuring using these radioactivity, fluorescence or enzyme activity, or a step of reacting a component to be measured with a capture antigen or antibody immobilized on a carrier, for a captured detection target Further, there are a method including a step of reacting a detection substance labeled with radioactivity, fluorescence or enzyme activity and a step of measuring using the radioactivity, fluorescence or enzyme activity.

  As a form of a conventional analysis chip, there is a reaction chamber inside the analysis chip, a carrier (solid particles) is accommodated in the reaction chamber, a weir part for damming the carrier is provided, and a component to be measured is immobilized on the carrier, A chip is known in which the immobilized measurement target component is photoanalyzed, or the immobilized measurement target component is reacted with a color reagent solution, and the color reagent solution thus colored is photoanalyzed in the flow path downstream of the reaction chamber. (For example, refer to Patent Document 1).

  In addition, a reaction chamber is provided in a microchip made of glass, a silicon substrate, etc., and a carrier such as a glass bead or polystyrene having a diameter of 1 mm or less as a reaction solid phase for immune antigen-antibody reaction (polymer solid). An immunoassay chip in which the reaction between the antigen and labeled antibody introduced from the microchannel inflow part is carried out on the carrier, unreacted substances are washed, and the reaction products are analyzed by photothermal conversion analysis in the reaction chamber. Is known (see, for example, Patent Document 2).

In these analysis chips, the filling of the carrier is often non-uniform, and when reacted with the component to be measured, the production of the reaction product often differs depending on the location of the reaction chamber. Therefore, when analyzing a component to be measured in a reaction chamber, the measurement value varies depending on the analysis location, and as a result, it is difficult to perform reproducible measurement and further quantitative measurement. there were. In addition, when the carrier filled in the reaction chamber has autofluorescence, there is a limit to the carrier that can be used for measurement because the autofluorescence affects the measurement.
JP 2004-132820 A JP 2001-4628 A

  As described above, reproducible measurement cannot be performed with a conventional analysis chip, or the use of a carrier having autofluorescence as a carrier packed in a reaction chamber is limited, or sufficient detection sensitivity is obtained. There was a problem that could not. The present invention seeks to solve these problems.

In order to solve the above problems, the present invention has the following configuration.
(1) Reaction that has a liquid inlet and a liquid outlet, and stores a carrier on which a selective binding substance that selectively binds to a measurement target component in a sample solution introduced from the liquid inlet is immobilized on the surface An analysis chip having a chamber therein, wherein the analysis chip has a liquid storage tank connected to a downstream side of the reaction chamber through a flow path.
(2) The analysis chip according to (1), wherein a solution containing a reaction product is stored in a liquid storage tank and analyzed in the liquid storage tank.
(3) The analysis chip according to (1), wherein the liquid storage tank is branched and connected from a flow path connecting the reaction chamber and the liquid outlet.
(4) The analysis chip according to (1), wherein a valve for opening and closing the flow path is provided in each flow path connecting the reaction chamber, the liquid outlet, and the reaction chamber and the storage tank.
(5) Using the analysis chip according to (1), selective binding to the measurement target component in the sample solution is performed on the carrier on which the selective binding substance that selectively binds to the measurement target component is immobilized. An analysis method characterized by binding a sex substance and immunologically measuring the component to be measured.

  According to the present invention, the following excellent effects can be obtained.

  Since the solution containing the reaction product can be stored together in the storage tank, the reaction product concentration can be made uniform. By measuring the reaction product in the liquid storage tank, the reaction product can be quantitatively and reproducibly measured. In the optical analysis, by measuring in the liquid storage tank, it is possible to secure a wider area for detecting the optical signal than in the case of measuring in the flow path, so that higher detection sensitivity can be obtained. Further, the carrier to be used is not limited, and a carrier having autofluorescence can also be used.

[Analysis chip]
The present invention accommodates a carrier having a liquid inlet and a liquid outlet, and a selective binding substance that selectively binds to a component to be measured in a sample solution introduced from the liquid inlet is immobilized on the surface thereof. An analysis chip having a reaction chamber therein, wherein the analysis chip has a liquid storage tank on the downstream side of the reaction chamber.

  1 and 2 show an embodiment of the present invention. FIG. 1 is a schematic plan view, and FIG. 2 is an exploded perspective view.

  As shown in FIG. 2, the analysis chip of the present embodiment is configured by laminating an analysis chip lid 2 on a substantially flat plate-shaped analysis chip substrate 1 and integrating them by bonding. A flow path and the like are formed in the analysis chip substrate 1. A reaction chamber 4 is formed between the flow paths 5 and 6 on the liquid inlet 8 side and the liquid outlet 9 side, and a carrier 7 is sealed inside the reaction chamber. Further, a blocking means 3 for the carrier 7 is formed between the reaction chamber 4 and the flow path 5 on the liquid outlet 9 side. Further, a branch portion 13 is provided in the flow path 5 on the liquid outlet 9 side, and a liquid storage tank 10 is provided connected to a flow path different from the liquid outlet 9. Valves 11 and 12 are provided between the branch path 13, the liquid outlet 9 and the liquid storage tank 10.

[Liquid storage tank]
The analysis chip of the present invention has a liquid storage tank. In this liquid storage tank, a solution containing a reaction product reacted in the reaction chamber is stored, and analysis can be performed in the liquid storage tank.

  The shape and size of the liquid storage tank can be appropriately set according to the amount of the reaction product. For example, when the liquid storage tank is cylindrical, the diameter is preferably 0.05 mm or more and 10 mm or less from the viewpoint of the liquid amount of the reaction product and the measurement range, and the depth (column height) is 0 from the same viewpoint. It is preferable that the distance is 0.05 mm or more and 5 mm or less. Furthermore, the volume of the liquid storage tank is preferably 0.01 μl or more and 10 μl or less from the viewpoint of obtaining the amount and handling of the reaction product solution to be stored and sufficient detection sensitivity.

  A valve capable of switching between liquid feeding and liquid feeding stop by opening and closing the flow path may be provided in each of the flow path connecting the reaction chamber and the liquid outlet and the flow path connecting the liquid storage tank. When a valve is provided, during the reaction of the component to be measured and the selective binding substance, the valve on the liquid outlet side downstream of the reaction chamber is opened, the valve on the liquid storage tank side is closed, and the reaction product is released in the reaction chamber. After the production, the valve on the liquid discharge port side is closed and the valve on the liquid storage tank side is opened, and the reaction product solution can be stored in the liquid storage tank by the liquid feeding means for analysis. As the valve for opening and closing the flow path, a known valve capable of switching between liquid feeding / liquid feeding stop may be used. Moreover, you may use what can switch opening and closing of flow paths like a valve instead of a valve. Alternatively, a mechanism such as a three-way cock for switching the liquid feed from the reaction chamber to the liquid outlet or the liquid storage tank may be provided in the flow path branching section connecting the reaction chamber, the liquid outlet and the liquid storage tank.

  The liquid storage tank may be provided with a mechanism for stirring the reaction product such as a stirring bar.

[Liquid feeding means]
The liquid feeding means used in the analysis chip of the present invention is for circulating the measurement target component solution, the selective binding substance solution, and the reaction product solution in the flow path, at a flow rate of microliter / minute to milliliter / minute. A liquid pump such as a micro pump or a syringe type capable of flowing a liquid can be used. Further, the control means for controlling the flow rate of the liquid feeding is preferably provided with a computer and can be automatically controlled according to a preset program, but may be manually controllable.

[Carrier]
The carrier packed in the analysis chip of the present invention is housed in a reaction chamber, and a selective binding substance is immobilized on the surface of the carrier.

  The size of the carrier packed in the analysis chip of the present invention is not particularly limited as long as the carrier can be enclosed in the reaction chamber. The shape of the carrier is preferably granular, and more preferably spherical, from the viewpoint of increasing the specific area for binding of the selective binding substance per unit volume. When the support is spherical, the diameter of the sphere is preferably 0.1 μm or more and 1000 μm or less, and more preferably 10 μm or more and 500 μm from the viewpoint that the support is blocked by the blocking means and the liquid passes through the reaction chamber without any problem. The following is preferable.

  Examples of the shape of the carrier include a granular shape, a film shape, a sponge shape, and a fiber shape. Examples of the carrier material include glass, agarose, silica gel, acrylamide and the like in the case of a granular form, and PVDF and cellulose in the case of a film, and nylon, silk, amyloid fiber and the like in the case of a fibrous form. .

[Selective binding substance]
In the present invention, the selective binding substance is a substance that specifically binds to the component to be measured. Specifically, antibody, antibody fragment, ligand, antigen, hapten, receptor, metal ion, heparin, streptavidin, biotin, benzamidine, glutathione, arginine, calmodulin, gelatin, lysine, mannose, DNA, 2'5'-adenosine -2-phosphate, nucleic acid, enzyme substrate, enzyme inhibitor and the like.

Polyclonal antibodies, monoclonal antibodies, synthetic antibodies, recombinant antibodies, multispecific antibodies (including bispecific antibodies), single-chain antibodies, antibody fragments include Fab fragments, F (ab ′) ₂ fragments, etc. Is included. Polyclonal antibodies can be prepared as specific antibodies by the so-called absorption method, which involves binding a purified polypeptide to a bound affinity column. These selective binding substances can be used as capture selective binding substances that are immobilized on a carrier and capture a measurement component, or they are labeled with some labeling substance and reacted with the measurement component to detect this. It can also be used as a selective binding substance for detection used in the above.

  In addition, the binding mode of the selective binding substance conjugate to the carrier is not particularly limited, and any mode may be employed as long as the selective binding agent conjugate is covalently or non-covalently bound to the surface or inside of the carrier. Alternatively, a selective binding substance conjugate may be mixed as a carrier composition. That is, it is sufficient that the selective binding substance conjugate is not peeled off from the carrier under the environment used in general immunoassay.

[Measuring component]
The measurement target component to be analyzed using the analysis chip of the present invention is not particularly limited, and examples thereof include proteins, nucleic acids, lipids, low molecular compounds, etc. Among them, proteins are desirable. A protein refers to a compound having a structure in which a plurality of amino acids are linked by peptide bonds, and includes natural products, synthetic products, and short-chain peptides. In addition to amino acids, sugars, nucleic acids, and lipids may be included as components.

  The protein that can be analyzed using the analysis chip of the present invention is not particularly limited, and may be a naturally derived product or a synthetic product, but includes components other than amino acids such as nucleoprotein, glycoprotein, and lipoprotein. It may be what you have. Among these, water-soluble proteins are particularly preferably used.

[Analysis method]
The analysis method of the present invention uses the analysis chip of the present invention to select a component to be measured in a sample solution on a carrier on which a selective binding substance that selectively binds to the component to be measured is immobilized. A binding substance is bound and the measurement target component is immunologically measured.

  That is, the analysis method according to the present invention includes a first step of binding a measurement target component and a selective binding substance that selectively reacts with the component to be measured, and a coloring property that is covalently or non-covalently bound to the selective binding substance. Second, analyze the component to be measured by immunological measurement that measures the amount of color, luminescence, radiation, fluorescence intensity, enzyme activity, etc. produced by a luminescent, radioactive, fluorescent or enzymatically active labeling substance Process.

Moreover, as an analysis method using the analysis chip of the present invention, the following methods (1) to (5) can be adopted, but are not limited thereto.
(1) An analysis method in which a sample solution is introduced into a reaction chamber, a measurement target component (A) in the sample solution is bound to a carrier, and a measurement target component (A) bound to the carrier is photoanalyzed. Introduced into the reaction chamber, the measurement target component (A) in the sample solution is bound to the carrier, a solution of the substance (B) that selectively binds to the measurement target component is introduced, and the measurement target component ( Analytical method in which the substance (B) is bound to A) and the substance (B) bound to the carrier via the component (A) is photoanalyzed. (3) The sample solution is introduced into the reaction chamber, and the measurement in the sample solution is performed. The target (A) was bound to the carrier, a solution of the reaction substrate (B) capable of reacting with the measurement target component was introduced, and the target (A) was generated by the reaction of the measurement target component (A) bound to the carrier surface with the reaction substrate (B). Analysis method for photoanalyzing the product (C) (4) Sample solution is introduced into the reaction chamber, The measurement target (A) is bound to the carrier, the substance (B) that selectively binds to the measurement target component (A) is subsequently bound, and the substance (C) that selectively binds to the substance (B) is subsequently bound. Analysis method for photoanalyzing substance (C) bound to carrier via component (A) and substance (B) (5) Component to be measured (A) for selective binding substance bound to carrier Component (A ′) that competitively binds to a sample and a sample, the mixed solution is introduced into a reactant, and the component (A ′) is photoanalyzed.

  Here, the component (A ′) can be a component (A) or a component obtained by binding a labeling substance to a part of the component (A), but is not limited thereto.

[Measuring component]
Components to be analyzed by the analysis chip of the present invention include sputum, saliva, urine, stool, semen, blood, tissues, organs, other body fluids of these subjects, or these body fluids, which are clinical specimens used for diagnosis of diseases. And the like are used. Here, examples of the subject include mammals such as, but not limited to, humans, monkeys, dogs, cats, cows, horses, pigs, sheep, goats, mice, rats and the like.

  Similarly, food, drinking water, soil, drainage, river water, seawater, environmental wiping liquid, environmental wiping cotton exudate, etc. are used as test materials. Moreover, the culture solution of a microbial cell and the microbial cell (colony) cultured on the solid medium can also be used.

[Flow path]
The flow path in the analysis chip of the present invention connects a liquid inlet, a reaction chamber, a liquid outlet, and a water storage tank.

  The flow path in the present invention can be produced by joining a plate-like base material in which a recess is formed with another base material. Alternatively, it can also be produced by joining a slit-shaped thin film penetrating and at least two base materials sandwiching the thin film. The substrate may be a sheet, plate, film, rod, tube, coating film, cylinder, or other complex shaped product, but is not limited thereto. In view of processability and ease of handling, a sheet shape, a plate shape, and a film shape are preferable.

  The width and depth of the flow path of the analysis chip can be appropriately set depending on the size of the analysis chip, the purpose of use, and the like. In the case of a fine flow path, specifically, the width of the flow path is preferably 0.1 μm or more and 1000 μm or less, and more preferably 10 μm or more and 500 μm or less. Specifically, the depth of the fine channel is preferably 0.1 μm or more and 1000 μm or less, and more preferably 10 μm or more and 500 μm or less.

  The length of the flow path can be appropriately selected according to the size of the analysis chip and the compound to be analyzed.

[Reaction room]
The reaction chamber of the present invention contains a carrier on which a selective binding substance is immobilized, and is used for performing a reaction by selectively binding components to be measured. The size of the reaction chamber may be any size as long as the carrier is enclosed. Specifically, the width is preferably 0.1 μm or more and 10,000 μm or less, and more preferably 10 μm or more and 5000 μm or less. The length of the reaction chamber is preferably 10 μm or more and 50 cm or less, and more preferably 100 μm or more and 10 cm or less. The depth of the reaction chamber is appropriately set depending on the size of the analysis chip, the purpose of use, and the like. Specifically, it is preferably 0.1 μm or more and 10,000 μm or less, and more preferably 10 μm or more and 5000 μm or less.

[Liquid inlet and outlet]
The sizes of the liquid inlet and the liquid outlet can be appropriately set according to the volume of the sample. For example, when the liquid inlet and the liquid outlet are cylindrical, the diameter is preferably 0.05 mm or more and 4 mm or less from the viewpoint of sample introduction operation. The depth is preferably 0.05 mm or more and 5 mm or less from the same viewpoint.

[Analysis chip materials]
As the base material used in the analysis chip of the present invention, any of inorganic materials and organic materials such as synthetic resins and natural resins can be used.

  In the case of inorganic materials, for example, iron, nickel, copper, zinc, aluminum, silicon, titanium, tantalum, magnesium, molybdenum, tungsten, rhodium, palladium, silver, gold, platinum, stainless steel, brass, brass, bronze, phosphor bronze, aluminum Metals such as copper alloy, aluminum magnesium alloy, aluminum magnesium silicon alloy, aluminum zinc magnesium copper alloy and iron nickel alloy; metal oxides such as silica gel, alumina, titania, zirconia and tantalum oxide; metal nitriding such as SiN, TiN and TaN Articles: Metal carbides such as SiC and WC; Carbon materials such as diamond and graphite; Glasses such as soda glass, borosilicate glass, Pyrex (registered trademark), and quartz glass.

  As the synthetic resin, either a thermoplastic resin or a thermosetting resin can be used. Examples of these synthetic methods include various known methods, but the resin material of the present invention includes synthetic resins obtained by any of these methods. For example, (1) addition polymer: homopolymer or copolymer of a monomer selected from the group consisting of olefins, vinyl compounds other than olefins, vinylidene compounds and other compounds having a carbon-carbon double bond, or Mixtures or modifications of these homopolymers or copolymers, (2) polycondensates: polymers such as polyesters and polyamides, or mixtures or modifications of these polymers, (3) addition condensates: phenolic resins , A polymer such as urea resin, melamine resin, xylene resin, or a mixture or modification of these polymers, (4) polyaddition product: a polymer such as polyurethane or polyurea, or a mixture of these polymers or Modified product, (5) Ring-opening polymer: cyclopropane, ethylene oxide, propylene oxide, lactone, lacquer (6) Cyclopolymers: divinyl compounds (for example, 1,4-pentadiene) and diyne compounds (e.g., homopolymers or copolymers, or mixtures or modifications of these homopolymers or copolymers) (E.g. 1,6-heptadiyne) or other homopolymers or copolymers, or mixtures or modifications of these homopolymers or copolymers, (7) isomerized polymers: e.g. alternating copolymers of ethylene and isobutene (8) Electropolymers: homopolymers or copolymers such as pyrrole, aniline, acetylene, or mixtures or modifications of these homopolymers or copolymers, (9) aldehyde or ketone polymers, (10) Polyethersulfone, (11) Polypeptide, and the like.

  Examples of natural resins include celluloses, proteins, polysaccharides and the like alone or as a mixture, and modified products thereof.

  In addition, the base resin used in the analysis chip of the present invention includes materials obtained by mixing with glass, metal, carbon material and the like.

  The glass transition temperature of the base material used in the analysis chip of the present invention is not particularly limited, but is preferably 80 ° C. or higher and 200 ° C. or lower from the viewpoint of mass production by injection molding and processing into a chip having a flow path.

[Blackening of analysis chip]
In the analysis chip of the present invention, a black material may be contained or coated on a resin as a base material. Here, black means that the spectral reflectance of the black portion does not have a specific spectral pattern (such as a specific peak) in the visible light (wavelength is 400 nm to 800 nm) range, and is a uniformly low value, and The spectral transmittance of the black portion also has a specific spectral pattern and is uniformly low.

  As the values of the spectral reflectance and the spectral transmittance, the spectral reflectance in the range of visible light (wavelength is 400 nm to 800 nm) is 7% or less, and the spectral transmittance in the same wavelength range is 2% or less. Is preferred. Note that the spectral reflectance here refers to the spectral reflectance when regular reflected light from the substrate is captured in an illumination / light-receiving optical system conforming to JIS Z 8722 Condition C.

  The means for blackening can be achieved by adding a black substance to the base material or insulating material. The black material is not particularly limited as long as it is difficult to reflect or transmit light, but preferred examples include carbon black, graphite, titanium black, aniline black, or Ru, Mn, Ni, Cr, Fe. , Co and / or Cu oxides, or black materials such as Si, Ti, Ta, Zr and / or Cr carbides.

  These black materials can be contained alone or in combination of two or more. For example, when the base material or the insulating material is a polymer such as polyethylene terephthalate, cellulose acetate, polycarbonate, polystyrene, polymethyl methacrylate, or silicon resin, carbon black, graphite, titanium black, and aniline black are preferably included among the black substances. In particular, carbon black can be preferably used. In the case of an inorganic material such as glass or ceramic, an oxide of Ru, Mn, Ni, Cr, Fe, Co and / or Cu, or a carbide of Si, Ti, Ta, Zr and / or Cr can be preferably contained.

[Method for producing analysis chip]
The analysis chip of the present invention can be produced by joining a base material in which a recess is formed with another base material. Or it can produce by pinching | interposing the thin film which has the slit shape penetrated by several base materials, and joining. Examples of a method for obtaining a concave or penetrating slit shape include injection molding, hot embossing, solvent casting, cutting, turning, grinding, etching, punching, photolithography, lift-off, vapor deposition, or a combination thereof. It is not limited. As a bonding method, surface activation by plasma treatment, a method involving heating, a method of applying pressure, a method of applying an electric field, a method of using an adhesive, a method of using a solvent, or a combination thereof may be used. it can.

  The present invention will be described more specifically with reference to the following examples. However, the scope of the present invention is not limited to these examples.

Example 1
In this example, the analysis chip of the present invention was prepared, and human interleukin-8 protein (hIL-8) was measured as a measurement target component.

  By using injection molding, another analysis chip lid 2 made of polymethacryl methacrylate is joined to the analysis chip substrate 1 made of polymethacryl methacrylate having recesses formed in the pattern shown in FIG. A chip was produced.

  The analysis chip produced as described above was immersed in a polyethylene glycol aqueous solution having molecular weights of 500,000 and 2,000 ppm as a hydrophilic polymer. The soaked polymethylmethacrylate plate was sealed and grafted by irradiation with gamma rays of 2.5 kGy.

  As a carrier having a hydrophilic polymer on its surface, 7.5 μl of polystyrene beads (PolyScience) were washed with a phosphate buffer, and 200 μl of 10 μg / ml anti-hIL-8 antibody (Kamakura Technoscience) 0.05 Phosphate buffer containing% Tween 20 was added and allowed to react overnight at 4 ° C. After permeation, the polystyrene beads were washed with 0.05% Tween 20-containing phosphate buffer, 200 μl BlockAce (Dainippon Pharmaceutical Co., Ltd.) was added, and the mixture was allowed to stand for 1 hour for blocking. The blocked polystyrene beads were washed with a phosphate buffer containing 0.05% Tween 20, and 0.1 μl (10 mm in reaction chamber length) was filled in the flow path of the analysis chip.

  After the valve 12 was closed and the valve 11 was opened, the inside of the flow path of the chip was washed with a phosphate buffer containing 0.05% tween 20 and then phosphoric acid containing 0.25% bovine serum albumin and 0.05% tween 20 20 μl of 500 pg / ml hIL-8 (Kamakura Technoscience) dissolved in a buffer solution was injected into the flow path using a syringe pump at a flow rate of 4 μl / min, and subsequently phosphorous containing 0.05% Tween 20 was used. The flow path was washed for 10 minutes by injecting an acid buffer at a flow rate of 15 μl / min. After washing, 20 μl of 8.2 μg / ml HRP (horseradish peroxidase) -labeled anti-hIL-8 antibody (Kamakura Technoscience) dissolved in a phosphate buffer containing 0.25% BSA and 0.05% Tween 20 was added. Injected into the channel using a syringe pump at a flow rate of 4 μl / min, reacted with the previously injected hIL-8, and further added 0.05% Tween 20-containing phosphate buffer at a flow rate of 20 μl / min. It was poured into and washed for 10 minutes. After washing, 0.1 M Tris-HCl buffer (pH 7.5) containing 20 μM hydrogen peroxide solution and 13 μg / ml Amplex Red (Molecular Probes) was introduced into the channel using a syringe pump at a flow rate of 4 μl / min. Injected and allowed to stand for 5 minutes. After standing, the valve 12 is opened, the valve 11 is closed, the generated resorufin is sent to the liquid storage tank 10, and the amount of resorufin generated in the flow path is analyzed using a fluorescence microscope IX-71 (Olympus). did. The measurement conditions were excitation wavelengths of 510 to 560 nm and 575 to 650 nm, and the exposure time was 0.5 seconds.

  Under these measurement conditions, the fluorescence intensity can be measured in the range of 0 to 4096, and usually 1000 to 4000 is a practical range. Therefore, it can be said that there is reproducibility if the variation in fluorescence intensity is about 50 or less.

  Table 1 shows the results of examining the reproducibility of the analysis when the above operation was repeated 5 times. The variation in Example 1 was 32, and it was confirmed that each measurement result was reproducible.

Comparative Example 1
In this comparative example, measurement of human interleukin-8 protein (hIL-8) was performed using the analysis chip shown in FIG. 3 that was prepared in the same manner as in Example 1 and was not provided with a liquid storage tank downstream of the reaction chamber. Went.

  As a carrier having a hydrophilic polymer on its surface, polystyrene beads (PolyScience) are washed with a phosphate buffer, and the same amount of 0.1 μg / ml anti-hIL-8 antibody (Kamakura Technoscience) as polystyrene beads. A phosphate buffer containing 0.05% Tween 20 was added and permeated overnight at 4 ° C. After permeation, the polystyrene beads were washed with a phosphate buffer containing 0.05% Tween 20, and 0.1 μl (10 mm in reaction chamber length) was filled in the flow path of the analysis chip.

  500 pg / ml hIL dissolved in a phosphate buffer containing 0.25% bovine serum albumin and 0.05% Tween 20 after the inside of the chip channel was washed with a phosphate buffer containing 0.05% Tween 20 -8 (Kamakura Techno Science Co., Ltd.) 20 μl was injected into the flow path using a syringe pump at a flow rate of 4 μl / min, and then 0.05% Tween 20-containing phosphate buffer was injected at a flow rate of 15 μl / min. By doing so, the inside of the flow path was washed for 10 minutes. After washing, 20 μl of 0.6 μg / ml HRP (horseradish peroxidase) -labeled anti-hIL-8 antibody (Kamakura Technoscience) dissolved in a phosphate buffer containing 0.25% BSA and 0.05% Tween 20 was added. Injected into the channel using a syringe pump at a flow rate of 4 μl / min, reacted with the previously injected hIL-8, and further added 0.05% Tween 20-containing phosphate buffer at a flow rate of 15 μl / min. It was poured into and washed for 10 minutes. After washing, 0.1 M Tris-HCl buffer (pH 7.5) containing 20 μM hydrogen peroxide solution and 13 μg / ml Amplex Red (Molecular Probes) was introduced into the channel using a syringe pump at a flow rate of 4 μl / min. Injected and allowed to stand for 5 minutes. After standing, the amount of resorufin produced in the reaction chamber was analyzed using a fluorescence microscope IX-71 (Olympus). As shown in FIG. 4, the analysis was performed at measurement points of 2.0, 4.0, 6.0, 8.0, and 10 mm from the upstream end 14 filled with the carrier in the reaction chamber. The measurement conditions were the same as in Example 1.

  Table 2 shows the analysis results when the above operation was repeated 5 times.

  The variation when the measurement at each measurement point was repeated 5 times was in the range of 162 to 715, and no reproducibility was observed. Moreover, the dispersion | variation in each measurement point in each measurement became 436-638.

  From this result, when measurement is performed in the reaction chamber using an analysis chip without a liquid storage tank, spots are generated in the reaction chamber, so the analysis results differ depending on the measurement location, and reproducible analysis is performed. I knew that I couldn't.

It is a plane schematic diagram of one embodiment of the analysis chip of the present invention. It is a decomposition | disassembly schematic diagram of the analysis chip shown in FIG. 6 is a schematic plan view of an analysis chip without a liquid storage tank in Comparative Example 1. FIG. It is an enlarged view of the reaction chamber part of the analysis chip of this invention shown in FIG.

Explanation of symbols

1 analysis chip substrate 2 analysis chip lid 3 damming means 4 reaction chamber 5 flow path (liquid outlet side)
6 Channel (liquid inlet side)
7 Carrier 8 Liquid inlet 9 Liquid outlet 10 Storage tank 11 Valve (liquid outlet side)
12 Valve (Liquid storage tank side)
13 Branching portion 14 Upstream end of carrier in reaction chamber

Claims (5)

  A reaction chamber having a liquid inlet and a liquid outlet and containing a carrier on which a selective binding substance that selectively binds to a measurement target component in a sample solution introduced from the liquid inlet is fixed. An analysis chip comprising: a storage tank connected to a downstream side of a reaction chamber through a flow path.   2. The analysis chip according to claim 1, wherein a solution containing a reaction product is stored in a liquid storage tank and analyzed in the liquid storage tank.   2. The analysis chip according to claim 1, wherein the storage tank is branched and connected from a flow path connecting the reaction chamber and the liquid outlet.   2. The analysis chip according to claim 1, wherein each of the flow paths connecting the reaction chamber and the liquid outlet and the reaction chamber and the liquid storage tank has a valve for opening and closing the flow path.   Using the analysis chip according to claim 1, on the carrier on which the selective binding substance that selectively binds to the measurement target component is immobilized on the surface, the measurement target component and the selective binding substance in the sample solution And analyzing the component to be measured immunologically.

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