JP2008268194A - Analysis method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To effectively prevent the back flow of a reaction liquid or a waste liquid in an analysis method, using a liquid feed chip that utilizes centrifugal force. <P>SOLUTION: The analysis method includes the process of rotating a chip, which has a columnar reaction chamber housing a carrier, having a selective bondable substance bonded there to, and the reagent-specimen reservoir communicating with the reaction chamber, with respect to a rotary shaft which is substantially in the vertical direction provided outside the chip to analyze a substance to be inspected. The angle θ, formed by the rotary shaft for revolution and the reservoir-reaction chamber axis of the chip during stop, is such that 0°≤θ≤80°. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to an analysis method, and more particularly to an analysis method capable of accurate and sensitive analysis.

  Currently, most of the analysis of trace molecules related to clinical diagnosis, food hygiene, and environmental analysis is performed by clinical testing companies and analysis companies. As a trend in the world, simple and rapid diagnosis at the bedside, The importance of conducting analysis and measurement at each processing and import site to prevent accidents in advance and analyzing hazardous substances in rivers and wastes at sites such as rivers and waste treatment plants is drawing attention. ing. Therefore, in recent years, the development of detection methods and analyzers that can be measured simply, quickly, inexpensively and with high sensitivity has been regarded as important.

  In particular, in the analysis for clinical diagnosis, the analysis time is shortened and the amount of the sample required for the analysis is reduced, and at the same time, in order to detect the disease state at an early stage, it is detected with a high sensitivity using a small amount of sample. This is an important issue. In order to solve these problems, use of a small chip called Lab-on-chip (μTAS) has been proposed in immunoassay for analyzing a small amount of a test substance in a specimen. In recent years, a technique related to a chip for feeding liquid using centrifugal force due to such rotation has been developed.

  For example, Patent Document 1, Patent Document 2, Patent Document 3, and Patent Document 4 have a first set of two or more microchannel structures, each having a specific structure. By rotating a disk-type microfluidic device connected by a micro-conduction unit, centrifugal force is used to flow liquid from the reagent / specimen reservoir to the reaction chamber. Techniques for measuring, synthesizing and preparing for disassembly are described. Further, Patent Document 5 describes a technique for rotating a micro system platform in which a fine channel is embedded and inducing fluid motion on the platform by using centripetal force generated thereby.

JP 2005-507762 A JP-T-2006-521558 JP-T-2004-529336 JP-T-2006-524816 JP 2000-514928 gazette

  However, in the above-described prior art, since the structure of the chip is a CD-like disk shape, the reagent / sample reservoir and the reaction chamber are arranged on a substantially horizontal plane. For this reason, there is a problem that when the reagent solution is fed at low speed or stopped, the reaction solution may flow backward from the reaction chamber to the reagent / sample reservoir, resulting in inaccurate measurement. In particular, when a large volume of reagent or sample is used, the possibility of causing a back flow from the reaction chamber to the reagent / sample reservoir increases, so there is a substantial limit to the volume of reagent or sample that can be used.

  Further, in the above-described prior art, the reaction chamber and the waste liquid tank communicating with the reaction chamber are arranged on a substantially horizontal plane, and therefore, when the reagent liquid is fed, the waste liquid from the waste liquid tank to the reaction chamber is stopped when stopped or stopped. There was also a problem that measurement might become inaccurate due to backflow. In particular, when a large volume of reagent or specimen is used, the possibility of causing a back flow from the waste liquid tank to the reaction chamber is increased, so there is a substantial limit on the volume of the reagent or specimen that can be used.

  In view of such conventional problems, the present invention provides an analysis method that can effectively prevent backflow of a reaction solution from a reaction chamber to a reagent / sample reservoir in an analysis method using a liquid-feeding chip utilizing centrifugal force. Another object of the present invention is to provide an analysis method capable of effectively preventing the backflow of waste liquid from the waste liquid tank to the reaction chamber. It is another object of the present invention to provide an analysis method capable of using a reagent or specimen having a large capacity with respect to the volume of the reaction chamber.

The present invention provides the following [1] to [19].
[1] A chip having a column-shaped reaction chamber containing a carrier to which a selective binding substance is bound and a reagent / specimen reservoir communicating with the reaction chamber with respect to a rotation axis in a substantially vertical direction outside the chip. An analysis method including a step of analyzing a test substance by rotating, wherein an angle θ formed by the rotation axis and the reservoir / reaction chamber axis of the stopped chip is 0 ° ≦ θ ≦ 80 °. Analysis method.
[2] A column-shaped reaction chamber containing a carrier to which a selective binding substance is bound, a reagent / specimen reservoir communicating with the reaction chamber, and a waste liquid tank communicating with the reaction chamber on the opposite side of the reagent / sample reservoir The analysis method includes a step of analyzing a test substance by rotating a chip having a rotation axis with respect to a rotation axis in a substantially vertical direction outside the chip, and a reaction chamber of the chip that is stopped The angle θ ′ formed by the waste liquid tank axis is 0 ° ≦ θ ′ ≦ 80 °.
[3] The chip has a waste tank connected to the reaction chamber on the side opposite to the reagent / sample reservoir, and an angle θ ′ formed between the rotating shaft and the reaction chamber / waste tank axis of the stopped chip is 0 ° ≦ The analysis method according to [1], wherein θ ′ ≦ 80 °.
[4] The analysis method according to any one of [1] to [3], wherein a volume ratio of the reagent / sample reservoir to the reaction chamber is 50 or more.
[5] The analysis method according to any one of [1] to [4], wherein the volume ratio of the reagent / sample reservoir to the reaction chamber is 50 to 5 × 10 ⁸ .
[6] The ratio of the projected sectional area of the reagent / specimen reservoir in the liquid feeding direction to the projected sectional area of the reaction chamber in the liquid feeding direction is 50 or more, [1] to [5] The analysis method as described in any one of.
[7] The ratio of the maximum cross-sectional area parallel to the liquid feeding direction of the reagent / sample reservoir and the maximum cross-sectional area parallel to the liquid feeding direction of the reaction chamber is 2 to 400, The analysis method according to any one of [1] to [6].
[8] A step of allowing a liquid containing a test substance to pass through the reaction chamber using centrifugal force generated by rotating the chip, and binding the test substance to a carrier to which the selective binding substance is bound, and The analysis method according to any one of [1] to [7], further including a step of allowing a solution containing a labeling substance to pass through a reaction chamber using a centrifugal force generated by rotating the chip.
[9] Using a centrifugal force generated by rotating the chip, a liquid mixture containing a test substance and a liquid containing a labeling substance is passed through the reaction chamber, and the complex of the test substance and the labeling substance is passed through the reaction chamber. The analysis method according to any one of [1] to [7], which comprises a step of binding to a carrier to which a selective binding substance is bound.
[10] Any one of [1] to [9], wherein in the reaction chamber, the test substance bound to the carrier bound with the selective binding substance is detected using optical means. Analysis method described in 1.
[11] The analysis method according to [10], wherein the optical means is fluorescence measurement.
[12] The analysis method according to [10], wherein the optical means is luminescence measurement.
[13] The analysis method according to [10], wherein the optical means is absorbance measurement.
[14] Any one of [1] to [9], wherein in the reaction chamber, the test substance bound to the carrier to which the selective binding substance is bound is detected using a radiation detection means. Analysis method described in 1.
[15] The analysis method according to any one of [1] to [14], wherein the selective binding substance is an antigen or an antibody.
[16] The analysis method according to any one of [8] to [15], wherein the labeling substance is a labeled antibody.
[17] The analysis method according to any one of [1] to [16], wherein the test substance is a protein.
[18] The analysis method according to any one of [1] to [17], wherein the test substance is a cytokine and / or a chemokine.
[19] The analysis method according to any one of [1] to [18], wherein the test substance is IL-6, IL-8, or TNF.

  According to the analysis method of the present invention, by keeping the reservoir / reaction chamber axis of the chip under analysis at a predetermined angle, as an idea not in the prior art, gravity is used to transfer the reaction chamber to the reagent / sample reservoir. It becomes possible to effectively prevent the reaction solution from flowing backward, and the measurement accuracy is improved. In addition, by maintaining the reaction chamber / waste tank axis of the chip under analysis at a predetermined angle, as an idea not in the prior art, the backflow of waste liquid from the waste tank to the reaction chamber is effectively prevented by using gravity. Measurement accuracy can be improved. In addition, since it becomes possible to effectively prevent the backflow in this way, it is possible to use a reagent or sample having a large capacity with respect to the volume of the reaction chamber, which was substantially difficult with the prior art. Become. By using a large volume of reagents and specimens relative to the reaction chamber volume, it is possible to concentrate the test substance and / or labeling substance in the reaction chamber, resulting in improved measurement sensitivity and detection of trace amounts of test substances. It becomes possible to do.

  In the present invention, the analysis method means a method of analyzing a liquid containing a test substance (hereinafter sometimes referred to as a specimen) using a selective binding substance. The selective binding substance in the present invention means a substance having a property of selectively binding to a test substance directly or indirectly, and representative examples include nucleic acids such as DNA and RNA, Examples include antigens, antibodies, avidin, proteins such as ligands and receptors, and compounds such as biotin and metals. A single-stranded nucleic acid having a specific base sequence selectively hybridizes and binds to a complementary base sequence, and thus corresponds to the selective binding substance referred to in the present invention. Since a nucleic acid having a specific base sequence is known to selectively bind to a protein that recognizes the sequence, it corresponds to the selective binding substance referred to in the present invention. Since an antibody or an antigen selectively binds to the corresponding antigen or antibody, it corresponds to a selective binding substance. Since avidin and streptavidin selectively bind to biotin, they correspond to the selective binding substance in the present invention. Since ligands and receptors also selectively bind to the corresponding receptors and ligands, they correspond to the selective binding substances in the present invention. Biotin is also a selective binding substance because it binds selectively to avidin and streptavidin. Metals such as nickel and cobalt are substances that selectively bind to peptides known as histidine tags, and thus correspond to selective binding substances.

  Among them, the selective binding substance in the present invention is preferably a protein such as an antigen, an antibody, avidin, a ligand or a receptor, and more preferably an antigen or an antibody. When the selective binding substance in the present invention is an antigen or an antibody, the analysis method in the present invention means an immunoassay method using an antigen-antibody reaction, and representatively, ELISA (Enzyme-Linked Immunosorbent Assay) is used. Examples thereof include solid phase enzyme immunoassay (RIA), radioimmunoassay radioimmunoassay (RIA), FIA (fluorescence immunoassay fluorescence immunoassay), and FLISA (fluorescence-linked immunoassay solid phase immunofluorescence assay). When the selective binding substance is an antigen or an antibody, the analysis chip in the present invention is defined as an immunoassay chip, and the analysis method is defined as an immunoassay method.

As an immunoassay method, 1) a direct method for directly recognizing and detecting a target substance with a labeled antibody,
2) An indirect method of recognizing a target substance with an antibody and recognizing and detecting an antibody bound to the target substance with a labeled antibody,
3) Competition method,
4) A two-antibody sandwich method in which a target substance is captured by an immobilized antibody and further detected by another labeled antibody,
5) A three-antibody sandwich method in which a target substance is captured by an immobilized antibody, a target substance is recognized by another antibody, and an antibody that has recognized the target substance is detected by a labeled antibody.
Etc.
Further, a method of detecting a test substance using avidin, streptavidin or the like, such as ABC method, may be used.

  The test substance in the analysis method may be a substance having a property of binding to a selective binding substance such as protein, sugar, lipid, nucleic acid, glycoprotein, glycolipid, or the like. When the selective binding substance is an antigen or an antibody, the test substance in the present invention may be any substance that specifically binds to the antigen or antibody. Examples include cytokines, chemokines, interleukins, allergens, DNA, RNA, antibodies, lipids, enzymes, and other chemical substances. In particular, the test substance in the present invention is preferably a protein that binds to an antigen or an antibody, more preferably a cytokine / chemokine. More preferred are IL-6, IL-8, and TNF. The organism from which the test substance is derived does not matter. The test substance may be one type or two or more types.

  Further, the purpose of the analysis is not particularly limited, and examples include detection of the presence or absence of a test substance in a specimen, quantification of the test substance, and the like. The analysis in the present invention can be used for analysis in clinical tests, food tests, environmental tests and the like.

  The specimen refers to a sample that may contain the test substance, and is preferably a liquid. For example, liquids collected from living bodies including body fluids such as blood, urine, spinal fluid, saliva, sputum, and cell suspension can be exemplified.

  In the analysis method of the present invention, a chip having a column-like reaction chamber containing a carrier that binds a selective binding substance and a reagent / sample reservoir communicating with the reaction chamber (hereinafter sometimes referred to as an analysis chip). ) Includes a step (hereinafter referred to as step (I)) of analyzing a test substance by rotating a rotation axis in a substantially vertical direction outside the chip.

  The analysis chip used in the present invention has a column-shaped reaction chamber that accommodates a carrier that binds a selective binding substance. The shape and size of the reaction chamber need only accommodate a carrier that binds a selective binding substance. The shape is preferably columnar, that is, tubular, and the cross section of the tube is not particularly limited, such as a circle or a polygon. The size of the reaction chamber is desirably narrow to some extent so that the analyte in the sample can be concentrated and detected, and the minor axis of the cross section is usually 0.1 to 1 mm, preferably 0.2 to 0.5 mm. The length is usually 0.5 to 10 mm, preferably 0.5 to 5 mm.

  The reaction chamber contains a carrier to which a selectively binding substance is bound. The number of carriers to be accommodated may be one or more, and it is preferable to accommodate a plurality of carriers from the viewpoint of increasing the efficiency of analysis.

The shape of the carrier may be a so-called microrod such as a cylinder or a polygonal column, or a plate-like microplate, in addition to spherical or elliptical microbeads. Moreover, a porous body may be sufficient.
The size of the carrier depends on the size of the reaction chamber, but it is preferred that the minor axis is 1-1000 μm, preferably the minor axis is 10-200 μm, regardless of the shape of the carrier.

The material of the carrier is not particularly limited, and glass, ceramic (for example, yttrium partially stabilized zirconia), metal (for example, gold, platinum, stainless steel), resin (for example, nylon, polystyrene, polyvinyl alcohol, polymethyl methacrylate, polyacrylamide, etc.), Agarose or the like can be used, and among these, resins, particularly polystyrene, are preferable.
When a plurality of carriers are stored in the reaction chamber, the shape, size, and material of each carrier may be uniform or varied. Further, it is not necessary that the selective binding substance is bound to all the carriers stored in the reaction chamber, and a part of the carrier that does not bind anything may be included.

  When the selective binding substance is an antigen or antibody, the antigen or antibody to be bound to the carrier may be various antibodies, antigen-binding fragments of antibodies such as Fab fragments and F (ab ′) 2 fragments, and various antigens. Among them, an antigen or an antibody that specifically binds to a test substance in a sample in an immunoassay can be appropriately selected, and may be one kind or plural kinds. There are no particular restrictions on the binding density, number of bindings, binding mode, etc. of the antigen or antibody to the carrier.

  There is no particular limitation on the method for binding the selective binding substance to the carrier. For example, when the selective binding substance is an antigen or an antibody, the carrier and the antigen or antibody can be mixed and brought into contact in a solution such as a buffer solution. The contact can be carried out usually under conditions of 1 hour to 24 hours (days) at a low temperature, generally 4 to 37 ° C., with stirring as necessary. The obtained carrier may be washed with a buffer solution, a washing solution or the like before use. The binding method is not limited to this. For example, a method of chemically binding an antigen or antibody and a carrier using a crosslinking agent containing a hydrophilic polymer (polyethyleneimine, polyethylene glycol, polyvinyl alcohol, sodium polysulfonate, etc.). Etc. can also be used. Alternatively, avidin and biotin may be used to bind to the carrier.

A liquid containing a test substance for analysis and a liquid containing a labeling substance are fed into the reaction chamber.
The label in the present invention means an enzyme such as a fluorescent substance, a luminescent substance, a light absorbing substance, a radioactive substance, horseradish peroxidase, alkaline phosphatase, luciferase, beta galactosidase, glucose oxidase and the like.

The labeling substance in the present invention means a substance having a label and capable of binding directly / indirectly to a test substance or a selective binding substance. Preferably, a labeled antibody such as an enzyme-labeled antibody, a radioactive substance-labeled antibody, or a fluorescent-labeled antibody is used. When an enzyme-labeled antibody is used, a solution containing an enzyme substrate is fed by rotation, and fluorescence, light absorption, luminescence, and the like generated as a result of the enzyme reaction can be detected.
Further, the test substance may have a label, and can be bound to the selective binding substance competitively with the test substance whose concentration in the sample is unknown. The test substance is as described above. In addition, a cleaning solution or the like can be passed as necessary.

  In the step (I), a liquid containing a test substance or a solution containing a labeling substance is sent to the reaction chamber by rotating the above-described chip around a rotation axis in a substantially vertical direction outside the chip.

  In the analysis method of the present invention, the rotation axis refers to a rotation axis in a substantially vertical direction outside the analysis chip. In other words, the rotation axis is outside the chip and points in a substantially vertical direction, but at least the reaction chamber of the analysis chip needs to rotate (revolve) along a trajectory centered on the rotation axis. The trajectory may be substantially circular, and may be an elliptical trajectory. Here, the “substantially vertical direction” means the same direction as the gravitational direction or a direction having a minute difference (for example, 0 to 5 °).

  The reagent / sample reservoir in the analysis method of the present invention means an area for holding a reagent or sample, a cleaning solution, a labeling substance, a substrate, or the like that is sent to the reaction chamber by centrifugal force. The reagent / specimen reservoir is connected to the reaction chamber directly or via a flow path, and has a “reservoir outlet” on the reaction chamber side for feeding the reaction chamber.

  In the analysis method of the present invention, the liquid flows into the reaction chamber of the analysis chip from the reagent / specimen reservoir through the “reservoir outlet” and directly or via a flow path by centrifugal force. The inflowing liquid passes through the reaction chamber and is discharged from the “reaction chamber outlet”.

  The analysis chip used in the analysis method of the present invention may have a waste liquid tank communicating with the reaction chamber on the side opposite to the reagent specimen reservoir. In this case, the reaction chamber and the waste liquid tank can be connected directly from the “reaction chamber outlet” or via a flow path.

  The waste liquid tank in the analysis method of the present invention means a region that holds a solution such as a reagent or a specimen, a cleaning liquid, a labeling substance, and a substrate that are sent from a reaction chamber through a “reaction chamber outlet” by centrifugal force. . The waste liquid tank is connected to the “reaction chamber outlet” directly or via a flow path, and has a “waste liquid tank inlet” on the reaction chamber side in order to allow the solution sent from the reaction chamber to flow in.

  The “reservoir / reaction chamber axis” in the present invention means an axis connecting the “reservoir outlet” and the “reaction chamber outlet” of the chip whose rotation is stopped. For example, the “reservoir / reaction chamber shaft” 103 in the chips J, K, L, M, N, and P in the rotation stop state illustrated in FIGS. 11 to 17 connects the “reservoir outlet” 101 and the “reaction chamber outlet” 102. Expressed as an axis.

  In the present invention, the angle θ formed between the “reservoir / reaction chamber axis” of the chip whose rotation is stopped and the rotation axis in the substantially vertical direction is adjusted to 0 ° ≦ θ ≦ 80 °. This makes it possible to effectively prevent the reaction solution in the reaction chamber from flowing back into the reservoir by using gravity as an idea not found in the prior art, and as a result, the measurement accuracy can be improved. Become. Furthermore, since it is possible to effectively prevent backflow even if a large amount of reagent or sample is used with respect to the volume of the reaction chamber, the test substance can be concentrated in the reaction chamber, so the measurement sensitivity is high. It is possible to improve and detect a very small amount of a test substance.

  The angle θ formed by the “reservoir / reaction chamber axis” and the rotation axis in the substantially vertical direction is preferably 0 ° ≦ θ ≦ 60 °, so that more effective backflow prevention can be achieved.

  In the present invention, the “reaction chamber / waste liquid tank axis” means an axis connecting the “reaction chamber outlet” and the “waste liquid tank inlet” of the chip whose rotation is stopped. For example, the “reaction chamber / waste liquid tank axis” 106 in the rotation-stopped chip P shown in FIG. 17 is represented as an axis connecting the “reaction chamber outlet” 102 and the “waste liquid tank inlet” 105.

  In the present invention, when an analysis chip having a waste liquid tank is used, the angle θ ′ formed between the “reaction chamber / waste liquid tank axis” and the rotation axis in the substantially vertical direction is adjusted to 0 ° ≦ θ ′ ≦ 80 °. It is preferable to do. Thereby, as an idea not in the prior art, by utilizing gravity, it is possible to effectively prevent the waste liquid in the waste liquid tank from flowing back into the reaction chamber. As a result, measurement accuracy can be improved. Furthermore, since it is possible to effectively prevent backflow even if a large amount of reagent or sample is used with respect to the volume of the reaction chamber, the test substance can be concentrated in the reaction chamber, so the measurement sensitivity is high. It is possible to improve and detect a very small amount of a test substance.

  The angle θ ′ formed between the “reaction chamber / waste liquid tank axis” and the rotation axis in the substantially vertical direction is preferably 0 ° ≦ θ ′ ≦ 60 °, because it is possible to prevent backflow more effectively.

  Furthermore, when a tip designed to satisfy the above angle ranges is used when the tip is mounted at an angle of a commercially available centrifuge, the present invention can be easily implemented using a commercially available centrifuge. it can.

  Further, the angle θ formed by the “reservoir / reaction chamber axis” of the chip whose rotation is stopped and the rotation axis in the substantially vertical direction is 0 ° ≦ θ ≦ 80 °, and is substantially the same as the “reaction chamber / waste liquid tank axis” whose rotation is stopped. By setting the angle θ ′ formed by the vertical rotation axis to 0 ° ≦ θ ′ ≦ 80 °, it is expected that the backflow prevention effect is further enhanced.

  The angle in the analysis method of the present invention will be described with reference to FIGS. 11 to 17 are diagrams showing an example of a chip used in the analysis method of the present invention. The chips J, K, L, M, N, and P in FIGS. 11 to 17 all have a column-shaped reaction chamber 11 and a reagent / sample reservoir 12, and the reaction chamber 11 and the reagent / sample reservoir 12 are , “Reservoir outlet” 101.

  FIGS. 11A and 11B are diagrams illustrating a “reservoir / reaction chamber axis” 103 defined by a “reservoir outlet” 101 and a “reaction chamber outlet” 102 in the stopped chip J, and a substantially vertical position outside the chip. The positional relationship with the rotating shaft 104 in the direction is shown, and the angle between the two is 0 ° ≦ θ ≦ 80 °. FIG. 11A shows a state in which the “reservoir / reaction chamber axis” 103 and the rotation axis 104 are parallel to each other, so that an angle θ between them is θ = 0 °. On the other hand, FIG. 11-2 shows a state in which the angle θ formed by the “reservoir / reaction chamber axis” 103 and the rotation shaft 104 forms an angle of 0 ° <θ ≦ 80 °.

  FIG. 12 shows the positions of the “reservoir / reaction chamber axis” 103 defined by the “reservoir outlet” 101 and the “reaction chamber outlet” 102 in the stopped chip K, and the substantially vertical rotation axis 104 outside the chip. This shows the relationship, in which the angle θ between them is 0 ° ≦ θ ≦ 80 °.

  FIG. 13 shows the positions of the “reservoir / reaction chamber axis” 103 defined by the “reservoir outlet” 101 and the “reaction chamber outlet” 102 in the stopped chip L, and the substantially vertical rotation axis 104 outside the chip. Since the “reservoir / reaction chamber axis” 103 and the rotation axis 104 are parallel to each other, the angle θ formed by both is θ = 0 °.

  FIG. 14 shows the positions of the “reservoir / reaction chamber axis” 103 defined by the “reservoir outlet” 101 and the “reaction chamber outlet” 102 in the stopped chip M, and the rotation axis 104 in the substantially vertical direction outside the chip. This shows the relationship, in which the angle θ between them is 0 ° ≦ θ ≦ 80 °.

  FIG. 15 shows the positions of the “reservoir / reaction chamber axis” 103 defined by the “reservoir outlet” 101 and the “reaction chamber outlet” 102 in the stopped chip N, and the rotation axis 104 in the substantially vertical direction outside the chip. This shows the relationship, in which the angle θ between them is 0 ° ≦ θ ≦ 80 °.

  FIG. 16 shows the positions of the “reservoir / reaction chamber axis” 103 defined by the “reservoir outlet” 101 and the “reaction chamber outlet” 102 in the stopped chip M, and the substantially vertical rotation axis 104 outside the chip. FIG. 14 shows a relationship in which the angle θ between the two is different from the direction of the chip M in FIG. 14 but is in the range of 0 ° ≦ θ ≦ 80 °.

  FIG. 17 shows the positions of the “reservoir / reaction chamber axis” 103 defined by the “reservoir outlet” 101 and the “reaction chamber outlet” 102 in the stopped chip P, and the rotation axis 104 in the substantially vertical direction outside the chip. This shows the relationship, in which the angle θ between them is 0 ° ≦ θ ≦ 80 °. Further, the “reaction chamber / waste liquid tank axis” 106 defined by the “reaction chamber outlet” 102 and the “waste liquid tank inlet” 105 in the stopped chip P, and the substantially vertical rotation shaft 104 outside the chip. The positional relationship is shown, and the angle between the two is θ′0 ° ≦ θ ′ ≦ 80 °.

  The flow path in the analysis method of the present invention means a flow path having a length of 1 μm or more, preferably 10 μm or more.

  The most preferable chip as described above has a reaction chamber containing a carrier to which a selective binding substance is bound, a reagent / specimen reservoir communicating with the reaction chamber, an angle rotor and / or a swing of a centrifuge An analysis chip that can be attached to a rotor is an example.

  As the first type of the analysis chip, one that can be inserted and fitted into the centrifuge tube can be cited. That is, it may be a type that can be attached to the angle rotor and / or swing rotor of the centrifuge while being fitted inside the centrifuge tube. In the case of this type of tip, when the tip of the present invention is put in the centrifuge tube, the tip is fixed to the inner wall surface of the centrifuge tube. In other words, any point or surface on the outer surface of the analysis chip is fixed at any point or surface on the inner wall surface of the centrifuge tube. Typical examples include analysis chips A, B, C, D, F, and Q shown in FIGS. 1 to 4, 7, and 18, and analysis chips J, K, and L shown in FIGS. , M, N. These tips can be used by being inserted into a centrifuge tube G as shown in FIG. 8 and fitted in such a manner that the corner 15 on the side surface of the tip is stretched and fixed to the inner wall of the centrifuge tube.

  On the other hand, the second type of the analysis chip includes a type that can be directly mounted on a chip mounting site such as an angle rotor of a centrifuge or a centrifuge tube of a swing rotor. Typical examples include an analysis chip E shown in FIGS. 5 and 6 and an analysis chip P shown in FIG.

  Here, the centrifuge tube in the present invention means a so-called test tube (usually a cap tube) that can be applied to a centrifuge, and includes all those called centrifuge tubes, conical tubes, Eppendorf tubes, and the like. The centrifuge is not particularly limited as long as it is compatible with a centrifuge tube, and a small desktop centrifuge may be used as long as the centrifuge tube can be attached.

  The shape and size of the reaction chamber of the analysis chip need only be able to accommodate a carrier to which a selective binding substance is bound. The shape is preferably tubular, and the cross section of the tube is not particularly limited, such as a circle or a polygon. The smaller the reaction chamber size, the smaller the amount of the carrier to which the selective binding substance is bound, the cost can be reduced, and the volume ratio with the reservoir can be easily increased, so that it can be mounted on a commercially available centrifuge. A large concentration effect can be obtained by size. The volume of the reaction chamber is usually 1 nL to 100 μL, preferably 10 nL to 10 μL. More preferably, it is 100 nL-1 microliter.

  The reaction chamber in the analysis chip will be described based on the examples shown in FIGS. The analysis chip A in FIG. 1 has a reaction chamber 11 connected to a later-described reagent / sample reservoir 12 via a “reservoir outlet” 101, and is provided on the opposite side of the opening 12 A of the reagent / sample reservoir 12. It has a “reaction chamber outlet” 102. The reaction chamber 11 contains a carrier 13 for binding a selectively binding substance. The reaction chamber 11 of the analysis chip E in FIGS. 5 and 6 is provided adjacent to the central tube wall of the analysis chip E, and the “reaction chamber” is located on the opposite side of the opening 12A of the reagent / sample reservoir 12. It has an “exit” 102.

  In the analysis chip, a carrier damming means 11A ′ can be provided at the outlet of the reaction chamber, so that the carrier can be kept from leaking from the chip. As the damming means, for example, a wire mesh or a filter can be used. In the case of a wire mesh, a wire mesh of an appropriate size (for example, an opening having a size of 20 μm × 20 μm) can be pressed into the opening to form a damming means. In the case of a filter, it can be obtained by press-fitting a cellulose acetate filter or the like into the opening. The damming means is not limited to a wire mesh, and a filter, a cap, or the like can be used.

  A case where the filter is blocked with a filter as a carrier blocking means will be described with reference to the embodiments shown in FIGS. FIG. 2 is a diagram schematically showing a longitudinal section of an analysis chip B preferably used in the present invention. The carrier blocking means 11A ′ is wider than the reaction chamber 11, and a filter is press-fitted into this portion. Also, the same damming means 11A ′ is provided in the analysis chips D and F of FIGS. In addition, it is not necessary that the width is wide when providing the damming means. The “reaction chamber outlet” 102 such as the analysis chip A in FIG. 1 or the “reaction chamber outlet” of the analysis chip E shown in FIGS. ”102 or the“ reaction chamber outlet ”102 of the analysis chips J, K, L, M, N, and P shown in FIGS. 11 to 17 can be provided with a damming means (not shown) by pressing the wire mesh. .

  In the analysis chip, a carrier in which a selective binding substance is bound is accommodated in a reaction chamber. The definitions of the selective binding substance, the carrier and the like are as described above. The method for accommodating the carrier having the selectively binding substance bound in the reaction chamber of the chip body can be performed, for example, as follows. That is, the beads to which the selective binding substance is bound are suspended in a buffer solution containing a surfactant and / or a blocking agent, and this suspension is poured into the sample / reagent reservoir of the chip body, and then the chip body. Is inserted into a centrifuge tube and centrifuged in a centrifuge for 10 seconds to 1 minute.

  Since the analysis chip includes a reagent / sample reservoir, the reaction can be performed simply by holding the liquid containing the test substance or the liquid containing the labeling substance in the reagent / sample reservoir and then applying the centrifuge. By making the reaction chamber narrower, the storage density of the carrier can be improved and the efficiency of analysis can be increased.

  The analysis chip A in FIG. 1 is provided with a reagent / sample reservoir 12 connected to the reaction chamber 11 via a “reservoir outlet” 101, and the reagent / sample reservoir 12 has an opening 12A on the outside. Similarly, in the analysis chip E of FIGS. 5 and 6, the reagent / sample reservoir 12 is similarly connected to the reaction chamber 11 through a “reservoir outlet” 101.

The volume (size) of the reagent / sample reservoir is preferably larger than that of the reaction chamber. This is because the reaction chamber is sufficiently smaller than the reservoir to provide a concentration effect of the test substance, and it is easy to hold the reagent and specimen in the reservoir. Specifically, for example, the volume ratio of the reagent / sample reservoir to the reaction chamber is usually 50 or more, preferably 50 to 5 × 10 ⁸ , particularly preferably 100 to 3000. In the conventional technology in which the analysis mechanism is integrated on the disk, the limit is about 15 times. By setting the above range, the concentration effect of the test substance is very large compared to the conventional technology. It becomes.
The volume of the reagent / sample reservoir is generally 30 μL to 500 mL, and preferably 30 μL to 1000 μL (1 mL).

  On the other hand, the ratio of the projected sectional area of the reagent / sample reservoir in the liquid feeding direction (area when projected onto a plane perpendicular to the liquid feeding direction) to the projected sectional area of the reaction chamber in the liquid feeding direction is usually 50. Or more, preferably 100 or more. Moreover, generally an upper limit shall be 10,000 or less. In the case of the conventional technology in which the analysis mechanism is integrated on the disk, the entire apparatus has a two-dimensional structure, so that the projected cross-sectional area ratio in the liquid feeding direction between the reservoir and the reaction chamber can only differ by a factor of about 10 times at most. However, since the analysis chip used in the present invention has a three-dimensional structure, the area ratio can be increased and the concentration efficiency of the test substance can be remarkably improved.

Further, the ratio of the maximum cross-sectional area parallel to the liquid feeding direction of the reagent / sample reservoir and the maximum cross-sectional area parallel to the liquid feeding direction of the reaction chamber is determined from the viewpoint of obtaining the concentration effect of the test substance. 2 to 400 is preferable. The maximum cross-sectional area parallel to the liquid feeding direction of the reagent-analyte reservoir is preferably a 10 mm ² to 200 mm ^2. The maximum cross-sectional area parallel to the liquid feeding direction of the reaction chamber is preferably set to 0.5 mm ² to 5 mm ^2.

  The shape of the reagent / sample reservoir is not particularly limited, and can be appropriately selected from various shapes such as a cylindrical shape and a polygonal shape. However, from the viewpoint of smoothly feeding the reagent or sample into the reaction chamber by centrifugal force, the area of the cross section However, it is preferable that the shape gradually narrows toward the side connected to the reaction chamber. For example, the shape of the reagent / sample reservoir 12 in the analysis chip A shown in FIG. 1 is basically a rectangular parallelepiped, with four corners on the reaction chamber 11 side being rounded. On the other hand, the shape of the reagent / sample reservoir 12 in each of the immunoassay chips B, C, and D shown in FIGS. 2, 3 and 4 is basically a rectangular parallelepiped, and the four side surfaces are the reaction chamber side. It is squeezed towards. The shape of the reagent / sample reservoir 12 of the analysis chips E and Q of FIGS. 5, 6 and 18 is the same as that of the embodiment shown in FIGS.

  The reagent / sample reservoir may be directly connected to the reaction chamber, or may be connected via a flow path. For example, the reagent / sample reservoir 12 and the reaction chamber 11 may be directly connected as in the analysis chip A of FIG. 1, the chip L of FIG. 13, the chip M of FIGS. 14 and 16, the chip N of FIG. As in the chip P of FIG. 17, the reservoir outlet 101 of the reagent / sample reservoir 12 and the reaction chamber 11 may be connected by a flow path 200. The reagent / sample reservoir and the reaction chamber may be coupled so that the central axes in the liquid feeding directions are parallel to each other, or may be coupled at an angle. For example, in the chip K of FIG. 12, the reaction chamber 11 is connected to the reagent / sample reservoir 12 at an angle at which the “reaction chamber outlet” 102 opens toward the outer peripheral side of the rotation shaft.

  The reagent / sample reservoir has an opening on the side opposite to the reaction chamber. Although the shape of the opening to the outside is not particularly limited, it can be appropriately determined in such a size that the injected reagent or specimen does not leak out. For example, the minor axis can be appropriately determined to be in the range of 1 mm to 100 mm, preferably 1 to 20 mm.

  The analysis chip may further include a waste liquid tank. By providing the waste liquid tank, it is possible to prevent the waste liquid from being scattered in the centrifuge. The waste liquid tank is connected to the reaction chamber directly or through a flow path. When the damming means is provided as described above, the waste liquid tank is connected to the tip of the damming means.

  For example, in the case of an analysis chip of a type that can be inserted and fitted into the centrifuge tube as shown in FIGS. 1 to 4, when it is attached to the centrifuge tube as shown in FIG. The formed space can be the waste liquid tank 16. On the other hand, although not shown in FIGS. 1 to 4, it is possible to provide a damming means for the carrier at the “reaction chamber outlet” 102 and attach a bag or container to form a waste liquid tank. In the case of the chip P in FIG. 17, the reaction chamber 11 and the waste liquid tank inlet 105 of the waste liquid tank 16 are connected by a flow path 200A.

  In the case of an analysis chip of the type that can be directly mounted on an angle rotor or swing rotor as shown in FIGS. 5, 6, and 17, the waste liquid tank 16 can be provided in the chip.

  In addition, in the case of a chip that can be inserted and fitted into the centrifuge tube among the above analysis chips, a protrusion may be further provided on the outer wall. When the tip is fitted to the centrifuge tube by providing the protrusion, the tip is fitted with the protrusion in contact with the wall of the centrifuge tube, and a space as a waste liquid tank as described above is formed at the bottom of the centrifuge tube. Can be secured. In addition, the chip can be easily removed from the centrifuge tube. Furthermore, when the chip is taken out from the centrifuge tube, the chip can be taken out and analyzed in a state where the waste liquid remains in the centrifuge tube, so that the influence of measurement noise derived from the waste liquid can be eliminated.

  The protrusion may be provided on the outer wall of the chip, and its shape and position are not specified. A position and shape that can be fixed at an arbitrary point or surface on the inner wall surface of the centrifuge tube when the tip is placed in the centrifuge tube can be appropriately determined. Examples of the manner of fixing on the inner wall surface of the centrifuge tube include a mode in which the tip is suspended and fixed, and a mode in which the tip is raised to the bottom of the centrifuge tube via a scaffold.

  For example, the protruding portion can be provided on the side surface of the tip, and the tip can be suspended and fixed by the contact or contact surface between the inner wall surface of the centrifuge tube and the protruding portion. Specifically, an ear protruding in the vertical direction can be provided around the opening of the reagent / sample reservoir. In the analysis chip D of FIG. 4, an ear 14A is provided around the opening 12A. The shape and size of the ear can be determined as appropriate in relation to the size of the centrifuge tube.

  Moreover, a protrusion can be provided on the bottom surface of the chip and extended to the bottom surface of the centrifuge tube to fix the chip. Specifically, a leg extending from the bottom surface of the reagent / sample reservoir can be provided. In the analysis chip F of FIG. 7, two legs 14B from which the chip extends from the bottom surface of the chip are provided. When the analysis chip F is attached to the centrifuge tube, the bottom is raised by the two legs 14B, and the space between the bottom surface of the centrifuge tube and the chip (mainly the space 14C between the legs 14B) is secured as a waste liquid tank.

  In the case of a chip of the type that fits inside the centrifuge tube among the analysis chips, it is preferable to detachably fit the centrifuge tube for the convenience of subsequent operations.

The size and shape of the chip body of the analysis chip can be determined appropriately depending on the type.
That is, in the case of a chip that is detachably fitted to the centrifuge tube, the size of the chip body can be determined in consideration of the fact that it can be inserted and fitted into the centrifuge tube as described above. The size of the centrifuge tube generally used is 8 to 40 mm in short diameter and 5 to 120 mm in height. Therefore, for example, the short diameter of the chip body is usually 6 to 40 mm and the height is usually 5 to 120 mm. It can be determined within the range. Since a centrifuge tube having a volume of 0.5 ml to 2.5 ml, such as an Eppendorf tube, which is preferable as a centrifuge tube because of its smaller size, its size is 8 to 10 mm. The size can be determined in the range of 6 to 10 mm for the minor axis, 5 to 30 mm for the height, and more preferably 5 to 15 mm for the height. On the other hand, in the case of a type of chip that can be directly mounted on the centrifuge tube mounting portion of the centrifuge, the size may be adjusted to the size of the rotor of the mounting centrifuge.

  Further, the shape of the chip body of the analysis chip can be determined in consideration of the ease of molding. For example, in the case of a chip that is detachably fitted to a centrifuge tube, it can be selected from a polygonal column shape such as a triangular column or a quadrangular column, a cylindrical shape, a pyramid shape, a conical shape, or the like. In the embodiments shown in FIGS. 1 to 4 and 18, all have a cubic shape. On the other hand, in the case of a tip that can be directly attached to the centrifuge tube's angle rotor or swing rotor's centrifuge tube mounting site, the shape matches the size of the centrifuge's rotor, that is, the same shape as the centrifuge tube. If it is.

  The material of the analysis chip is not particularly limited, and examples thereof include resin and glass. In particular, from the viewpoint of facilitating observation of the reaction chamber from the outside, it is preferable that at least a part of the reaction chamber is transparent. By making at least a part of the reaction chamber transparent, a more concentrated test substance can be easily detected. Therefore, it is preferable to use a transparent material for a part of the reaction chamber, and it is particularly preferable to form the whole from a transparent material. Further, the surface of the reaction chamber made of a transparent material may be a flat surface or a lens shape (concave surface). The chip shown in FIG. 18 includes a chip part A 107 that can hold the shape and a chip part B 108 that is a thin transparent material. It is possible to reduce auto-fluorescence from the material by thinning the chip component B, and it is easier to detect from the outside by observing from the chip component B 108 side as shown on the right side of FIG. It is possible. In addition, the material of the chip component A located on the opposite side to the observation side with respect to the reaction chamber 11 may reduce autofluorescence by mixing carbon black or the like with resin.

  Examples of the transparent material include various organic materials and inorganic materials, such as polymethyl methyl methacrylate (PMMA), polycarbonate, polypropylene, polyethylene, polymethylpentene, polystyrene, polytetrafluoroethylene, ABS resin, polydimethyl. Resins such as siloxane and silicon, copolymers or composites containing such polymer compounds; quartz glass, pyrex (registered trademark) glass, soda glass, borate glass, silicate glass, borosilicate glass, and the like; The composite; a metal whose surface is coated with an insulating material, a composite thereof, a ceramic, a composite thereof, and the like are preferably used. Of these, polymethyl methyl methacrylate (PMMA) is particularly preferably used.

  Moreover, it is preferable that the reaction chamber and the surface of the reagent / specimen reservoir are subjected to an adsorption suppression treatment for suppressing nonspecific adsorption of biomolecules. Adsorption suppression treatment methods include a coating treatment that electrostatically adsorbs hydrophilic polymer material to the surface, a method of irradiating high-energy rays and covalently bonding the hydrophilic polymer to the resin surface to immobilize it firmly. Is used.

In the analysis method of the present invention, the above-described step (I), that is, the step of analyzing the test substance by rotating the above-described chip with respect to the rotation axis in the substantially vertical direction outside the chip includes the following ( It is preferable to carry out by the two steps a) and (b).
(A) passing a liquid containing a test substance through the reaction chamber using centrifugal force generated by rotating the chip, and binding the test substance to a carrier to which the selective binding substance is bound; and (B) A step of passing a solution containing a labeling substance through a reaction chamber using centrifugal force generated by rotating the chip.

Moreover, the above-mentioned process (I) may be implemented by the following 1 process (A).
(A) Using a centrifugal force generated by rotating the chip, a liquid mixture containing a test substance and a liquid containing a labeling substance is passed through the reaction chamber, and the complex of the test substance and the labeling substance is passed through the reaction chamber. A step of binding a selective binding substance to a bound carrier

  In the analysis method of the present invention, after the above-described step (I), the test substance bound to the carrier to which the selective binding substance is bound can be subsequently detected in the reaction chamber of the chip. The detection of the test substance is preferably performed using an optical means, a radiation detection means, or the like, and particularly preferably performed using an optical means. Examples of the optical means include fluorescence measurement, luminescence measurement, and absorbance measurement, and preferably fluorescence measurement.

  Hereinafter, in the analysis method of the present invention, an analyzer having a reaction chamber containing a carrier bound with a selective binding substance is rotated with respect to a rotating shaft outside the analyzer, whereby the specimen and reagent are reacted with the reaction. An example of a procedure for measuring the amount of a test substance in the reaction chamber (procedure for obtaining each measurement result) by analyzing the cytokine by the ELISA method using the analysis chip The explanation will be given with reference to FIG. In the reaction chamber of the analysis chip, primary antibody adsorption beads of cytokine are accommodated.

  When the analysis method of the present invention is performed using the analysis chip, it can be performed as follows. Hereinafter, a description will be given with reference to FIG.

When the step (I) of the present invention is performed by the two steps (a) and (b), it is performed as follows.
For step (a), first, a liquid (specimen) containing a test substance is injected into the specimen / reagent reservoir ((1) in FIG. 9), and then the specimen is transferred to the reaction chamber by centrifugal force (FIG. 9). (2) An antigen-antibody reaction is carried out with a selective binding substance on the bead carrier ((3) in FIG. 9). That is, the analysis chip after sample injection is inserted into a centrifuge tube, and this centrifuge tube is set in a centrifuge and rotated. By this process, the sample is transferred from the sample / reagent reservoir to the reaction chamber by centrifugal force, and the reaction is performed at the same time.

  Subsequently, for step (b), after injecting a solution containing a labeled antibody having an enzyme label or a fluorescent label into the specimen / reagent reservoir ((1) in FIG. 9), this substrate is similarly subjected to a reaction chamber by centrifugal force. (2 in FIG. 9) and reacted with the test substance bound to the selective binding substance on the bead carrier ((3) in FIG. 9). That is, in the transfer of the sample, centrifugation is performed in the same manner except that the sample is replaced with a solution containing a labeled antibody.

  After the step (b), the test substance bound to the carrier to which the selective binding substance is bound is detected in the reaction chamber. That is, the fluorescence intensity in the reaction chamber is measured by a fluorescence detection device (fluorescence microscope or the like) while the chip is taken out from the centrifuge tube or the chip is inserted into the centrifuge tube. When the label of the labeled antibody is an enzyme label, a reagent serving as an enzyme substrate is transferred to the reaction chamber by centrifugal force, and a fluorescent substance or a luminescent substance is generated by the action of the enzyme. The luminescence or fluorescence intensity is measured. In the case of detecting the presence or absence of a cytokine in a sample as a test substance, if the fluorescence intensity can be measured, it is confirmed that the cytokine is present in the sample. On the other hand, in the case of immunoassay for the purpose of quantifying cytokines in a sample, the concentration of cytokines is specified in comparison with a calibration curve prepared by measuring the concentrations of cytokines in advance in the same manner.

  On the other hand, the step (I) of the present invention is the step (A), a liquid obtained by adding a labeled antibody to a liquid containing a test substance using a centrifugal force generated by rotating the chip, and a liquid containing a test substance. And a liquid mixture containing a labeling substance are allowed to pass through the reaction chamber and the complex of the test substance and the labeling substance is bound only to the carrier to which the selective binding substance is bound. In the description of the step (a), it can be carried out by injecting a liquid added with a labeling substance (labeled antibody) or the like into the specimen instead of the specimen.

  As described above, in the analysis method of the present invention, the operations in the case of using the above-described analysis chip are as follows: (1) Sample / reagent introduction, (2) Liquid feeding by centrifugal force, ( 3) The three steps of the ELISA reaction in the bead portion can be repeated. In addition, after the sample injection and the substrate injection, a washing solution or a buffer solution may be injected into the reservoir as necessary for washing the reaction chamber, and the centrifugal treatment may be similarly performed. In the present invention, such a measurement procedure may be repeated twice or more with different centrifugal forces to obtain measurement results of two or more test substances.

Example 1
An analysis chip was manufactured as follows. FIG. 2 shows a cross-sectional view of the chip. The analysis chip is composed of a cylindrical chip body, and includes a tubular reaction chamber 11 and a cylindrical sample / reagent reservoir 12 having a throttle shape on the reaction chamber side, and both are connected by a “reservoir outlet” 101. Each of the sample / reagent reservoir and the reaction chamber has an opening to the outside, and an opening protruding outside is provided at the opening of the sample / reagent reservoir. Since the reaction chamber is filled with a bead carrier to which a primary antibody is bound, a filter press-fitting hole for damming the beads is provided.

  First, a chip body was manufactured by cutting a resin (made by Kuraray) made of polymethyl methacrylate. A cellulose acetate filter having a diameter of 2 mm and a length of 2 mm was press-fitted through a filter press-fitting hole to produce an analysis chip.

Subsequently, the reaction chamber of the analysis chip was filled with a bead carrier.
Polystyrene beads (Polyscience, particle size: 25 μm) were washed with phosphate buffer, and the same amount of 0.1 μg / ml anti-hIL-6 antibody 0.05% Tween 20 phosphate buffer as polystyrene beads was added. Shake overnight at 4 ° C. After shaking, 2 μl of polystyrene beads were suspended in 150 μl of a phosphate buffer containing 0.05% Tween 20. After pouring this suspension into the reservoir of the above analysis chip, the chip is put into a centrifuge tube (trade name: Eppen microtube, manufacturer name: Eppendorf, size: 1.5 mL), and centrifuged (trade name: micro high-speed cooling). Centrifuge, manufacturer name: Tommy Seiko Co., Ltd., model name: MX-300) Rotor (product) so that the angle between the reservoir / reaction chamber axis of the chip and the rotation axis in the almost vertical direction is 45 ° Name: rotor, manufacturer name: Tommy Seiko Co., Ltd., model name: TMP-21), and centrifuged at 2000 G for 30 seconds. The density of antibody-bound polystyrene beads relative to the reaction chamber volume was 9 × 10 ⁴ pieces / mm ³ .

To the reservoir of the analysis chip, 100 μl of 0.05% Tween 20-containing phosphate buffer was poured, and centrifuged at 2000 G for 60 seconds in a centrifuge to wash the beads. Thereafter, 50 μl of hIL-6 (human interleukin-6, Kamakura Technoscience) dissolved in a phosphate buffer containing 0.25% bovine serum albumin (BSA) and 0.05% Tween 20 and 0.25% 50 μl of 0.6 μg / ml HRP (horseradish peroxidase) -labeled anti-hIL-6 antibody (Kamakura Technoscience) dissolved in a phosphate buffer containing BSA and 0.05% Tween 20 was mixed. Injected into reservoir. The chip was then reacted by centrifugation at 500 G for 600 seconds. After the reaction, 100 μl of 0.05% Tween 20-containing phosphate buffer was injected into the reservoir, and the chip was centrifuged at 2000 G for 60 seconds to wash the beads again. After washing, 100 μl of 0.1 M Tris-HCl buffer (pH 7.5) containing 20 μM hydrogen peroxide solution and 13 μg / ml Amplex Red (Molecular Probes) was poured into a reservoir and centrifuged at 2000 G for 10 seconds to send the solution. Thereafter, the chip was taken out from the centrifuge tube, and the amount of resorufin produced in the reaction chamber was measured using a fluorescence microscope IX-71 (Olympus). The measurement results of the fluorescence intensity of hIL-6 at each concentration are shown in FIG. The measurement conditions were an excitation wavelength of 510-560 nm, an emission wavelength of 575-650 nm, and an exposure time of 5 milliseconds.
The angle θ formed between the rotating shaft and the reservoir / reaction chamber axis of the stopped chip was 45 °.

  As is clear from Table 1 and FIG. 10, there was no waste liquid backflow from the reaction chamber to the reagent / specimen reservoir after each reagent feeding, and good measurement results could be obtained.

FIG. 1 is a perspective view schematically showing an example of a chip used in the present invention. FIG. 2 is a longitudinal sectional view schematically showing an example of a chip used in the present invention. FIG. 3 is a longitudinal sectional view schematically showing an example of a chip used in the present invention. FIG. 4 is a top view and a longitudinal sectional view schematically showing an example of a chip used in the present invention. FIG. 5 is a longitudinal sectional view schematically showing an example of a chip used in the present invention. FIG. 6 is a top view and a longitudinal sectional view schematically showing an example of a chip used in the present invention. FIG. 7 is a longitudinal sectional view schematically showing an example of a chip used in the present invention. FIG. 8 is a perspective view schematically showing a state where a chip that can be used in the present invention is inserted into a centrifuge tube. FIG. 9 is an explanatory diagram showing an example of the procedure of the analysis method of the present invention. FIG. 10 is a graph showing the results of the example. FIG. 11A is a longitudinal sectional view schematically showing an example of a chip used in the present invention together with a rotation axis. FIG. 11-2 is a longitudinal sectional view schematically showing an example of a chip used in the present invention together with a rotation axis. FIG. 12 is a longitudinal sectional view schematically showing an example of a chip used in the present invention together with a rotation axis. FIG. 13 is a longitudinal sectional view schematically showing an example of a chip used in the present invention together with a rotation axis. FIG. 14 is a longitudinal sectional view schematically showing an example of a chip used in the present invention together with a rotation axis. FIG. 15 is a longitudinal sectional view schematically showing an example of a chip used in the present invention together with a rotation axis. FIG. 16 is a longitudinal sectional view schematically showing an example of a chip used in the present invention together with a rotation axis. FIG. 17 is a longitudinal sectional view schematically showing an example of a chip used in the present invention together with a rotation axis. FIG. 18 is a longitudinal sectional view schematically showing an example of a chip used in the present invention.

Explanation of symbols

11 Reaction chamber 11A ′ Carrier damming means 12 Reagent / specimen reservoir 12A Opening portion 13 Carrier 14A to which antigen and / or antibody is bound Ear 14B Foot 14C Space 15 Corner 16 on side surface of immunoassay chip Waste tank 101 Reservoir outlet 102 Reaction Chamber outlet 103 Reservoir / reaction chamber shaft 104 Rotating shaft 105 Waste liquid tank inlet 106 Reaction chamber / waste liquid tank shaft 200, 200A Channels A to F, J to M, P, Q Tip G Centrifuge tube 107 Tip component A
108 Chip part B

Claims (19)

  A chip having a column-shaped reaction chamber containing a carrier to which a selective binding substance is bound and a reagent / sample reservoir communicating with the reaction chamber is rotated with respect to a rotation axis in a substantially vertical direction outside the chip. An analysis method comprising a step of analyzing a test substance, wherein an angle θ formed by the rotation axis and the reservoir / reaction chamber axis of the stopped chip is 0 ° ≦ θ ≦ 80 ° Method.   A chip having a column-shaped reaction chamber containing a carrier to which a selective binding substance is bound, a reagent / sample reservoir communicating with the reaction chamber, and a waste liquid tank communicating with the reaction chamber on the opposite side of the reagent / sample reservoir Analysis method including a step of analyzing a test substance by rotating about a rotation axis in a substantially vertical direction outside the chip, the reaction chamber / waste tank axis of the chip being stopped and the chip being stopped An analysis method characterized in that an angle θ ′ formed by: 0 ° ≦ θ ′ ≦ 80 °.   The chip has a waste tank connected to the reaction chamber on the side opposite to the reagent / sample reservoir, and an angle θ ′ formed between the rotating shaft and the reaction chamber / waste tank axis of the stopped chip is 0 ° ≦ θ ′ ≦ The analysis method according to claim 1, wherein the angle is 80 °.   The analysis method according to any one of claims 1 to 3, wherein a volume ratio of the reagent / sample reservoir to the reaction chamber is 50 or more. 5. The analysis method according to claim 1, wherein a volume ratio of the reagent / specimen reservoir to the reaction chamber is 50 to 5 × 10 ⁸ .   The ratio of the projected sectional area of the reagent / specimen reservoir in the liquid feeding direction to the projected sectional area of the reaction chamber in the liquid feeding direction is 50 or more, 6. Analysis method described in 1.   The ratio of the maximum cross-sectional area parallel to the liquid feeding direction of the reagent / sample reservoir and the maximum cross-sectional area parallel to the liquid feeding direction of the reaction chamber is 2 to 400. Item 7. The analysis method according to any one of Items 1 to 6. A step of passing a liquid containing a test substance through the reaction chamber using a centrifugal force generated by rotating the chip, and binding the test substance to a carrier to which the selective binding substance is bound; and The analysis method according to any one of claims 1 to 7, further comprising a step of allowing a solution containing a labeling substance to pass through a reaction chamber using centrifugal force generated by rotation.   Using a centrifugal force generated by rotating the chip, a liquid mixture containing a test substance and a liquid containing a labeling substance is passed through the reaction chamber, and the complex of the test substance and the labeling substance is selectively bonded. The analysis method according to any one of claims 1 to 7, further comprising a step of binding to a carrier to which a sex substance is bound.   The analysis method according to any one of claims 1 to 9, wherein in the reaction chamber, a test substance bound to a carrier bound with a selective binding substance is detected using optical means. .   The analysis method according to claim 10, wherein the optical means is fluorescence measurement.   The analysis method according to claim 10, wherein the optical means is luminescence measurement.   The analysis method according to claim 10, wherein the optical means is absorbance measurement.   10. The analysis method according to claim 1, wherein in the reaction chamber, detection of a test substance bound to a carrier to which a selectively binding substance is bound is performed using a radiation detection means. .   15. The analysis method according to claim 1, wherein the selective binding substance is an antigen or an antibody.   The analysis method according to any one of claims 8 to 15, wherein the labeling substance is a labeled antibody.   The analysis method according to any one of claims 1 to 16, wherein the test substance is a protein.   The analysis method according to any one of claims 1 to 17, wherein the test substance is a cytokine and / or a chemokine.   The analysis method according to any one of claims 1 to 18, wherein the test substance is IL-6, IL-8, or TNF.

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