WO2015098967A1 - Reagent container, reagent-filled reagent container, reaction unit, and analysis system - Google Patents

Reagent container, reagent-filled reagent container, reaction unit, and analysis system Download PDF

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Publication number
WO2015098967A1
WO2015098967A1 PCT/JP2014/084151 JP2014084151W WO2015098967A1 WO 2015098967 A1 WO2015098967 A1 WO 2015098967A1 JP 2014084151 W JP2014084151 W JP 2014084151W WO 2015098967 A1 WO2015098967 A1 WO 2015098967A1
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WO
WIPO (PCT)
Prior art keywords
reagent
reaction
syringe
container
plunger
Prior art date
Application number
PCT/JP2014/084151
Other languages
French (fr)
Japanese (ja)
Inventor
荒川　智
悟史 八幡
秀和 長峯
Original Assignee
東亜ディーケーケー株式会社
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 東亜ディーケーケー株式会社 filed Critical 東亜ディーケーケー株式会社
Priority to CN201480070820.6A priority Critical patent/CN105849531B/en
Priority to JP2015535636A priority patent/JP5979318B2/en
Publication of WO2015098967A1 publication Critical patent/WO2015098967A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/49—Blood

Definitions

  • the present invention relates to a reagent container, a reagent-containing reagent container, a reaction unit, and an analysis system.
  • the present invention claims priority based on Japanese Patent Application No. 2013-265683 filed in Japan on December 24, 2013, the contents of which are incorporated herein by reference.
  • Patent Document 3 proposes a technique in which a reagent is fixed in a container, and the reagent is dissolved and reacted with the introduced sample solution.
  • Patent Document 3 it is proposed to use a container having a plurality of reaction chambers partitioned by a diaphragm in order to sequentially perform a step of releasing a luminescent substrate and a step of adding a luminescent reagent (FIG. 8, etc. of Patent Document 3). ).
  • a hole is made in the diaphragm with a needle or the like, and the sample solution is fixed to the sample solution through this hole. It is described that it is dropped into a reaction chamber and reacted with a luminescent reagent.
  • Patent Document 3 also proposes to use a container containing a reagent containing cup in which a reagent activated by microbial contaminants is fixed, a synthetic substrate on the ceiling, and a luminescent reagent on the bottom.
  • a container containing a reagent containing cup in which a reagent activated by microbial contaminants is fixed, a synthetic substrate on the ceiling, and a luminescent reagent on the bottom.
  • the present invention has been made in view of the above circumstances, and includes a reagent container that can hold a liquid unstable reagent in a sealed state until immediately before the reaction with the sample liquid, and a reagent containing the reagent held in the reagent container. It is an object to provide a reagent container. It is another object of the present invention to provide a reaction unit that uses the above-described reagent container to allow a stably held reagent to react with almost the entire amount of a sample solution and to easily perform a two-stage reaction. It is another object of the present invention to provide an analysis system that can analyze a sample solution with high sensitivity and can be easily performed using the reaction unit, and that can also perform analysis by a two-stage reaction.
  • a syringe in which one end in the axial direction is a first opening end and the other end is a second opening end, and a plunger slidable in the syringe,
  • the plunger has a sliding contact portion that is in sliding contact with the inner surface of the syringe, and a reagent holding portion that is not in sliding contact with the inner surface of the syringe and is surrounded by the sliding contact portion,
  • the reagent container wherein the reagent holding part is movable from the inside of the syringe to the outside of the first opening end of the syringe.
  • a reagent supply hole is formed in the side surface of the syringe, and the reagent supply hole can communicate with the reagent holding unit in a state where the reagent holding unit is located inside the syringe [1].
  • the reagent container according to 1. [3] The reagent container according to [1] or [2], wherein the syringe has a protruding portion that protrudes inwardly in an axial intermediate portion. [4] Further, an elastic member is provided, When the reagent holding part is moved outside the first open end of the syringe, The reagent container according to any one of [1] to [3], wherein the elastic member applies an urging force for moving the plunger toward the second opening end.
  • a process of A sample solution analysis system configured to sequentially perform the step of measuring the sample solution after reaction with the reagent by the optical measuring means.
  • a liquid unstable reagent can be held in a sealed state until immediately before the reaction with the sample liquid. Further, according to the reaction unit of the present invention, the stably held reagent can be reacted with almost the entire amount of the sample solution, and a two-stage reaction can be easily performed.
  • the analysis system of the present invention it is possible to analyze a sample solution with high sensitivity and simplicity, and it is also possible to easily perform analysis by a two-stage reaction.
  • FIG. 1 shows a reagent container
  • FIG.1 shows one aspect
  • 2A is a perspective view
  • FIG. 2B is a side view
  • FIG. 2C is a cross-sectional view taken along line II-II in FIG. 2B.
  • FIGS. 3A and 3B are diagrams for explaining a method of holding a reagent in the reaction unit of FIG. 1,
  • FIG. 3A shows a method of holding the second reagent R2 in the reagent container 10, and FIG. Each method for retaining R1 will be described.
  • FIG. 4A is a diagram showing an analysis system using the reaction unit of FIG.
  • FIG. 5A is a perspective view
  • FIG. 5B is a side view
  • FIG. 5C is a side view seen from a different direction from FIG. 5B
  • FIG. d) is a VV cross-sectional view of FIG.
  • FIGS. 6A and 6B are views showing another modification of the plunger, in which FIG. 6A is a perspective view, FIG. 6B is a side view, and FIG. 6C is a side view as seen from a different direction from FIG.
  • FIG. 7A is a perspective view
  • FIG. 7B is a side view
  • FIG. 7C is a cross-sectional view taken along the line VII-VII in FIG. 7B.
  • reaction unit 1A and 1B show a reaction unit according to an embodiment of the present invention, in which FIG. 1A shows an embodiment of a reagent container, and FIG. 1B shows an embodiment of a reaction container.
  • the reagent container 10 shown in FIG. 1A is schematically constituted by a syringe 20, a plunger 30, and a spring 40 (elastic member).
  • the syringe 20 has one end in the axial direction as a first opening end 20a, the other end as a second opening end 20b, an opening 21a in the first opening end 20a, and a second opening end 20b. Each has an opening 21b.
  • the second opening end 20b is provided with a flange 22 that protrudes radially outward over the entire circumferential direction.
  • the protrusion part 23 which protrudes over the whole circumferential direction is provided in the inner surface 20c of the syringe 20 at the radial inside.
  • the protrusion 23 is an intermediate portion in the axial direction of the syringe 20 and is provided on the inner surface 20c of a portion slightly closer to the first opening end 20a than the center in the axial direction.
  • a sealing material 24 is attached to the radially inner side of the protrusion 23.
  • the sealing material 24 includes a tubular portion 24a and a flange portion 24b that protrudes radially outward on one end side of the tubular portion 24a.
  • the tubular portion 24 a is interposed between the protruding portion 23 and the plunger shaft 31 of the plunger 30 and constitutes a sliding surface with the plunger 30.
  • the flange portion 24 b is in close contact with the second opening end 20 b side of the protruding portion 23, and serves as a buffer between the spring 40 and the protruding portion 23.
  • a reagent supply hole 25 is formed on the side surface of the syringe 20.
  • the reagent supply hole 25 is an intermediate portion in the axial direction of the syringe 20 and is formed in a portion closer to the first opening end 20a than a portion where the protruding portion 23 is provided.
  • the plunger 30 includes a plunger shaft 31 and a gasket 32 screwed to the tip of the plunger shaft 31.
  • the plunger shaft 31 is provided with a columnar shaft body 31a and one end of the shaft body 31a, and a substantially disk-shaped push plate having a larger diameter than the shaft body 31a.
  • 31 b and a male screw portion 31 c that is provided at the other end of the shaft main body 31 a and is inserted into the gasket 32.
  • the shaft body 31a, the push plate 31b, and the male screw portion 31c are integrally formed of a rigid material, for example, a resin such as polypropylene, glass, or metal.
  • the gasket 32 has a truncated cone part 32a, a first large diameter part 32b, a small diameter part 32c, a second large diameter part 32d, and a tip part 32e from the side where the plunger shaft 31 is inserted, and these are integrally formed. ing.
  • the outer diameters of the first large diameter portion 32b and the second large diameter portion 32d are substantially the same as the inner diameter of the portion of the syringe 20 where the protruding portion 23 is not provided, and these side surfaces are in sliding contact with the inner surface of the syringe 20, respectively.
  • a first sliding contact portion 32A and a second sliding contact portion 32B are provided.
  • the outer diameter of the small diameter part 32c is smaller than the outer diameters of the first large diameter part 32b and the second large diameter part 32d, and does not slide on the inner surface of the syringe 20.
  • the side surface of the small diameter portion 32c is a concave shape surrounded by the first sliding contact portion 32A and the second sliding contact portion 32B together with the surfaces of the first large diameter portion 32b and the second large diameter portion 32d on the small diameter portion 32c side.
  • the reagent holding unit 32C is configured. Both ends of the small diameter portion 32c gradually increase in diameter, and a curved surface is formed at each of the boundary between the first large diameter portion 32b and the boundary between the second large diameter portion 32d.
  • the reagent supply hole 25 is formed at a position communicating with the reagent holding portion 32C when the plunger 30 is pulled toward the second opening end 20b until the gasket 32 contacts the protruding portion 23 of the syringe 20.
  • the truncated cone portion 32a has a truncated cone shape having a smaller diameter than the first large diameter portion 32b and a larger diameter on the first large diameter portion 32b side.
  • the tip 32e has a shape with a smaller volume when the sphere is cut off by a surface that does not pass through the center.
  • the cut surface is a circle having the same diameter as the second large diameter portion 32d, and the tip end portion 32e is connected to the second large diameter portion 32d on the cut surface side.
  • a female screw 32D is formed from the truncated cone part 32a to the middle of the small diameter part 32c to which the male screw part 31c of the plunger shaft 31 is screwed.
  • the gasket 32 is made of an elastomer such as elastic rubber.
  • the material of the gasket 32 is preferably one having chemical resistance and cold resistance and low gas permeability. Specific examples include fluororubbers such as butyl rubber and tetrafluoroethylene.
  • the spring 40 is arranged so that the plunger shaft 31 is inserted inside.
  • the spring 40 is compressed between the protruding portion 23 and the push plate 31b when the reagent holding portion 32C is moved to the outside of the first opening end 20a, and the second opening end 20b side with respect to the plunger 30 The urging force to move to is given.
  • a reaction vessel 50 shown in FIG. 1 (b) includes a bottomed cylindrical reaction vessel main body 51 whose upper end is an open end 51a, and a flange 52 that protrudes radially outward over the entire circumferential direction of the open end 51a.
  • FIG. 1B shows a state in which the opening 53 of the opening end 51 a of the reaction vessel 50 is closed with a lid 54.
  • the lid body 54 was provided at the distal end of the insertion portion 54b, a lid portion 54a having an outer diameter substantially the same as that of the flange portion 52, an insertion portion 54b inserted into the opening 53 of the reaction vessel 50 in a liquid-tight and air-tight manner.
  • a plurality (two) of leg portions 54c are formed integrally.
  • the inside of the leg portion 54c is a substantially cylindrical concave portion 54d, and the leg portion 54c can be deformed to the concave portion 54d side, so that the leg portion 54c and the insertion portion 54b are inserted into the opening 53 of the reaction vessel 50. Is easy.
  • the reagent container 10 can be inserted into the reaction container 50 from the first opening end 20a side. Further, when the flange portion 22 of the syringe 20 abuts on the flange portion 52 of the reaction vessel 50, the first open end 20 a of the syringe 20 remains at the position in the middle portion in the axial direction of the reaction vessel 50. It does not reach the bottom. 4B, with the reagent container 10 inserted into the reaction container 50, the gasket 32 of the plunger 30 is moved to the outside of the first open end 20a, and the gasket 32 is moved to the reaction container 50. It can be made to reach to the bottom or near the bottom.
  • FIG. 3 is a view for explaining a method of holding the reagent in the reaction unit of FIG. 1, FIG. 3 (a) shows a method of holding the second reagent R 2 in the reagent container 10, and FIG. 3 (b) shows a reaction container 50. Each of the methods for retaining the first reagent R1 will be described.
  • the holding operation of the second reagent R2 is performed in a state in which the reagent container 10 is placed horizontally with the reagent supply hole 25 on the upper side.
  • the plunger 30 is pulled to the second opening end 20b side until the gasket 32 comes into contact with the protruding portion 23 of the syringe 20, and the reagent supply hole 25 communicates with the reagent holding portion 32C.
  • a holding operation of the second reagent R2 first, a solution in which the second reagent R2 is dissolved in an appropriate solvent (for example, water) is filled from the reagent supply hole 25 toward the reagent holding unit 32C. Thereafter, the second reagent R2 can be held in the reagent holding unit 32C in a dry state by lyophilization while the reagent supply hole 25 is in communication with the reagent holding unit 32C.
  • an appropriate solvent for example, water
  • the reagent holding portion 32C causes the first sliding contact portion 32A and the second sliding contact portion 32B surrounding the reagent holding portion 32C, and the inner surface 20c of the syringe 20 to move. Therefore, the held second reagent R2 can be stored in a sealed state. Note that after lyophilization, the second reagent R2 may adhere not only to the reagent holding part 32C but also to the inner surface 20c of the syringe 20. However, when the plunger 30 is moved to the position shown in FIG. 1A, the second reagent R2 attached to the inner surface 20c of the syringe 20 is scraped off by the first sliding contact portion 32A.
  • the above operation for holding the second reagent R2 is preferably performed in a vacuum or an inert gas atmosphere.
  • the holding operation of the first reagent R1 is performed with the reaction vessel 50 placed in a vertical state with the open end 51a on the upper side.
  • a holding operation of the first reagent R1 first, the lid 54 is removed, and a solution in which the first reagent R1 is dissolved in an appropriate solvent (for example, water) is filled from the opening 53. Thereafter, the lid 54 is lyophilized with the lid 54 inserted into the opening 53 so that a part of the leg 54c is exposed to the outside of the opening end 51a so as to keep the opening 53 in communication with the outside.
  • One reagent R1 can be held in the reaction vessel 50 in a dry state. Thereafter, when the lid 54 is pushed into the position shown in FIG. 1B, the first reagent R1 held in the reaction vessel 50 can be stored in a sealed state.
  • the above operation for holding the first reagent R1 is preferably performed in a vacuum or an inert gas atmosphere.
  • FIG. 4 is a diagram showing an analysis system using the reaction unit of FIG. 1 and a method for optically measuring a sample solution after the two-stage reaction using this analysis system.
  • the analysis system shown in FIG. 4 includes the reaction unit shown in FIG.
  • the optical measuring means 55 includes a light detection device that detects light (including fluorescence and bioluminescence) emitted from the reaction container 50 of the reaction unit, a light source, and a light receiving device that detects light transmitted through the reaction container 50.
  • a scattered light detection device comprising a light-absorbance detection device, a light source, and a light-receiving device that detects light scattered in the reaction vessel 50 can be appropriately employed.
  • the analysis system of FIG. 4 may include a heating means for appropriately heating the reaction vessel 50 in order to promote the progress of the reaction.
  • the operation of the analysis system in FIG. 4 is controlled by a control device (not shown), and the following steps are sequentially performed on the reaction unit.
  • first reaction step A step of reacting the sample solution S introduced into the reaction vessel 50 with the first reagent R1 (hereinafter referred to as “first reaction step”).
  • second reaction step a step of reacting the second reagent R2 (hereinafter referred to as “second reaction step”).
  • second reaction step A step of measuring the sample solution S after the reaction with the second reagent R2 by the optical measuring means 55 (hereinafter referred to as “measurement step”).
  • the following preparation is required prior to the first reaction step.
  • the reaction container 50 in which the first reagent R1 is held and sealed by the lid 54, and the second reagent R2 are held in the reagent holding part 32C, and the reagent holding part 32C is sealed in the state shown in FIG.
  • the reagent container 10 (reagent-containing reagent container) accommodated in the space thus prepared is prepared.
  • the lid 54 is removed from the reaction vessel 50 in which the first reagent R1 is held, and the sample solution S is put therein.
  • the reagent container 10 is inserted into the reaction container 50 and set in the analysis system.
  • the operation so far is preferably performed manually, but may be automated by adding a sampling mechanism to the analysis system.
  • the sample solution S is introduced into the reaction vessel 50, the first reagent R1 is dissolved by the introduced sample solution S, and the sample solution S and the first reagent R1 can react.
  • the first reaction step is a step from setting the reaction unit to the analysis system to starting the next second reaction step.
  • the time of the first reaction step is appropriately set in consideration of the reaction time necessary for reacting the sample solution S introduced into the reaction vessel 50 with the first reagent R1.
  • the reaction vessel 50 can be appropriately heated to promote the reaction.
  • the push plate 31b of the plunger 30 is pushed by the push rod (not shown) to push the plunger 30 down.
  • the gasket 32 is moved from the inside of the syringe 20 to the outside of the first open end 20a, and the tip of the gasket 32 is made to reach the bottom of the reaction vessel 50 as shown in FIG. In this way, the entire reagent holding part 32C is immersed in the sample solution S after the reaction with the first reagent R1.
  • the second reagent R2 When the reagent holding part 32C is immersed in the sample solution S after reacting with the first reagent R1, the second reagent R2 is dissolved by the sample solution S, and the sample solution S and the second reagent R2 can react.
  • the second reagent R2 is not only directly fixed to the reagent holding part 32C, but can also be held in a region surrounded by the reagent holding part 32C and the inner surface 20c of the syringe 20 facing the reagent holding part 32C. However, the second reagent R2 in this region can be pushed out by the first sliding contact portion 32A, and almost the entire amount can be put into the sample solution S after the reaction with the first reagent R1 to be reacted.
  • the time for keeping the plunger 30 in the state shown in FIG. 4 (b) only needs to be sufficient to dissolve the second reagent R2. If the solubility of the second reagent R2 is high, the plunger 30 is pushed down. Thereafter, the measurement process can be immediately performed. For example, in the case of a reaction system in which the generation of light to be detected is completed in a short time after the start of the reaction between the sample solution S and the second reagent R2, such as fluorescence measurement or bioluminescence measurement, the plunger 30 is shown in FIG. It is preferable to immediately shift to the measuring step after pushing down to the state shown in b). When time is required for the dissolution of the second reagent R2 in the sample solution S and the reaction between the sample solution S and the second reagent R2, the process proceeds to the measurement step after an appropriate waiting time has elapsed.
  • the pressing of the push plate 31b of the plunger 30 by a push rod (not shown) is released. Then, as shown in FIG. 4C, the pressing plate 31b is pushed up to the second opening end 20b side by the urging force of the spring 40 compressed in FIG. 4B. Thereby, the gasket 32 that obstructs the optical path is separated from the bottom of the reaction vessel 50, and the second reagent R ⁇ b> 2 and the sample solution S after the reaction can be measured by the optical measuring means 55.
  • the optical measurement means 55 optically measures the second reagent R2 and the sample solution S after the reaction.
  • the type of optical measurement is not particularly limited, and fluorescence detection, bioluminescence detection, absorbance detection, scattered light detection, and the like can be appropriately employed.
  • the above operation is sequentially performed on each reaction unit.
  • the conditions of optical measurement can be easily uniformed by analyzing the sample liquid introduced into each reaction unit according to a predetermined time table. Therefore, it is easy to eliminate detection errors due to variations in measurement conditions.
  • almost all of the first reagent R1 and the second reagent R2 are used for the reaction in the reaction vessel 50. Further, after the sample solution S is put in the reaction vessel 50, it is not necessary to move it to another place. Therefore, even if the sample liquid S is small, it can be analyzed with high accuracy and good reproducibility.
  • the plunger used for the reagent container has the structure shown in FIG. 2, but the specific structure of the plunger is not particularly limited.
  • the plunger shown in FIGS. 60, 70, and 80 may be used.
  • the plunger 60 shown in FIG. 5 includes a plunger shaft 61 and a gasket 62 screwed onto the tip of the plunger shaft 61.
  • the plunger shaft 61 is provided at a cylindrical shaft main body 61a, one end of the shaft main body 61a, a substantially disc-shaped push plate 61b having a larger diameter than the shaft main body 61a, and the other end of the shaft main body 61a. And a male thread portion 61c inserted into the gasket 62.
  • the shaft main body 61a, the push plate 61b, and the male screw portion 61c are integrally formed, and the same material as the shaft main body 31a, the push plate 31b, and the male screw portion 31c in FIG. 2 can be used.
  • the gasket 62 has a truncated cone part 62a, a first large diameter part 62b, a side wall part 62c, a second large diameter part 62d, and a tip part 62e from the side where the plunger shaft 61 is inserted, and these are integrally formed. ing.
  • the outer diameters of the first large diameter portion 62b and the second large diameter portion 62d are substantially the same as the inner diameter of the portion of the syringe 20 where the protruding portion 23 is not provided, and these side surfaces are in sliding contact with the inner surface of the syringe 20, respectively.
  • a first sliding contact portion 62A and a second sliding contact portion 62B are provided.
  • the side wall part 62c has a shape with a smaller volume when a cylinder having the same diameter as the first large diameter part 62b and the second large diameter part 62d is cut by a vertical surface that does not pass through the center of the bottom surface. .
  • the portion corresponding to the remaining side surface of the cylinder is a third sliding contact portion 62 ⁇ / b> C that is in sliding contact with the inner surface of the syringe 20. Further, the side surface corresponding to the cut surface of the cylinder does not slide in contact with the inner surface of the syringe 20.
  • This side surface is surrounded by the first sliding contact portion 62A, the second sliding contact portion 62B, and the third sliding contact portion 62C together with the surfaces of the first large diameter portion 62b and the second large diameter portion 62d on the side wall portion 62c side.
  • the concave reagent holding part 62D is formed.
  • the reagent supply hole 25 is positioned to communicate with the reagent holding portion 62D when the plunger 60 is pulled toward the second opening end 20b until the gasket 62 comes into contact with the protruding portion 23 of the syringe 20.
  • the truncated cone part 62a has a truncated cone shape having a smaller diameter than the first large diameter part 62b and a larger diameter on the first large diameter part 62b side.
  • the distal end portion 62e has a shape with a smaller volume when the sphere is cut off by a surface that does not pass through the center.
  • the cut surface is circular with the same diameter as the second large diameter portion 62d, and the tip end portion 62e is connected to the second large diameter portion 62d on the cut surface side.
  • a female screw 62E into which the male screw portion 61c of the plunger shaft 61 is screwed is formed in the truncated cone portion 62a.
  • the gasket 62 can be made of an elastomer similar to the gasket 32 of FIG.
  • the plunger 70 in FIG. 6 includes a plunger shaft 71 and a gasket 72 that is screwed onto the tip of the plunger shaft 71.
  • the plunger shaft 71 is provided at a columnar shaft main body 71a, one end of the shaft main body 71a, a substantially disk-shaped push plate 71b having a larger diameter than the shaft main body 71a, and the other end of the shaft main body 71a. And a male threaded portion 71c inserted into the gasket 72.
  • the shaft body 71a, the push plate 71b, and the male screw portion 71c are integrally formed, and the same material as the shaft main body 31a, the push plate 31b, and the male screw portion 31c in FIG. 2 can be used.
  • the gasket 72 has a truncated cone part 72a, a through-hole forming part 72b, and a tip part 72c from the side where the plunger shaft 71 is inserted, and these are integrally formed.
  • the through-hole forming portion 72b has a structure in which a cylindrical through-hole 72d is formed in a radial direction in a column that is substantially the same as the inner diameter of a portion where the protruding portion 23 of the syringe 20 is not provided.
  • the side surface of the portion other than the through hole 72 d of the through hole forming portion 72 b is a sliding contact portion 72 A that is in sliding contact with the inner surface of the syringe 20.
  • the inner surface of the through hole forming portion 72b where the through hole 72d is formed is a reagent holding portion 72B that does not slide in contact with the inner surface of the syringe 20, and is surrounded by the sliding contact portion 72A.
  • the reagent supply hole 25 is configured such that when the plunger 70 is pulled toward the second opening end 20 b until the gasket 72 comes into contact with the protruding portion 23 of the syringe 20, It is formed at a position communicating with 72B (through hole 72d).
  • reagent supply holes communicating with the reagent holding part 72B (through hole 72d) may be respectively formed at two opposing positions in the radial direction of the syringe 20.
  • the truncated cone part 72a has a truncated cone shape having a smaller diameter than the through hole forming part 72b and a larger diameter on the through hole forming part 72b side.
  • the tip 72c has a shape with a smaller volume when the sphere is cut off by a cut surface that does not pass through the center.
  • the cut surface is a circle having the same diameter as the through-hole forming portion 72b, and the distal end portion 72c is connected to the through-hole forming portion 72b on the cut surface side.
  • a female screw 72D into which the male screw portion 71c of the plunger shaft 71 is screwed is formed in the truncated cone portion 72a.
  • the gasket 72 can be made of the same elastomer as the gasket 32 of FIG.
  • the plunger shaft 81 includes a cylindrical shaft main body 81a, an O-ring mounting plate 81c provided near one end of the shaft main body 81a, and an O-ring mounting plate 81d provided at one end of the shaft main body 81a. And have.
  • Each of the O-ring mounting plate 81c and the O-ring mounting plate 81d has a substantially disc shape having a surface perpendicular to the axial direction of the shaft main body 81a, and a groove for mounting the O-ring is formed on the peripheral surface.
  • the shaft main body 81a, the O-ring mounting plate 81c, and the O-ring mounting plate 81d are integrally formed, and the same material as the shaft main body 31a, the push plate 31b, and the male screw portion 31c in FIG. 2 can be used.
  • a female screw 81b is formed at the other end of the shaft body 81a.
  • the push plate 83 has a substantially disk-like push plate main body 83a having a diameter larger than that of the shaft main body 81a, and a male screw portion 83b that is suspended from the push plate main body 83a and screwed into the female screw 81b.
  • the push plate main body 83a and the male screw portion 83b are integrally formed, and the same material as the shaft main body 31a, the push plate 31b, and the male screw portion 31c in FIG. 2 can be used.
  • the O-rings 82a and 82b can be made of the same material as the gasket 32c of FIG.
  • the O-rings 82a and 82b are attached to O-ring attachment plates 81c and 81d, respectively.
  • the O-rings 82a and 82b are configured such that the outer diameter of the O-rings 82a and 82b is substantially the same as the inner diameter of the portion of the syringe 20 where the protrusion 23 is not provided. Yes.
  • the outer periphery of the O-ring 82a is a first sliding contact portion 82A that is in sliding contact with the inner surface of the syringe 20
  • the outer periphery of the O-ring 82b is a second sliding contact portion 82B that is in sliding contact with the inner surface of the syringe 20.
  • a portion sandwiched between the first sliding contact portion 82A and the second sliding contact portion 82B (the surrounded portion) is a reagent holding portion 82C.
  • the reagent holding part 82C does not slide in contact with the inner surface of the syringe 20.
  • the reagent supply hole 25 is formed when the plunger 80 is pulled toward the second opening end 20 b until the O-ring attachment plate 81 c contacts the protruding portion 23 of the syringe 20. It is formed at a position communicating with the reagent holding part 82C.
  • the plunger shaft 81 is inserted from the first opening end 20a side of the syringe 20 with the push plate 83 removed from the plunger shaft 81, and then the push plate 83 is moved to the plunger 20 What is necessary is just to screw to the axis
  • the reagent holding portion is formed over the entire circumferential direction, but as can be understood from the example of the plunger 60 and the plunger 70, the reagent holding portion does not have to extend over the entire circumferential direction. Moreover, in each said plunger, a part or all of a sliding contact part exists in two places which pinch
  • the relationship between the reagent holding part and the sliding contact part is not limited to such an embodiment, the reagent holding part is surrounded by the sliding contact part, and between the reagent holding part and the inner wall of the syringe 20, What is necessary is just to be able to form a sealed space.
  • a vertical groove may be formed on the side surface of the side wall portion 62c of the plunger 60 at a position separated from the reagent holding portion 62D.
  • a vertical groove may be formed on the side surface of the through hole forming portion 72b of the plunger 70 at a position spaced from the through hole 72d.
  • the plunger 60, and the plunger 70 it was set as the structure attached to the plunger axis
  • the plunger 80 is configured such that the push plate 83 is attached to the plunger shaft 81 by screwing, but may be attached by fitting, for example.
  • the reagent supply hole is formed in the syringe, but the reagent supply hole is not essential.
  • the reagent solution is supplied toward the reagent holding part while part or all of the reagent holding part is moved to the outside of the first open end of the syringe, and is freeze-dried or the like. What is necessary is just to hold.
  • the structure which provides the protrusion part which protrudes inside in the intermediate part of a syringe continuously over the whole circumferential direction it is good also as a structure provided intermittently in a part of circumferential direction.
  • a configuration is possible in which convex portions are provided at 90 ° intervals at four locations in the circumferential direction.
  • the spring when the reagent holding part is moved to the outside of the first opening end, the spring can be compressed by fixing the position on one end side of the spring.
  • the protrusion part which protrudes inside in the intermediate part of a syringe is not essential. When this is not provided, alignment with the reagent supply hole may be performed visually. Further, as will be described below, it is not essential to compress the spring disposed so as to insert the plunger shaft 31 inside.
  • the elastic member that gives the urging force to move the plunger toward the second opening end side is arranged so that the plunger shaft 31 is inserted inside.
  • the spring 40 is not limited.
  • an elastic member such as a spring or rubber arranged so as to pull up the push plate 31b from above can be used. In this case, when these elastic members are extended, a biasing force for moving the plunger toward the second opening end side can be applied.
  • an elastic member that gives a biasing force to move the plunger toward the second opening end side is not essential.
  • the gasket 32 can be retracted, and the process can be shifted from the second reaction step to the measurement step.
  • the plunger shaft 81 of the plunger 80 shown in FIG. 7 is formed of a transparent material, only the O-rings 82a and 82b prevent light transmission. Does not substantially interfere with detection. In this case, the measurement process can be performed without hindrance without returning the plunger 80 upward.
  • the position which provides the collar part 22 is not limited to the 2nd opening end 20b,
  • the 2nd opening It may be provided in the vicinity of the end 20b (from the second opening end 20b to the first opening end 20a side).
  • the shape of the flange portion 22 is not particularly limited.
  • a key-like portion that hangs downward may be provided on the periphery of the disk so as to be fitted with the upper end of the reaction vessel 50. In this case, the reagent container 10 can be stably supported even if the reaction container 50 does not have the flange 52.
  • the collar part 22 of the syringe 20 is not essential.
  • the flange 22 When the flange 22 is not provided, it may be supported by a separate support means so that the first open end 20a of the reagent container 10 does not reach the bottom of the reaction container 50.
  • the first reagent R1 is held in the reaction vessel 50 and the two-stage reaction is performed. However, it is not essential to hold the reagent in the reaction vessel 50. When the reagent is not held in the reaction vessel 50, a one-step reaction between the sample solution S and the second reagent R2 can be performed. Further, the sample solution S may be previously reacted with the first reagent R1. Alternatively, both the sample solution S and the first reagent R1 may be manually introduced into the reaction vessel 50. In these cases, the two-stage reaction can be performed without holding the reagent in the reaction vessel 50.
  • the first reagent R1 retained in the reaction container is a factor C activated by binding with endotoxin And a reagent containing a luminescent synthetic substrate formed by binding a luminescent substrate to a peptide.
  • a reagent containing a luminescent enzyme and other compounds necessary for the luminescent reaction is used as the second reagent R2 held in the reagent container.
  • factor C activated by binding to endotoxin is used as the first reagent R1 held in the reaction vessel, and a reagent containing a luminescent synthetic substrate formed by binding a luminescent substrate to a peptide, a luminescent enzyme, and a luminescent reaction.
  • a reagent containing other necessary compounds may be used as the second reagent R2 held in the reagent container.
  • a horseshoe crab blood cell extract component (Limulus reagent) can be suitably used as a reagent containing Factor C activated by binding to endotoxin.
  • a luminescent synthetic substrate formed by binding a luminescent substrate to a peptide cleaves the bond between the luminescent substrate and the peptide by the action (protease activity) of at least one of active factor C, active factor B, and clotting enzyme. It has a structure.
  • the activated factor C is generated by activating factor C.
  • the activated factor B is generated by activating factor B by the action of activated factor C.
  • the clotting enzyme is produced by the activation of the procoagulase by the action of active factor B or active factor C.
  • aminoluciferin can be preferably used.
  • the peptide that binds to the luminescent substrate an amino acid sequence in which an amide bond with aminoluciferin at the C-terminus of the peptide is cleaved by at least one protease activity of active factor C, active factor B and coagulation enzyme Anything consisting of can be used.
  • NaCl may be added to the first reagent R1.
  • a luminescent enzyme is an enzyme that generates light by using bioluminescence of a luminescent substrate released from a luminescent synthetic substrate as a catalyst. When the luminescent substrate is aminoluciferin, the luminescent enzyme is luciferase, and other compounds required for the luminescent reaction are ATP and divalent metal ions.
  • the specific structure of the reagent container and reaction unit and the specific procedure of the analysis system can be suitably applied as described above.
  • the time from when the reagent holding part holding the second reagent R2 is immersed in the sample solution until the luminescence reaction is optically detected is shorter.
  • the operation of depressing the plunger 30 in the second reaction step and releasing the depressing to move to the measurement step is performed as quickly as possible.
  • the plunger 30 is pushed down after the container unit is arranged at a position where the light measurement reaction can be detected by the optical measuring means.
  • the first reagent R1 is a mixed reagent of a horseshoe crab blood cell extract component (Limulus reagent) and a synthetic substrate in which aminoluciferin is bound to a peptide
  • the second reagent R2 is a mixed reagent containing luciferase, ATP and a divalent metal ion. In some cases, it is preferable to incubate at 37 ° C. for about 15 to 30 minutes after first introducing the sample solution into the reaction vessel holding the first reagent R1.
  • the plunger 30 is pushed down to immerse the reagent holding part holding the second reagent R2 in the sample solution to cause a luminescence reaction (bioluminescence reaction) between luciferin and luciferase.
  • the temperature during the luminescence reaction can be, for example, room temperature (25 ° C.).
  • the reaction time of the luminescence reaction that is, the time from when the plunger 30 is depressed to when the depression is released and the luminescence reaction is optically detected is preferably 0 to 10 seconds.
  • the present invention can be suitably used for analysis using a liquid unstable reagent such as detection of microbial contaminants such as endotoxin and ⁇ -glucan.

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Abstract

Provided are the following: a reagent container that can hold a reagent, which is unstable in a liquid state, in an airtight state until immediately before a reaction, and that enables almost the entire amount of the reagent to be reliably used in the reaction; a reaction unit that uses the reagent container; and an analysis system. A reagent container (10) comprises: a syringe (20) in which, in the axial direction, one end is a first opening end (20a) and the other end is a second opening end (20b); and a plunger (30) that can slide inside of the syringe (20). The plunger (30) comprises: a first sliding contact part (32A) and a second sliding contact part (32B) that make sliding contact with the inner surface of the syringe; and a reagent holding part (32C) that does not make sliding contact with the inner surface of the syringe (20) and that is surrounded by the first sliding contact part (32A) and the second sliding contact part (32B). The reagent holding part (32C) can move from inside of the syringe (20) to the outside of the first opening end (20a) of the syringe (20).

Description

試薬容器、試薬入り試薬容器、反応ユニット、および分析システムReagent container, reagent-containing reagent container, reaction unit, and analysis system
　本発明は、試薬容器、試薬入り試薬容器、反応ユニット、および分析システムに関する。
　本発明は、２０１３年１２月２４日に日本国に出願された、特願２０１３－２６５６８３号に基づき優先権を主張し、その内容をここに援用する。 The present invention relates to a reagent container, a reagent-containing reagent container, a reaction unit, and an analysis system.
The present invention claims priority based on Japanese Patent Application No. 2013-265683 filed in Japan on December 24, 2013, the contents of which are incorporated herein by reference.
　エンドトキシン、βグルカン等の微生物夾雑物を高感度で検出する方法として、試料液に、微生物夾雑物によって活性化する試薬、および活性化された試薬により発光基質を遊離する合成基質、を反応させる工程と、発光試薬を作用させて発光基質が遊離したか否かを検出する工程を順次行う技術が提案されている（特許文献１、２）。
　この技術では、少なくとも発光基質を遊離させる工程と発光試薬の添加工程の各工程を順次行う二段反応とすることが求められ、少なくとも２～３回のピペット操作が必要である。そのため、試料液に外部の微生物夾雑物の混入が起こるおそれがあり、高感度測定には不向きであると共に、操作も煩雑であった。また、発光試薬等は、時間経過と共に失活するため用時調製が必要であり、特許文献１、２のような測定方法は自動化も困難であった。 As a method for detecting microbial contaminants such as endotoxin and β-glucan with high sensitivity, a step of reacting a sample solution with a reagent that is activated by microbial contaminants and a synthetic substrate that liberates a luminescent substrate by the activated reagent In addition, techniques for sequentially performing a process of detecting whether or not a luminescent substrate is released by the action of a luminescent reagent have been proposed (Patent Documents 1 and 2).
This technique requires a two-step reaction in which at least the steps of releasing the luminescent substrate and adding the luminescent reagent are sequentially performed, and at least two to three pipetting operations are required. Therefore, there is a possibility that external microbial contaminants may be mixed in the sample solution, which is not suitable for high-sensitivity measurement and complicated in operation. In addition, since the luminescent reagent and the like are deactivated with the passage of time, preparation at the time of use is necessary, and it is difficult to automate the measurement methods such as Patent Documents 1 and 2.
　そこで、特許文献３では、容器内に試薬を固定し、導入した試料液で試薬を溶解して反応させる技術が提案されている。特許文献３では、発光基質を遊離させる工程と発光試薬の添加工程を順次行うため、隔膜で仕切られた複数の反応室を有する容器を用いることが提案されている（特許文献３の図８等）。そして、まず、発光基質を遊離させるために必要な試薬を固定した反応室に試料液を導入して反応させた後、隔膜に針等で孔を空け、この孔を通して試料液を発光試薬が固定された反応室に落下させて発光試薬と反応させることが記載されている。 Therefore, Patent Document 3 proposes a technique in which a reagent is fixed in a container, and the reagent is dissolved and reacted with the introduced sample solution. In Patent Document 3, it is proposed to use a container having a plurality of reaction chambers partitioned by a diaphragm in order to sequentially perform a step of releasing a luminescent substrate and a step of adding a luminescent reagent (FIG. 8, etc. of Patent Document 3). ). First, after introducing the sample solution into a reaction chamber in which a reagent necessary for releasing the luminescent substrate is fixed and reacting, a hole is made in the diaphragm with a needle or the like, and the sample solution is fixed to the sample solution through this hole. It is described that it is dropped into a reaction chamber and reacted with a luminescent reagent.
　特許文献３には、また、内部に微生物夾雑物によって活性化する試薬を固定した試薬収容カップを配置すると共に、天井部分に合成基質を、底部に発光試薬を各々固定した容器を用いることが提案されている（特許文献３の図１０等）。そして、まず、試薬収容カップに試料液を導入して反応させた後、天地を逆転させて、合成基質が固定された天井部分に試料液を移動させ、その後再度天地を逆転させて、発光試薬が固定された容器底部に試料液を移動させ、順次反応を進めることが記載されている。 Patent Document 3 also proposes to use a container containing a reagent containing cup in which a reagent activated by microbial contaminants is fixed, a synthetic substrate on the ceiling, and a luminescent reagent on the bottom. (FIG. 10 etc. of Patent Document 3). First, after introducing and reacting the sample solution into the reagent containing cup, the top and bottom are reversed, the sample solution is moved to the ceiling portion on which the synthetic substrate is fixed, and then the top and bottom are reversed again, so that the luminescent reagent It is described that the sample solution is moved to the bottom of the container in which is fixed, and the reaction is sequentially advanced.
国際公開第０９／０６３８４０号International Publication No. 09/063840 特開２０１０－１８７６３４号公報JP 2010-187634 A 特開２０１２－１３２８７８号公報JP 2012-132878 A
　しかし、特許文献３の隔膜を使用する方法では、特に数１００μＬ程度の少量の試料液を用いる場合、隔膜に孔を空けても試料液が隔膜に付着等して孔から抜けきらない液量が無視できず、高感度な検出が困難であった。
　また、特許文献３の図１０等の容器は、内部に試薬収容カップを設けるため、構造が複雑となる上、反応を進めるために転倒動作が必要であるため、自動化も困難であった。さらに、数１００μＬ程度の少量の試料液を用いる場合、容器を転倒させても試料液が試薬収容カップに付着等して移動しきれない液量が無視できず、高感度な検出が困難であった。 However, in the method using the diaphragm of Patent Document 3, especially when a small amount of sample liquid of about several hundred μL is used, even if a hole is made in the diaphragm, the amount of liquid that the sample liquid does not come out of the hole due to adhesion to the diaphragm etc. It could not be ignored and it was difficult to detect with high sensitivity.
Further, the container shown in FIG. 10 and the like of Patent Document 3 is provided with a reagent storage cup, so that the structure is complicated, and an overturning operation is required to advance the reaction, so that automation is difficult. Furthermore, when a small amount of sample solution of about several hundred μL is used, the amount of solution that cannot be moved due to the sample solution adhering to the reagent storage cup even if the container is turned over cannot be ignored, and high-sensitivity detection is difficult. It was.
　本発明は、上記事情に鑑みてなされたものであって、液状では不安定な試薬を試料液との反応直前まで密閉状態で保持できる試薬容器と、当該試薬容器に試薬が保持された試薬入り試薬容器を提供することを課題とする。
　また、前記試薬容器を用い、安定に保持された試薬を試料液のほぼ全量と反応させることができ、かつ、二段反応も簡便に行うことができる反応ユニットを提供することを課題とする。
　また、前記反応ユニットを用い、試料液を高感度かつ簡便に分析することが可能で、かつ、二段反応による分析も簡便に行うことができる分析システムを提供することを課題とする。 The present invention has been made in view of the above circumstances, and includes a reagent container that can hold a liquid unstable reagent in a sealed state until immediately before the reaction with the sample liquid, and a reagent containing the reagent held in the reagent container. It is an object to provide a reagent container.
It is another object of the present invention to provide a reaction unit that uses the above-described reagent container to allow a stably held reagent to react with almost the entire amount of a sample solution and to easily perform a two-stage reaction.
It is another object of the present invention to provide an analysis system that can analyze a sample solution with high sensitivity and can be easily performed using the reaction unit, and that can also perform analysis by a two-stage reaction.
　上記の課題を達成するために、本発明は以下の構成を採用した。
［１］軸方向の一端が第一の開口端とされ、他端が第二の開口端とされているシリンジと、該シリンジ内を摺動可能なプランジャを備え、
　前記プランジャは、前記シリンジの内面に摺接する摺接部と、前記シリンジの内面に摺接せず、かつ前記摺接部に囲まれている試薬保持部とを有し、
　前記試薬保持部は、前記シリンジの内部から、前記シリンジの第一の開口端の外部まで移動可能とされていることを特徴とする試薬容器。
［２］前記シリンジの側面に試薬供給孔が形成され、該試薬供給孔は、前記試薬保持部が前記シリンジの内部に位置する状態で、前記試薬保持部と連通可能とされている［１］に記載の試薬容器。
［３］前記シリンジは、軸方向の中間部に内側に突出する突出部を有する［１］または［２］に記載の試薬容器。
［４］さらに、弾性部材を備え、
　前記試薬保持部を前記シリンジの第一の開口端の外部に移動させたとき、
　前記弾性部材は、前記プランジャに、前記第二の開口端側に移動させる付勢力を与える［１］～［３］のいずれか一項に記載の試薬容器。
［５］前記シリンジは、第二の開口端またはその近傍に、外側に突出する鍔部を有する［１］～［４］のいずれか一項に記載の試薬容器。
［６］［１］～［５］のいずれか一項に記載の試薬容器と、前記試薬保持部に保持された試薬を備えることを特徴とする試薬入り試薬容器。
［７］［１］～［５］のいずれか一項に記載の試薬容器と、上端が開口端とされている有底筒状の反応容器とを備え、
　前記試薬容器は、前記シリンジの第一の開口端が前記反応容器の軸方向中間部に達するように、前記第一の開口端側から前記反応容器に挿入可能とされていることを特徴とする反応ユニット。
［８］前記試薬容器の前記試薬保持部に試薬が保持されている［７］に記載の反応ユニット。
［９］前記反応容器内に第一試薬が、前記試薬容器の試薬保持部内に第二試薬が、各々保持されている［７］に記載の反応ユニット。
［１０］［８］に記載の反応ユニットと、前記反応容器内の試料液を光学的に測定する光学測定手段を備え、
　前記試薬容器を前記反応容器に挿入した状態で、前記試薬保持部を前記シリンジの第一の開口端の外部に移動させて予め前記反応容器に導入された試料液に接触させ、前記試薬を反応させる工程と、
　前記試薬と反応後の試料液を、前記光学測定手段により測定する工程とを順次行うように構成されたことを特徴とする試料液の分析システム。
［１１］［９］に記載の反応ユニットと、前記反応容器内の試料液を光学的に測定する光学測定手段を備え、
　前記反応容器に導入された試料液と、前記第一試薬を反応させる工程と、
　前記試薬容器を前記反応容器に挿入した状態で、前記試薬保持部を前記シリンジの第一の開口端の外部に移動させて前記第一試薬と反応後の試料液に接触させ、前記第二試薬を反応させる工程と、
　前記第二試薬と反応後の試料液を、前記光学測定手段により測定する工程とを順次行うように構成されたことを特徴とする試料液の分析システム。 In order to achieve the above object, the present invention employs the following configuration.
[1] A syringe in which one end in the axial direction is a first opening end and the other end is a second opening end, and a plunger slidable in the syringe,
The plunger has a sliding contact portion that is in sliding contact with the inner surface of the syringe, and a reagent holding portion that is not in sliding contact with the inner surface of the syringe and is surrounded by the sliding contact portion,
The reagent container, wherein the reagent holding part is movable from the inside of the syringe to the outside of the first opening end of the syringe.
[2] A reagent supply hole is formed in the side surface of the syringe, and the reagent supply hole can communicate with the reagent holding unit in a state where the reagent holding unit is located inside the syringe [1]. The reagent container according to 1.
[3] The reagent container according to [1] or [2], wherein the syringe has a protruding portion that protrudes inwardly in an axial intermediate portion.
[4] Further, an elastic member is provided,
When the reagent holding part is moved outside the first open end of the syringe,
The reagent container according to any one of [1] to [3], wherein the elastic member applies an urging force for moving the plunger toward the second opening end.
[5] The reagent container according to any one of [1] to [4], wherein the syringe has a flange protruding outward at or near the second opening end.
[6] A reagent container containing the reagent container according to any one of [1] to [5] and a reagent held in the reagent holding part.
[7] A reagent container according to any one of [1] to [5], and a bottomed cylindrical reaction container having an open end at the top,
The reagent container can be inserted into the reaction container from the first opening end side so that the first opening end of the syringe reaches the intermediate portion in the axial direction of the reaction container. Reaction unit.
[8] The reaction unit according to [7], wherein the reagent is held in the reagent holding portion of the reagent container.
[9] The reaction unit according to [7], in which the first reagent is held in the reaction container and the second reagent is held in the reagent holding part of the reagent container.
[10] The reaction unit according to [8] and an optical measurement unit that optically measures the sample solution in the reaction vessel,
With the reagent container inserted into the reaction container, the reagent holding part is moved to the outside of the first opening end of the syringe and brought into contact with the sample solution previously introduced into the reaction container to react the reagent. A process of
A sample solution analysis system configured to sequentially perform the step of measuring the sample solution after reaction with the reagent by the optical measuring means.
[11] The reaction unit according to [9] and an optical measurement unit that optically measures the sample solution in the reaction container,
Reacting the sample solution introduced into the reaction vessel with the first reagent;
With the reagent container inserted into the reaction container, the reagent holding part is moved to the outside of the first open end of the syringe to contact the first reagent and the sample solution after the reaction, and the second reagent Reacting with
A sample solution analysis system configured to sequentially perform the step of measuring the sample solution after reacting with the second reagent by the optical measuring means.
　本発明の試薬容器及び試薬入り試薬容器によれば、液状では不安定な試薬を試料液との反応直前まで密閉状態で保持できる。
　また、本発明の反応ユニットによれば、安定に保持された試薬を試料液のほぼ全量と反応させることができ、かつ、二段反応も簡便に行うことができる。
　また、本発明の分析システムによれば、試料液を高感度かつ簡便に分析することが可能で、かつ、二段反応による分析も簡便に行うことができる。 According to the reagent container and reagent-containing reagent container of the present invention, a liquid unstable reagent can be held in a sealed state until immediately before the reaction with the sample liquid.
Further, according to the reaction unit of the present invention, the stably held reagent can be reacted with almost the entire amount of the sample solution, and a two-stage reaction can be easily performed.
In addition, according to the analysis system of the present invention, it is possible to analyze a sample solution with high sensitivity and simplicity, and it is also possible to easily perform analysis by a two-stage reaction.
一実施形態に係る反応ユニットを示す縦断面図であり、図１（ａ）が試薬容器、図１（ｂ）が反応容器の一態様を示す。It is a longitudinal cross-sectional view which shows the reaction unit which concerns on one Embodiment, FIG. 1 (a) shows a reagent container, FIG.1 (b) shows one aspect | mode of a reaction container. 図１（ａ）のプランジャを示す図で、図２（ａ）が斜視図、図２（ｂ）が側面図、図２（ｃ）が図２（ｂ）のII－II断面図である。2A is a perspective view, FIG. 2B is a side view, and FIG. 2C is a cross-sectional view taken along line II-II in FIG. 2B. 図１の反応ユニットに試薬を保持させる方法を説明する図で、図３（ａ）が試薬容器１０に第二試薬Ｒ２を保持させる方法を、図３（ｂ）が反応容器５０に第一試薬Ｒ１を保持させる方法を、各々示す。FIGS. 3A and 3B are diagrams for explaining a method of holding a reagent in the reaction unit of FIG. 1, FIG. 3A shows a method of holding the second reagent R2 in the reagent container 10, and FIG. Each method for retaining R1 will be described. 図１の反応ユニットを用いた分析システムと、この分析システムを用いて、二段反応後の試料液を光学的に測定する方法を示す図で、図４（ａ）が第一反応工程を、図４（ｂ）が第二反応工程を、図４（ｃ）が測定工程を、各々示す。FIG. 4A is a diagram showing an analysis system using the reaction unit of FIG. 1 and a method for optically measuring a sample solution after the two-stage reaction using this analysis system, and FIG. FIG. 4B shows the second reaction step, and FIG. 4C shows the measurement step. プランジャの変形例を示す図で、図５（ａ）が斜視図、図５（ｂ）が側面図、図５（ｃ）が図５（ｂ）と異なる方向から見た側面図、図５（ｄ）が図５（ｂ）のＶ－Ｖ断面図である。FIG. 5A is a perspective view, FIG. 5B is a side view, FIG. 5C is a side view seen from a different direction from FIG. 5B, and FIG. d) is a VV cross-sectional view of FIG. プランジャの別の変形例を示す図で、図６（ａ）が斜視図、図６（ｂ）が側面図、図６（ｃ）が図６（ｂ）と異なる方向から見た側面図である。FIGS. 6A and 6B are views showing another modification of the plunger, in which FIG. 6A is a perspective view, FIG. 6B is a side view, and FIG. 6C is a side view as seen from a different direction from FIG. . プランジャの別の変形例を示す図で、図７（ａ）が斜視図、図７（ｂ）が側面図、図７（ｃ）が図７（ｂ）のＶII－ＶII断面図である。FIG. 7A is a perspective view, FIG. 7B is a side view, and FIG. 7C is a cross-sectional view taken along the line VII-VII in FIG. 7B.
［反応ユニット］
　図１は、本発明の一実施形態に係る反応ユニットで、図１（ａ）が試薬容器の一態様、図１（ｂ）が反応容器の一態様を示す。
　図１（ａ）に示す試薬容器１０は、シリンジ２０とプランジャ３０と、バネ４０（弾性部材）とから概略構成されている。 [Reaction unit]
1A and 1B show a reaction unit according to an embodiment of the present invention, in which FIG. 1A shows an embodiment of a reagent container, and FIG. 1B shows an embodiment of a reaction container.
The reagent container 10 shown in FIG. 1A is schematically constituted by a syringe 20, a plunger 30, and a spring 40 (elastic member).
　シリンジ２０は、軸方向の一端が第一の開口端２０aとされ、他端が第二の開口端２０ｂとされ、第一の開口端２０aには開口２１ａを、第二の開口端２０ｂには開口２１ｂを、各々有している。
　また、第二の開口端２０ｂには、周方向全体にわたって径方向外側に突出する鍔部２２が設けられている。また、シリンジ２０の内面２０ｃには、径方向内側に周方向全体にわたって突出する突出部２３が設けられている。突出部２３は、シリンジ２０の軸方向の中間部であって、軸方向の中心よりもやや第一の開口端２０ａに近い部分の内面２０ｃに設けられている。 The syringe 20 has one end in the axial direction as a first opening end 20a, the other end as a second opening end 20b, an opening 21a in the first opening end 20a, and a second opening end 20b. Each has an opening 21b.
The second opening end 20b is provided with a flange 22 that protrudes radially outward over the entire circumferential direction. Moreover, the protrusion part 23 which protrudes over the whole circumferential direction is provided in the inner surface 20c of the syringe 20 at the radial inside. The protrusion 23 is an intermediate portion in the axial direction of the syringe 20 and is provided on the inner surface 20c of a portion slightly closer to the first opening end 20a than the center in the axial direction.
　突出部２３の径方向内側には、シール材２４が取り付けられている。シール材２４は管状部２４ａと、管状部２４ａの一端側において径方向外側に突出する鍔部２４ｂとから構成されている。管状部２４ａは突出部２３とプランジャ３０のプランジャ軸３１との間に介在し、プランジャ３０との摺面を構成している。また、鍔部２４ｂは突出部２３の第二の開口端２０ｂ側に密着し、バネ４０と突出部２３との間の緩衝体となっている。
　また、シリンジ２０の側面には、試薬供給孔２５が形成されている。試薬供給孔２５は、シリンジ２０の軸方向の中間部であって、突出部２３が設けられている部分よりも第一の開口端２０ａに近い部分に形成されている。 A sealing material 24 is attached to the radially inner side of the protrusion 23. The sealing material 24 includes a tubular portion 24a and a flange portion 24b that protrudes radially outward on one end side of the tubular portion 24a. The tubular portion 24 a is interposed between the protruding portion 23 and the plunger shaft 31 of the plunger 30 and constitutes a sliding surface with the plunger 30. Further, the flange portion 24 b is in close contact with the second opening end 20 b side of the protruding portion 23, and serves as a buffer between the spring 40 and the protruding portion 23.
A reagent supply hole 25 is formed on the side surface of the syringe 20. The reagent supply hole 25 is an intermediate portion in the axial direction of the syringe 20 and is formed in a portion closer to the first opening end 20a than a portion where the protruding portion 23 is provided.
　プランジャ３０は、プランジャ軸３１と、プランジャ軸３１の先端に螺合されたガスケット３２とから構成されている。
　図２を用いて詳述するように、プランジャ軸３１は、円柱状の軸本体３１ａと、軸本体３１ａの一方の端部に設けられ、軸本体３１ａよりも大径の略円盤状の押し板３１ｂと、軸本体３１ａの他方の端部に設けられ、ガスケット３２内に挿入される雄ねじ部３１ｃとを有している。
　軸本体３１ａと、押し板３１ｂと、雄ねじ部３１ｃは、剛性の材質、例えばポリプロピレン等の樹脂、ガラス、金属により一体成形されている。 The plunger 30 includes a plunger shaft 31 and a gasket 32 screwed to the tip of the plunger shaft 31.
As will be described in detail with reference to FIG. 2, the plunger shaft 31 is provided with a columnar shaft body 31a and one end of the shaft body 31a, and a substantially disk-shaped push plate having a larger diameter than the shaft body 31a. 31 b and a male screw portion 31 c that is provided at the other end of the shaft main body 31 a and is inserted into the gasket 32.
The shaft body 31a, the push plate 31b, and the male screw portion 31c are integrally formed of a rigid material, for example, a resin such as polypropylene, glass, or metal.
　ガスケット３２は、プランジャ軸３１が挿入される側から、円錐台部３２ａ、第一大径部３２ｂ、小径部３２ｃ、第二大径部３２ｄ、先端部３２ｅを有し、これらが一体に形成されている。
　第一大径部３２ｂと第二大径部３２ｄの外径は、シリンジ２０の突出部２３が設けられていない部分の内径と略同一で、これらの側面は、各々シリンジ２０の内面と摺接する第一摺接部３２Ａと第二摺接部３２Ｂとなっている。 The gasket 32 has a truncated cone part 32a, a first large diameter part 32b, a small diameter part 32c, a second large diameter part 32d, and a tip part 32e from the side where the plunger shaft 31 is inserted, and these are integrally formed. ing.
The outer diameters of the first large diameter portion 32b and the second large diameter portion 32d are substantially the same as the inner diameter of the portion of the syringe 20 where the protruding portion 23 is not provided, and these side surfaces are in sliding contact with the inner surface of the syringe 20, respectively. A first sliding contact portion 32A and a second sliding contact portion 32B are provided.
　また、小径部３２ｃの外径は、第一大径部３２ｂと第二大径部３２ｄの外径より小さくシリンジ２０の内面と摺接しない。この小径部３２ｃの側面が、第一大径部３２ｂと第二大径部３２ｄの各々の小径部３２ｃ側の面と共に、第一摺接部３２Ａと第二摺接部３２Ｂに囲まれた凹状の試薬保持部３２Ｃを構成している。なお、小径部３２ｃの両端は、漸次拡径し、第一大径部３２ｂとの境界と第二大径部３２ｄとの境界の各々において、曲面が形成されている。
　試薬供給孔２５は、プランジャ３０をガスケット３２がシリンジ２０の突出部２３に当接するまで第二の開口端２０ｂ側に引いた際に、この試薬保持部３２Ｃと連通する位置に形成されている。 Moreover, the outer diameter of the small diameter part 32c is smaller than the outer diameters of the first large diameter part 32b and the second large diameter part 32d, and does not slide on the inner surface of the syringe 20. The side surface of the small diameter portion 32c is a concave shape surrounded by the first sliding contact portion 32A and the second sliding contact portion 32B together with the surfaces of the first large diameter portion 32b and the second large diameter portion 32d on the small diameter portion 32c side. The reagent holding unit 32C is configured. Both ends of the small diameter portion 32c gradually increase in diameter, and a curved surface is formed at each of the boundary between the first large diameter portion 32b and the boundary between the second large diameter portion 32d.
The reagent supply hole 25 is formed at a position communicating with the reagent holding portion 32C when the plunger 30 is pulled toward the second opening end 20b until the gasket 32 contacts the protruding portion 23 of the syringe 20.
　円錐台部３２ａは第一大径部３２ｂより小径で、かつ第一大径部３２ｂ側の方の径が大きい円錐台状となっている。
　先端部３２ｅは、球体を、中心を通らない面で切り取った際の体積の小さい方の形状とされている。この切断面は、第二大径部３２ｄと同径の円形とされ、先端部３２ｅは、この切断面側で第二大径部３２ｄに接続している。
　また、円錐台部３２ａから小径部３２ｃの中程にかけて、プランジャ軸３１の雄ねじ部３１ｃが螺合される雌ねじ３２Ｄが形成されている。
　ガスケット３２は、弾性ゴムのようなエラストマーで構成されている。ガスケット３２の材質は、耐薬品性、耐寒性を備え、ガス透過性が低いものが好ましい。具体的には、ブチルゴム、テトラフルオロエチレン等のフッ素ゴムが挙げられる。 The truncated cone portion 32a has a truncated cone shape having a smaller diameter than the first large diameter portion 32b and a larger diameter on the first large diameter portion 32b side.
The tip 32e has a shape with a smaller volume when the sphere is cut off by a surface that does not pass through the center. The cut surface is a circle having the same diameter as the second large diameter portion 32d, and the tip end portion 32e is connected to the second large diameter portion 32d on the cut surface side.
A female screw 32D is formed from the truncated cone part 32a to the middle of the small diameter part 32c to which the male screw part 31c of the plunger shaft 31 is screwed.
The gasket 32 is made of an elastomer such as elastic rubber. The material of the gasket 32 is preferably one having chemical resistance and cold resistance and low gas permeability. Specific examples include fluororubbers such as butyl rubber and tetrafluoroethylene.
　バネ４０は、内側にプランジャ軸３１を挿通するように配置されている。バネ４０は、試薬保持部３２Ｃを第一の開口端２０ａの外部に移動させた際、突出部２３と押し板３１ｂとの間で圧縮され、プランジャ３０に対して、第二の開口端２０ｂ側に移動させる付勢力を与えるようになっている。 The spring 40 is arranged so that the plunger shaft 31 is inserted inside. The spring 40 is compressed between the protruding portion 23 and the push plate 31b when the reagent holding portion 32C is moved to the outside of the first opening end 20a, and the second opening end 20b side with respect to the plunger 30 The urging force to move to is given.
　図１（ｂ）に示す反応容器５０は、上端が開口端５１ａとされた有底筒状の反応容器本体５１と、開口端５１ａの周方向全体にわたって径方向外側に突出する鍔部５２とを有している。
　図１（ｂ）では、反応容器５０の開口端５１ａの開口５３を、蓋体５４で閉塞した状態を示している。蓋体５４は、鍔部５２と略同一の外径の蓋部５４ａと、反応容器５０の開口５３に、液密かつ気密に挿入される挿入部５４ｂと、挿入部５４ｂの先端に設けられた複数本（２本）の脚部５４ｃを有し、これらが一体に形成されている。脚部５４ｃの内側は、略円柱状の凹部５４ｄとなっており、脚部５４ｃが凹部５４ｄ側に変形可能であることにより、反応容器５０の開口５３への脚部５４ｃと挿入部５４ｂの挿入が容易となっている。 A reaction vessel 50 shown in FIG. 1 (b) includes a bottomed cylindrical reaction vessel main body 51 whose upper end is an open end 51a, and a flange 52 that protrudes radially outward over the entire circumferential direction of the open end 51a. Have.
FIG. 1B shows a state in which the opening 53 of the opening end 51 a of the reaction vessel 50 is closed with a lid 54. The lid body 54 was provided at the distal end of the insertion portion 54b, a lid portion 54a having an outer diameter substantially the same as that of the flange portion 52, an insertion portion 54b inserted into the opening 53 of the reaction vessel 50 in a liquid-tight and air-tight manner. A plurality (two) of leg portions 54c are formed integrally. The inside of the leg portion 54c is a substantially cylindrical concave portion 54d, and the leg portion 54c can be deformed to the concave portion 54d side, so that the leg portion 54c and the insertion portion 54b are inserted into the opening 53 of the reaction vessel 50. Is easy.
　試薬容器１０は、図４（ａ）に示すように、第一の開口端２０a側から反応容器５０に挿入可能とされている。また、シリンジ２０の鍔部２２が反応容器５０の鍔部５２に当接することにより、シリンジ２０の第一の開口端２０aは、反応容器５０の軸方向中間部の位置に留まり、反応容器５０の底部まで到達しないようになっている。
　また、図４（ｂ）に示すように、試薬容器１０を反応容器５０に挿入した状態で、プランジャ３０のガスケット３２を、第一の開口端２０aの外部まで移動させ、ガスケット３２を反応容器５０の底部または底部近傍まで到達させられるようになっている。 As shown in FIG. 4A, the reagent container 10 can be inserted into the reaction container 50 from the first opening end 20a side. Further, when the flange portion 22 of the syringe 20 abuts on the flange portion 52 of the reaction vessel 50, the first open end 20 a of the syringe 20 remains at the position in the middle portion in the axial direction of the reaction vessel 50. It does not reach the bottom.
4B, with the reagent container 10 inserted into the reaction container 50, the gasket 32 of the plunger 30 is moved to the outside of the first open end 20a, and the gasket 32 is moved to the reaction container 50. It can be made to reach to the bottom or near the bottom.
［試薬の保持］
　図３は図１の反応ユニットに試薬を保持させる方法を説明する図で、図３（ａ）が試薬容器１０に第二試薬Ｒ２を保持させる方法を、図３（ｂ）が反応容器５０に第一試薬Ｒ１を保持させる方法を、各々示す。 [Reagent retention]
FIG. 3 is a view for explaining a method of holding the reagent in the reaction unit of FIG. 1, FIG. 3 (a) shows a method of holding the second reagent R 2 in the reagent container 10, and FIG. 3 (b) shows a reaction container 50. Each of the methods for retaining the first reagent R1 will be described.
　図３（ａ）に示すように、第二試薬Ｒ２の保持作業は、試薬容器１０を、試薬供給孔２５を上側とした横置きの状態として行う。また、プランジャ３０を、ガスケット３２がシリンジ２０の突出部２３に当接するまで第二の開口端２０ｂ側に引いた位置とし、試薬供給孔２５が、試薬保持部３２Ｃと連通する状態として行う。
　第二試薬Ｒ２の保持作業としては、まず、試薬供給孔２５から試薬保持部３２Ｃに向けて、第二試薬Ｒ２を適当な溶媒（例えば水）に溶解した溶液を充填する。その後、試薬供給孔２５を試薬保持部３２Ｃに連通させたままの状態で凍結乾燥することにより、第二試薬Ｒ２を乾燥状態で試薬保持部３２Ｃに保持させることができる。 As shown in FIG. 3A, the holding operation of the second reagent R2 is performed in a state in which the reagent container 10 is placed horizontally with the reagent supply hole 25 on the upper side. The plunger 30 is pulled to the second opening end 20b side until the gasket 32 comes into contact with the protruding portion 23 of the syringe 20, and the reagent supply hole 25 communicates with the reagent holding portion 32C.
As a holding operation of the second reagent R2, first, a solution in which the second reagent R2 is dissolved in an appropriate solvent (for example, water) is filled from the reagent supply hole 25 toward the reagent holding unit 32C. Thereafter, the second reagent R2 can be held in the reagent holding unit 32C in a dry state by lyophilization while the reagent supply hole 25 is in communication with the reagent holding unit 32C.
　その後、プランジャ３０を図１（ａ）に示す位置まで移動させると、試薬保持部３２Ｃは、試薬保持部３２Ｃを囲む第一摺接部３２Ａと第二摺接部３２Ｂ、及びシリンジ２０の内面２０ｃで密閉された空間内に収容されるので、保持された第二試薬Ｒ２を、密閉状態で保管することができる。
　なお、凍結乾燥後、第二試薬Ｒ２は、試薬保持部３２Ｃだけでなく、シリンジ２０の内面２０ｃにも付着しうる。しかし、プランジャ３０を図１（ａ）に示す位置まで移動させると、シリンジ２０の内面２０ｃにも付着した第二試薬Ｒ２は、第一摺接部３２Ａによって擦り取られる。そして、移動後の試薬保持部３２Ｃと、移動後の試薬保持部３２Ｃに対向するシリンジ２０の内面２０ｃに囲まれた領域まで移動するので、ほぼ全量を試薬保持部３２Ｃとその近傍に保持しておくことができる。
　第二試薬Ｒ２を保持させる上記作業は、真空又は不活性ガス雰囲気内で行うことが好ましい。 Thereafter, when the plunger 30 is moved to the position shown in FIG. 1A, the reagent holding portion 32C causes the first sliding contact portion 32A and the second sliding contact portion 32B surrounding the reagent holding portion 32C, and the inner surface 20c of the syringe 20 to move. Therefore, the held second reagent R2 can be stored in a sealed state.
Note that after lyophilization, the second reagent R2 may adhere not only to the reagent holding part 32C but also to the inner surface 20c of the syringe 20. However, when the plunger 30 is moved to the position shown in FIG. 1A, the second reagent R2 attached to the inner surface 20c of the syringe 20 is scraped off by the first sliding contact portion 32A. And since it moves to the area | region enclosed by the reagent holding part 32C after movement, and the inner surface 20c of the syringe 20 which opposes the reagent holding part 32C after movement, substantially the whole quantity is hold | maintained at the reagent holding part 32C and its vicinity. I can leave.
The above operation for holding the second reagent R2 is preferably performed in a vacuum or an inert gas atmosphere.
　図３（ｂ）に示すように、第一試薬Ｒ１の保持作業は、反応容器５０を、開口端５１ａを上側とした縦置きの状態として行う。
　第一試薬Ｒ１の保持作業としては、まず、蓋体５４を外し、開口５３から第一試薬Ｒ１を適当な溶媒（例えば水）に溶解した溶液を充填する。その後、開口５３が外部と連通した状態を保つように、脚部５４ｃの一部が開口端５１ａの外側に露出する程度に蓋体５４を開口５３に挿入した状態で凍結乾燥することにより、第一試薬Ｒ１を乾燥状態で反応容器５０内に保持することができる。
　その後、蓋体５４を図１（ｂ）に示す位置まで押し込むと、反応容器５０内に保持された第一試薬Ｒ１を、密閉状態で保管することができる。
　第一試薬Ｒ１を保持させる上記作業は、真空又は不活性ガス雰囲気内で行うことが好ましい。 As shown in FIG. 3B, the holding operation of the first reagent R1 is performed with the reaction vessel 50 placed in a vertical state with the open end 51a on the upper side.
As a holding operation of the first reagent R1, first, the lid 54 is removed, and a solution in which the first reagent R1 is dissolved in an appropriate solvent (for example, water) is filled from the opening 53. Thereafter, the lid 54 is lyophilized with the lid 54 inserted into the opening 53 so that a part of the leg 54c is exposed to the outside of the opening end 51a so as to keep the opening 53 in communication with the outside. One reagent R1 can be held in the reaction vessel 50 in a dry state.
Thereafter, when the lid 54 is pushed into the position shown in FIG. 1B, the first reagent R1 held in the reaction vessel 50 can be stored in a sealed state.
The above operation for holding the first reagent R1 is preferably performed in a vacuum or an inert gas atmosphere.
［分析システム］
　図４は図１の反応ユニットを用いた分析システムと、この分析システムを用いて、二段反応後の試料液を光学的に測定する方法を示す図である。
　図４の分析システムは、図１の反応ユニットと光学測定手段５５から構成されている。
　光学測定手段５５としては、反応ユニットの反応容器５０内から発せられる光（蛍光や生物発光を含む）を検出する光検出装置、光源と、反応容器５０を透過した光を検出する受光装置とからなる吸光度検出装置、光源と、反応容器５０内で散乱された光を検出する受光装置とからなる散乱光検出装置等を適宜採用できる。
　図４の分析システムは、反応の進行を促進するため、反応容器５０を適宜加温する加温手段を備えていてもよい。 [Analysis system]
FIG. 4 is a diagram showing an analysis system using the reaction unit of FIG. 1 and a method for optically measuring a sample solution after the two-stage reaction using this analysis system.
The analysis system shown in FIG. 4 includes the reaction unit shown in FIG.
The optical measuring means 55 includes a light detection device that detects light (including fluorescence and bioluminescence) emitted from the reaction container 50 of the reaction unit, a light source, and a light receiving device that detects light transmitted through the reaction container 50. A scattered light detection device comprising a light-absorbance detection device, a light source, and a light-receiving device that detects light scattered in the reaction vessel 50 can be appropriately employed.
The analysis system of FIG. 4 may include a heating means for appropriately heating the reaction vessel 50 in order to promote the progress of the reaction.
　図４の分析システムは、図示を省略する制御装置により、その動作が制御され、反応ユニットに対して、以下の工程を順次行うようになっている。
（ｉ）反応容器５０に導入された試料液Ｓと、第一試薬Ｒ１を反応させる工程（以下「第一反応工程」という。）。
（ii）試薬容器１０を反応容器５０に挿入した状態で、試薬保持部３２Ｃをシリンジ２０の第一の開口端２０ａの外部に移動させて第一試薬Ｒ１と反応後の試料液Ｓに接触させ、第二試薬Ｒ２を反応させる工程（以下「第二反応工程」という。）。
（iii）第二試薬Ｒ２と反応後の試料液Ｓを、光学測定手段５５により測定する工程（以下「測定工程」という。）。 The operation of the analysis system in FIG. 4 is controlled by a control device (not shown), and the following steps are sequentially performed on the reaction unit.
(I) A step of reacting the sample solution S introduced into the reaction vessel 50 with the first reagent R1 (hereinafter referred to as “first reaction step”).
(Ii) With the reagent container 10 inserted into the reaction container 50, the reagent holding part 32C is moved to the outside of the first open end 20a of the syringe 20 so as to contact the first reagent R1 and the sample liquid S after the reaction. And a step of reacting the second reagent R2 (hereinafter referred to as “second reaction step”).
(Iii) A step of measuring the sample solution S after the reaction with the second reagent R2 by the optical measuring means 55 (hereinafter referred to as “measurement step”).
　図４の分析システムで上記各工程を行って二段反応を利用した分析を行うためには、第一反応工程に先立ち、以下の準備が必要である。まず、第一試薬Ｒ１が保持され、蓋体５４により密閉された反応容器５０、並びに第二試薬Ｒ２が試薬保持部３２Ｃに保持され、図１（ａ）に示す状態で試薬保持部３２Ｃが密閉された空間に収容された試薬容器１０（試薬入り試薬容器）を用意する。
　次に、第一試薬Ｒ１が保持された反応容器５０から蓋体５４を外し、試料液Ｓを入れる。その後、図４（ａ）に示すように、反応容器５０に試薬容器１０を挿入した状態で、分析システムにセッティングする。
　ここまでの作業は人手により行うことが好ましいが、分析システムにサンプリング機構を追加して自動化してもよい。反応容器５０に試料液Ｓを導入すると、導入した試料液Ｓによって第一試薬Ｒ１が溶解され、試料液Ｓと第一試薬Ｒ１とが反応可能となる。 In order to perform an analysis using the two-stage reaction by performing the above steps with the analysis system of FIG. 4, the following preparation is required prior to the first reaction step. First, the reaction container 50 in which the first reagent R1 is held and sealed by the lid 54, and the second reagent R2 are held in the reagent holding part 32C, and the reagent holding part 32C is sealed in the state shown in FIG. The reagent container 10 (reagent-containing reagent container) accommodated in the space thus prepared is prepared.
Next, the lid 54 is removed from the reaction vessel 50 in which the first reagent R1 is held, and the sample solution S is put therein. Thereafter, as shown in FIG. 4A, the reagent container 10 is inserted into the reaction container 50 and set in the analysis system.
The operation so far is preferably performed manually, but may be automated by adding a sampling mechanism to the analysis system. When the sample solution S is introduced into the reaction vessel 50, the first reagent R1 is dissolved by the introduced sample solution S, and the sample solution S and the first reagent R1 can react.
　第一反応工程は、反応ユニットを分析システムにセッティングしてから、次の第二反応工程を開始するまでの間の工程である。
　第一反応工程の時間は、反応容器５０に導入された試料液Ｓと第一試薬Ｒ１とを反応させるために必要な反応時間を考慮して適宜設定する。試料液Ｓと第一試薬Ｒ１との反応が速い場合は、システムの動作上物理的に必要な時間のみをとって、次の第二反応工程に移行すればよい。第一反応工程では、反応を促進するため反応容器５０を適宜加温することができる。 The first reaction step is a step from setting the reaction unit to the analysis system to starting the next second reaction step.
The time of the first reaction step is appropriately set in consideration of the reaction time necessary for reacting the sample solution S introduced into the reaction vessel 50 with the first reagent R1. When the reaction between the sample solution S and the first reagent R1 is fast, only the time physically required for the operation of the system may be taken and the process proceeds to the next second reaction step. In the first reaction step, the reaction vessel 50 can be appropriately heated to promote the reaction.
　第二反応工程では、図示を省略する押棒により、プランジャ３０の押し板３１ｂを押してプランジャ３０を押し下げる。これにより、ガスケット３２をシリンジ２０の内部から第一の開口端２０ａの外部まで移動させ、図４（ｂ）に示すように、ガスケット３２の先端を反応容器５０の底部まで到達させる。このようにして、試薬保持部３２Ｃ全体を第一試薬Ｒ１と反応後の試料液Ｓ内に浸漬させる。 In the second reaction step, the push plate 31b of the plunger 30 is pushed by the push rod (not shown) to push the plunger 30 down. Thereby, the gasket 32 is moved from the inside of the syringe 20 to the outside of the first open end 20a, and the tip of the gasket 32 is made to reach the bottom of the reaction vessel 50 as shown in FIG. In this way, the entire reagent holding part 32C is immersed in the sample solution S after the reaction with the first reagent R1.
　試薬保持部３２Ｃが第一試薬Ｒ１と反応後の試料液Ｓ内に浸漬されると、試料液Ｓによって第二試薬Ｒ２が溶解され、試料液Ｓと第二試薬Ｒ２とが反応可能となる。なお、第二試薬Ｒ２は、試薬保持部３２Ｃに直接固定されるだけでなく、試薬保持部３２Ｃと試薬保持部３２Ｃに対向するシリンジ２０の内面２０ｃに囲まれた領域にも保持されうる。しかし、この領域内の第二試薬Ｒ２は、第一摺接部３２Ａによって押し出すことができ、ほぼ全量を第一試薬Ｒ１と反応後の試料液Ｓ内に入れ、反応させることができる。 When the reagent holding part 32C is immersed in the sample solution S after reacting with the first reagent R1, the second reagent R2 is dissolved by the sample solution S, and the sample solution S and the second reagent R2 can react. The second reagent R2 is not only directly fixed to the reagent holding part 32C, but can also be held in a region surrounded by the reagent holding part 32C and the inner surface 20c of the syringe 20 facing the reagent holding part 32C. However, the second reagent R2 in this region can be pushed out by the first sliding contact portion 32A, and almost the entire amount can be put into the sample solution S after the reaction with the first reagent R1 to be reacted.
　プランジャ３０を図４（ｂ）に示す状態に留めておく時間は、第二試薬Ｒ２が溶解されるのに充分であればよく、第二試薬Ｒ２の溶解性が高ければ、プランジャ３０を押し下げた後、直ちに測定工程に移行することができる。
　たとえば、蛍光測定や生物発光測定のように、試料液Ｓと第二試薬Ｒ２の反応開始後、検出すべき光の発生が短時間で終了するような反応系の場合、プランジャ３０を図４（ｂ）に示す状態に押し下げた後、直ちに測定工程に移行することが好ましい。
　試料液Ｓへの第二試薬Ｒ２の溶解や、試料液Ｓと第二試薬Ｒ２との反応に時間を要する場合は、適宜の待機時間経過後、測定工程に移行する。 The time for keeping the plunger 30 in the state shown in FIG. 4 (b) only needs to be sufficient to dissolve the second reagent R2. If the solubility of the second reagent R2 is high, the plunger 30 is pushed down. Thereafter, the measurement process can be immediately performed.
For example, in the case of a reaction system in which the generation of light to be detected is completed in a short time after the start of the reaction between the sample solution S and the second reagent R2, such as fluorescence measurement or bioluminescence measurement, the plunger 30 is shown in FIG. It is preferable to immediately shift to the measuring step after pushing down to the state shown in b).
When time is required for the dissolution of the second reagent R2 in the sample solution S and the reaction between the sample solution S and the second reagent R2, the process proceeds to the measurement step after an appropriate waiting time has elapsed.
　測定工程では、図示を省略する押棒によるプランジャ３０の押し板３１ｂの押圧を解除する。すると、図４（ｃ）に示すように、図４（ｂ）で圧縮されていたバネ４０の付勢力により押し板３１ｂが第二の開口端２０ｂ側に押し上げられる。これにより、光路を妨げるガスケット３２が反応容器５０の底部から離れ、第二試薬Ｒ２と反応後の試料液Ｓを光学測定手段５５により測定可能となる。
　測定工程では、光学測定手段５５により、第二試薬Ｒ２と反応後の試料液Ｓを光学的に測定する。
　光学的な測定の種類に特に限定はなく、蛍光検出、生物発光検出、吸光度検出、散乱光検出等を適宜採用できる。 In the measurement process, the pressing of the push plate 31b of the plunger 30 by a push rod (not shown) is released. Then, as shown in FIG. 4C, the pressing plate 31b is pushed up to the second opening end 20b side by the urging force of the spring 40 compressed in FIG. 4B. Thereby, the gasket 32 that obstructs the optical path is separated from the bottom of the reaction vessel 50, and the second reagent R <b> 2 and the sample solution S after the reaction can be measured by the optical measuring means 55.
In the measurement step, the optical measurement means 55 optically measures the second reagent R2 and the sample solution S after the reaction.
The type of optical measurement is not particularly limited, and fluorescence detection, bioluminescence detection, absorbance detection, scattered light detection, and the like can be appropriately employed.
　反応ユニットが複数ある場合、以上の操作を、各反応ユニットに対して順次行う。
　本実施形態によれば、各反応ユニットに導入した試料液について、所定のタイムテーブルに従って分析を行うことにより、光学的測定の条件を容易に均一化できる。そのため、測定条件の変動による検出誤差を排除しやすい。
　また、第一試薬Ｒ１と第二試薬Ｒ２は、ほぼ全量が反応容器５０内での反応に使用される。また、試料液Ｓは、反応容器５０に入れた後、他の場所に移動させる必要がない。そのため、試料液Ｓが少量であっても、高い精度でかつ再現性よく分析できる。 When there are a plurality of reaction units, the above operation is sequentially performed on each reaction unit.
According to this embodiment, the conditions of optical measurement can be easily uniformed by analyzing the sample liquid introduced into each reaction unit according to a predetermined time table. Therefore, it is easy to eliminate detection errors due to variations in measurement conditions.
In addition, almost all of the first reagent R1 and the second reagent R2 are used for the reaction in the reaction vessel 50. Further, after the sample solution S is put in the reaction vessel 50, it is not necessary to move it to another place. Therefore, even if the sample liquid S is small, it can be analyzed with high accuracy and good reproducibility.
［プランジャの変形例］
　上記実施形態では、試薬容器に用いるプランジャを図２に記載した構造としたが、プランジャの具体的構造に特に限定はなく、図２のプランジャ３０に代えて、例えば、図５～７に示すプランジャ６０、７０、８０を用いてもよい。 [Modification of plunger]
In the above embodiment, the plunger used for the reagent container has the structure shown in FIG. 2, but the specific structure of the plunger is not particularly limited. For example, the plunger shown in FIGS. 60, 70, and 80 may be used.
　図５のプランジャ６０は、プランジャ軸６１と、プランジャ軸６１の先端に螺合されたガスケット６２とから構成されている。
　プランジャ軸６１は、円柱状の軸本体６１ａと、軸本体６１ａの一方の端部に設けられ、軸本体６１ａよりも大径の略円盤状の押し板６１ｂと、軸本体６１ａの他方の端部に設けられ、ガスケット６２内に挿入される雄ねじ部６１ｃとを有している。
　軸本体６１ａと、押し板６１ｂと、雄ねじ部６１ｃは一体成形されており、図２の軸本体３１ａ、押し板３１ｂ、雄ねじ部３１ｃと同様の材質のものを使用できる。 The plunger 60 shown in FIG. 5 includes a plunger shaft 61 and a gasket 62 screwed onto the tip of the plunger shaft 61.
The plunger shaft 61 is provided at a cylindrical shaft main body 61a, one end of the shaft main body 61a, a substantially disc-shaped push plate 61b having a larger diameter than the shaft main body 61a, and the other end of the shaft main body 61a. And a male thread portion 61c inserted into the gasket 62.
The shaft main body 61a, the push plate 61b, and the male screw portion 61c are integrally formed, and the same material as the shaft main body 31a, the push plate 31b, and the male screw portion 31c in FIG. 2 can be used.
　ガスケット６２は、プランジャ軸６１が挿入される側から、円錐台部６２ａ、第一大径部６２ｂ、側壁部６２ｃ、第二大径部６２ｄ、先端部６２ｅを有し、これらが一体に形成されている。
　第一大径部６２ｂと第二大径部６２ｄの外径は、シリンジ２０の突出部２３が設けられていない部分の内径と略同一で、これらの側面は、各々シリンジ２０の内面と摺接する第一摺接部６２Ａと第二摺接部６２Ｂとなっている。 The gasket 62 has a truncated cone part 62a, a first large diameter part 62b, a side wall part 62c, a second large diameter part 62d, and a tip part 62e from the side where the plunger shaft 61 is inserted, and these are integrally formed. ing.
The outer diameters of the first large diameter portion 62b and the second large diameter portion 62d are substantially the same as the inner diameter of the portion of the syringe 20 where the protruding portion 23 is not provided, and these side surfaces are in sliding contact with the inner surface of the syringe 20, respectively. A first sliding contact portion 62A and a second sliding contact portion 62B are provided.
　側壁部６２ｃは、第一大径部６２ｂ及び第二大径部６２ｄと同径の円柱を、底面の中心を通らない縦方向の面で切断した際の体積の小さい方の形状とされている。残された円柱の側面に該当する部分は、シリンジ２０の内面と摺接する第三摺接部６２Ｃとなっている。また、円柱の切断面に該当する側面は、シリンジ２０の内面と摺接しない。この側面が、第一大径部６２ｂと第二大径部６２ｄの各々の側壁部６２ｃ側の面と共に、第一摺接部６２Ａと第二摺接部６２Ｂと第三摺接部６２Ｃに囲まれた凹状の試薬保持部６２Ｄを構成している。
　試薬供給孔２５は、プランジャ６０をガスケット６２がシリンジ２０の突出部２３に当接するまで第二の開口端２０ｂ側に引いた際に、この試薬保持部６２Ｄと連通する位置とされている。 The side wall part 62c has a shape with a smaller volume when a cylinder having the same diameter as the first large diameter part 62b and the second large diameter part 62d is cut by a vertical surface that does not pass through the center of the bottom surface. . The portion corresponding to the remaining side surface of the cylinder is a third sliding contact portion 62 </ b> C that is in sliding contact with the inner surface of the syringe 20. Further, the side surface corresponding to the cut surface of the cylinder does not slide in contact with the inner surface of the syringe 20. This side surface is surrounded by the first sliding contact portion 62A, the second sliding contact portion 62B, and the third sliding contact portion 62C together with the surfaces of the first large diameter portion 62b and the second large diameter portion 62d on the side wall portion 62c side. The concave reagent holding part 62D is formed.
The reagent supply hole 25 is positioned to communicate with the reagent holding portion 62D when the plunger 60 is pulled toward the second opening end 20b until the gasket 62 comes into contact with the protruding portion 23 of the syringe 20.
　円錐台部６２ａは第一大径部６２ｂより小径で、かつ第一大径部６２ｂ側の方の径が大きい円錐台状となっている。
　先端部６２ｅは、球体を、中心を通らない面で切り取った際の体積の小さい方の形状とされている。この切断面は、第二大径部６２ｄと同径の円形とされ、先端部６２ｅは、この切断面側で第二大径部６２ｄに接続している。
　また、円錐台部６２ａ内に、プランジャ軸６１の雄ねじ部６１ｃが螺合される雌ねじ６２Ｅが形成されている。
　ガスケット６２は、図２のガスケット３２と同様のエラストマーで構成することができる。 The truncated cone part 62a has a truncated cone shape having a smaller diameter than the first large diameter part 62b and a larger diameter on the first large diameter part 62b side.
The distal end portion 62e has a shape with a smaller volume when the sphere is cut off by a surface that does not pass through the center. The cut surface is circular with the same diameter as the second large diameter portion 62d, and the tip end portion 62e is connected to the second large diameter portion 62d on the cut surface side.
In addition, a female screw 62E into which the male screw portion 61c of the plunger shaft 61 is screwed is formed in the truncated cone portion 62a.
The gasket 62 can be made of an elastomer similar to the gasket 32 of FIG.
　図６のプランジャ７０は、プランジャ軸７１と、プランジャ軸７１の先端に螺合されたガスケット７２とから構成されている。
　プランジャ軸７１は、円柱状の軸本体７１ａと、軸本体７１ａの一方の端部に設けられ、軸本体７１ａよりも大径の略円盤状の押し板７１ｂと、軸本体７１ａの他方の端部に設けられ、ガスケット７２内に挿入される雄ねじ部７１ｃとを有している。
　軸本体７１ａと、押し板７１ｂと、雄ねじ部７１ｃは一体成形されており、図２の軸本体３１ａ、押し板３１ｂ、雄ねじ部３１ｃと同様の材質のものを使用できる。 The plunger 70 in FIG. 6 includes a plunger shaft 71 and a gasket 72 that is screwed onto the tip of the plunger shaft 71.
The plunger shaft 71 is provided at a columnar shaft main body 71a, one end of the shaft main body 71a, a substantially disk-shaped push plate 71b having a larger diameter than the shaft main body 71a, and the other end of the shaft main body 71a. And a male threaded portion 71c inserted into the gasket 72.
The shaft body 71a, the push plate 71b, and the male screw portion 71c are integrally formed, and the same material as the shaft main body 31a, the push plate 31b, and the male screw portion 31c in FIG. 2 can be used.
　ガスケット７２は、プランジャ軸７１が挿入される側から、円錐台部７２ａ、貫通孔形成部７２ｂ、先端部７２ｃを有し、これらが一体に形成されている。
　貫通孔形成部７２ｂは、外径がシリンジ２０の突出部２３が設けられていない部分の内径と略同一の円柱に、円柱状の貫通孔７２ｄが径方向に形成された構造とされている。
　貫通孔形成部７２ｂの貫通孔７２ｄが形成されている以外の部分の側面は、シリンジ２０の内面と摺接する摺接部７２Ａとなっている。貫通孔形成部７２ｂの貫通孔７２ｄが形成された内面は、シリンジ２０の内面と摺接しない試薬保持部７２Ｂとされており、摺接部７２Ａに囲まれている。 The gasket 72 has a truncated cone part 72a, a through-hole forming part 72b, and a tip part 72c from the side where the plunger shaft 71 is inserted, and these are integrally formed.
The through-hole forming portion 72b has a structure in which a cylindrical through-hole 72d is formed in a radial direction in a column that is substantially the same as the inner diameter of a portion where the protruding portion 23 of the syringe 20 is not provided.
The side surface of the portion other than the through hole 72 d of the through hole forming portion 72 b is a sliding contact portion 72 A that is in sliding contact with the inner surface of the syringe 20. The inner surface of the through hole forming portion 72b where the through hole 72d is formed is a reagent holding portion 72B that does not slide in contact with the inner surface of the syringe 20, and is surrounded by the sliding contact portion 72A.
　プランジャ３０に代えてプランジャ７０を用いる場合、試薬供給孔２５は、プランジャ７０をガスケット７２がシリンジ２０の突出部２３に当接するまで第二の開口端２０ｂ側に引いた際に、この試薬保持部７２Ｂ（貫通孔７２ｄ）と連通する位置に形成されている。
　なお、プランジャ７０を用いる場合、シリンジ２０の径方向の対向する２カ所に、試薬保持部７２Ｂ（貫通孔７２ｄ）と連通する試薬供給孔を各々形成してもよい。 When the plunger 70 is used instead of the plunger 30, the reagent supply hole 25 is configured such that when the plunger 70 is pulled toward the second opening end 20 b until the gasket 72 comes into contact with the protruding portion 23 of the syringe 20, It is formed at a position communicating with 72B (through hole 72d).
In the case where the plunger 70 is used, reagent supply holes communicating with the reagent holding part 72B (through hole 72d) may be respectively formed at two opposing positions in the radial direction of the syringe 20.
　円錐台部７２ａは貫通孔形成部７２ｂより小径で、かつ貫通孔形成部７２ｂ側の方の径が大きい円錐台状となっている。
　先端部７２ｃは、球体を、中心を通らない切断面で切り取った際の体積の小さい方の形状とされている。この切断面は、貫通孔形成部７２ｂと同径の円形とされ、先端部７２ｃは、この切断面側で貫通孔形成部７２ｂに接続している。
　また、円錐台部７２ａ内に、プランジャ軸７１の雄ねじ部７１ｃが螺合される雌ねじ７２Ｄが形成されている。
　ガスケット７２は、図２のガスケット３２と同様のエラストマーで構成することができる。 The truncated cone part 72a has a truncated cone shape having a smaller diameter than the through hole forming part 72b and a larger diameter on the through hole forming part 72b side.
The tip 72c has a shape with a smaller volume when the sphere is cut off by a cut surface that does not pass through the center. The cut surface is a circle having the same diameter as the through-hole forming portion 72b, and the distal end portion 72c is connected to the through-hole forming portion 72b on the cut surface side.
Further, a female screw 72D into which the male screw portion 71c of the plunger shaft 71 is screwed is formed in the truncated cone portion 72a.
The gasket 72 can be made of the same elastomer as the gasket 32 of FIG.
　図７のプランジャ８０は、プランジャ軸８１と、プランジャ軸８１の一方の端部側に取り付けられたＯリング８２a、８２ｂと、プランジャ軸８１の他方の端部に螺合された押し板８３とから構成されている。
　プランジャ軸８１は、円柱状の軸本体８１ａと、軸本体８１ａの一方の端部近傍に設けられたＯリング取り付け板８１ｃと、軸本体８１ａの一方の端部に設けられたＯリング取り付け板８１ｄとを有している。Ｏリング取り付け板８１ｃとＯリング取り付け板８１ｄは、各々軸本体８１ａの軸方向と垂直な面を有する略円盤状で、その周面にＯリングを取り付けるための溝が形成されている。
　軸本体８１ａと、Ｏリング取り付け板８１ｃと、Ｏリング取り付け板８１ｄは一体成形されており、図２の軸本体３１ａ、押し板３１ｂ、雄ねじ部３１ｃと同様の材質のものを使用できる。軸本体８１ａの他方の端部には、雌ねじ８１ｂが形成されている。
　また、押し板８３は、軸本体８１ａよりも大径の略円盤状の押し板本体８３ａと、押し板本体８３ａに垂設され、雌ねじ８１ｂと螺合する雄ねじ部８３ｂを有している。
　押し板本体８３ａと雄ねじ部８３ｂは一体成形されており、図２の軸本体３１ａ、押し板３１ｂ、雄ねじ部３１ｃと同様の材質のものを使用できる。
　Ｏリング８２a、８２ｂは、図２のガスケット３２ｃと同様の材質のものを使用できる。 7 includes a plunger shaft 81, O- rings 82 a and 82 b attached to one end side of the plunger shaft 81, and a push plate 83 screwed to the other end portion of the plunger shaft 81. It is configured.
The plunger shaft 81 includes a cylindrical shaft main body 81a, an O-ring mounting plate 81c provided near one end of the shaft main body 81a, and an O-ring mounting plate 81d provided at one end of the shaft main body 81a. And have. Each of the O-ring mounting plate 81c and the O-ring mounting plate 81d has a substantially disc shape having a surface perpendicular to the axial direction of the shaft main body 81a, and a groove for mounting the O-ring is formed on the peripheral surface.
The shaft main body 81a, the O-ring mounting plate 81c, and the O-ring mounting plate 81d are integrally formed, and the same material as the shaft main body 31a, the push plate 31b, and the male screw portion 31c in FIG. 2 can be used. A female screw 81b is formed at the other end of the shaft body 81a.
Further, the push plate 83 has a substantially disk-like push plate main body 83a having a diameter larger than that of the shaft main body 81a, and a male screw portion 83b that is suspended from the push plate main body 83a and screwed into the female screw 81b.
The push plate main body 83a and the male screw portion 83b are integrally formed, and the same material as the shaft main body 31a, the push plate 31b, and the male screw portion 31c in FIG. 2 can be used.
The O- rings 82a and 82b can be made of the same material as the gasket 32c of FIG.
　Ｏリング８２a、８２ｂは、各々Ｏリング取り付け板８１ｃ、８１ｄに取り付けられている。Ｏリング８２a、８２ｂは、Ｏリング取り付け板８１ｃ、８１ｄに取り付けられた状態において、その外径が、シリンジ２０の突出部２３が設けられていない部分の内径と略同一となるように構成されている。Ｏリング８２aの外周は、シリンジ２０の内面と摺接する第一摺接部８２Ａとなり、Ｏリング８２ｂの外周は、シリンジ２０の内面と摺接する第二摺接部８２Ｂとなっている。
　また、第一摺接部８２Ａと第二摺接部８２Ｂに挟まれた部分（囲まれた部分）は、試薬保持部８２Ｃとされている。試薬保持部８２Ｃはシリンジ２０の内面と摺接しない。 The O- rings 82a and 82b are attached to O- ring attachment plates 81c and 81d, respectively. The O- rings 82a and 82b are configured such that the outer diameter of the O- rings 82a and 82b is substantially the same as the inner diameter of the portion of the syringe 20 where the protrusion 23 is not provided. Yes. The outer periphery of the O-ring 82a is a first sliding contact portion 82A that is in sliding contact with the inner surface of the syringe 20, and the outer periphery of the O-ring 82b is a second sliding contact portion 82B that is in sliding contact with the inner surface of the syringe 20.
Further, a portion sandwiched between the first sliding contact portion 82A and the second sliding contact portion 82B (the surrounded portion) is a reagent holding portion 82C. The reagent holding part 82C does not slide in contact with the inner surface of the syringe 20.
　プランジャ３０に代えてプランジャ８０を用いる場合、試薬供給孔２５は、プランジャ８０をＯリング取り付け板８１ｃがシリンジ２０の突出部２３に当接するまで第二の開口端２０ｂ側に引いた際に、この試薬保持部８２Ｃと連通する位置に形成されている。
　プランジャ８０をシリンジ２０内に配置するためには、押し板８３をプランジャ軸８１から外した状態でプランジャ軸８１をシリンジ２０の第一の開口端２０a側から挿入し、その後、押し板８３をプランジャ軸８１に螺合すればよい。 When the plunger 80 is used instead of the plunger 30, the reagent supply hole 25 is formed when the plunger 80 is pulled toward the second opening end 20 b until the O-ring attachment plate 81 c contacts the protruding portion 23 of the syringe 20. It is formed at a position communicating with the reagent holding part 82C.
In order to dispose the plunger 80 in the syringe 20, the plunger shaft 81 is inserted from the first opening end 20a side of the syringe 20 with the push plate 83 removed from the plunger shaft 81, and then the push plate 83 is moved to the plunger 20 What is necessary is just to screw to the axis | shaft 81.
　プランジャ３０とプランジャ８０では、周方向全体にわたる試薬保持部が形成されているが、プランジャ６０とプランジャ７０の例から理解できるように、試薬保持部は、周方向全体にわたっていなくともよい。
　また、上記各プランジャでは、摺接部の一部または全部が、試薬保持部を軸方向において挟む二カ所にあり、各々が、周方向全体にわたって連続している。
　しかし、試薬保持部と摺接部との関係は、そのような態様には限定されず、試薬保持部が摺接部に囲まれており、試薬保持部とシリンジ２０の内壁との間に、密閉空間を形成できるようになっていればよい。例えばプランジャ６０の側壁部６２ｃの側面に、試薬保持部６２Ｄから離間した位置において、縦方向の溝が形成されていても差し支えない。また、プランジャ７０の貫通孔形成部７２ｂの側面に、貫通孔７２ｄから離間した位置において、縦方向の溝が形成されていても差し支えない。
　また、プランジャ３０、プランジャ６０、プランジャ７０では、ガスケットが螺合によりプランジャ軸に取り付けられた構成としたが、例えば嵌め合いにより取り付けられていてもよい。同様に、プランジャ８０では、押し板８３が螺合によりプランジャ軸８１に取り付けられた構成としたが、例えば嵌め合いにより取り付けられていてもよい。 In the plunger 30 and the plunger 80, the reagent holding portion is formed over the entire circumferential direction, but as can be understood from the example of the plunger 60 and the plunger 70, the reagent holding portion does not have to extend over the entire circumferential direction.
Moreover, in each said plunger, a part or all of a sliding contact part exists in two places which pinch | interpose a reagent holding | maintenance part in an axial direction, and each is continuing over the whole circumferential direction.
However, the relationship between the reagent holding part and the sliding contact part is not limited to such an embodiment, the reagent holding part is surrounded by the sliding contact part, and between the reagent holding part and the inner wall of the syringe 20, What is necessary is just to be able to form a sealed space. For example, a vertical groove may be formed on the side surface of the side wall portion 62c of the plunger 60 at a position separated from the reagent holding portion 62D. Further, a vertical groove may be formed on the side surface of the through hole forming portion 72b of the plunger 70 at a position spaced from the through hole 72d.
Moreover, in the plunger 30, the plunger 60, and the plunger 70, it was set as the structure attached to the plunger axis | shaft by screwing, However, For example, you may attach by fitting. Similarly, the plunger 80 is configured such that the push plate 83 is attached to the plunger shaft 81 by screwing, but may be attached by fitting, for example.
［その他の態様］
　上記実施形態では、シリンジに試薬供給孔が形成された構成としたが、試薬供給孔は必須ではない。試薬供給孔を形成しない場合、試薬保持部の一部または全部を、シリンジの第一の開口端の外部に移動させた状態で試薬の溶液を試薬保持部に向けて供給し、凍結乾燥等により保持させればよい。 [Other aspects]
In the above embodiment, the reagent supply hole is formed in the syringe, but the reagent supply hole is not essential. When the reagent supply hole is not formed, the reagent solution is supplied toward the reagent holding part while part or all of the reagent holding part is moved to the outside of the first open end of the syringe, and is freeze-dried or the like. What is necessary is just to hold.
　また、上記実施形態では、シリンジの中間部において内側に突出する突出部を周方向全体にわたって連続的に設ける構成としたが、周方向の一部に断続的に設ける構成としてもよい。例えば、周方向の４カ所に９０゜間隔で、凸部を設ける構成が考えられる。シリンジの中間部において内側に突出する突出部を連続的又は断続的に周方向に設けることにより、試薬保持部に試薬を保持させる際、試薬供給孔との位置合わせが容易となる。また、試薬保持部を第一の開口端の外部に移動させたとき、バネの一端側の位置を固定することによりバネを圧縮することができる。
　シリンジの中間部において内側に突出する突出部は必須ではない。これを設けない場合は、試薬供給孔との位置合わせは目視により行えばよい。また、以下に説明するように、内側にプランジャ軸３１を挿通するように配置したバネを圧縮することは必須でない。 Moreover, in the said embodiment, although it was set as the structure which provides the protrusion part which protrudes inside in the intermediate part of a syringe continuously over the whole circumferential direction, it is good also as a structure provided intermittently in a part of circumferential direction. For example, a configuration is possible in which convex portions are provided at 90 ° intervals at four locations in the circumferential direction. By continuously or intermittently providing a protruding portion that protrudes inward in the middle portion of the syringe in the circumferential direction, alignment with the reagent supply hole is facilitated when the reagent holding portion holds the reagent. Further, when the reagent holding part is moved to the outside of the first opening end, the spring can be compressed by fixing the position on one end side of the spring.
The protrusion part which protrudes inside in the intermediate part of a syringe is not essential. When this is not provided, alignment with the reagent supply hole may be performed visually. Further, as will be described below, it is not essential to compress the spring disposed so as to insert the plunger shaft 31 inside.
　試薬保持部をシリンジの第一の開口端の外部に移動させたとき、プランジャに、第二の開口端側に移動させる付勢力を与える弾性部材は、内側にプランジャ軸３１を挿通するように配置したバネ４０に限定されない。例えば、押し板３１ｂを上方から引き上げるように配置されたバネやゴム等の弾性部材を用いることができる。この場合、これらの弾性部材が伸張することにより、プランジャに、第二の開口端側に移動させる付勢力を与えることができる。 When the reagent holding part is moved to the outside of the first opening end of the syringe, the elastic member that gives the urging force to move the plunger toward the second opening end side is arranged so that the plunger shaft 31 is inserted inside. The spring 40 is not limited. For example, an elastic member such as a spring or rubber arranged so as to pull up the push plate 31b from above can be used. In this case, when these elastic members are extended, a biasing force for moving the plunger toward the second opening end side can be applied.
　また、試薬保持部をシリンジの第一の開口端の外部に移動させたとき、プランジャに、第二の開口端側に移動させる付勢力を与える弾性部材は必須ではない。例えば、押し板３１ｂを押し下げる押棒の先端を押し板３１ｂに固定し、この押棒を引き上げることにより、ガスケット３２を待避させ、第二反応工程から測定工程に移行することができる。 Also, when the reagent holding part is moved to the outside of the first opening end of the syringe, an elastic member that gives a biasing force to move the plunger toward the second opening end side is not essential. For example, by fixing the tip of the push rod that pushes down the push plate 31b to the push plate 31b and pulling up the push rod, the gasket 32 can be retracted, and the process can be shifted from the second reaction step to the measurement step.
　さらに、プランジャを押し下げたままでも測定工程に支障が生じなければ、測定工程を行うためにプランジャを上に戻す必要もない。例えば、図７のプランジャ８０のプランジャ軸８１を透明材料で形成した場合、光の透過を妨げるのはＯリング８２a、８２ｂのみとなるので、試薬保持部８２Ｃ近傍で発生する蛍光や生物発光等の検出を実質的に妨げない。この場合、プランジャ８０を上に戻さなくとも、支障なく測定工程を行える。 Furthermore, if there is no problem in the measurement process even if the plunger is held down, there is no need to return the plunger to perform the measurement process. For example, when the plunger shaft 81 of the plunger 80 shown in FIG. 7 is formed of a transparent material, only the O- rings 82a and 82b prevent light transmission. Does not substantially interfere with detection. In this case, the measurement process can be performed without hindrance without returning the plunger 80 upward.
　また、上記実施形態では、シリンジ２０の第二の開口端２０ｂに鍔部２２を設けたが、鍔部２２を設ける位置は、第二の開口端２０ｂに限定されず、例えば、第二の開口端２０ｂの近傍（第二の開口端２０ｂから、やや第一の開口端２０ａ側）に設けてもよい。
　また、鍔部２２の形状にも特に限定はなく、例えば、円盤の周縁に、下方に垂下する鍵状部を設け、反応容器５０の上端と勘合するようにしてもよい。この場合、反応容器５０に鍔部５２がなくとも、安定的に試薬容器１０を支持することができる。
　さらに、シリンジ２０の鍔部２２は必須ではない。鍔部２２がない場合は、別途の支持手段で、試薬容器１０の第一の開口端２０ａが反応容器５０の底部に達しないよう、支持すればよい。 Moreover, in the said embodiment, although the collar part 22 was provided in the 2nd opening end 20b of the syringe 20, the position which provides the collar part 22 is not limited to the 2nd opening end 20b, For example, the 2nd opening It may be provided in the vicinity of the end 20b (from the second opening end 20b to the first opening end 20a side).
Further, the shape of the flange portion 22 is not particularly limited. For example, a key-like portion that hangs downward may be provided on the periphery of the disk so as to be fitted with the upper end of the reaction vessel 50. In this case, the reagent container 10 can be stably supported even if the reaction container 50 does not have the flange 52.
Furthermore, the collar part 22 of the syringe 20 is not essential. When the flange 22 is not provided, it may be supported by a separate support means so that the first open end 20a of the reagent container 10 does not reach the bottom of the reaction container 50.
　また、上記実施形態では、反応容器５０に第一試薬Ｒ１を保持させ、二段反応を行わせる態様としたが、反応容器５０に試薬を保持させることは必須でない。反応容器５０に試薬を保持しない場合、試料液Ｓと第二試薬Ｒ２との一段反応をさせることができる。また、試料液Ｓは、予め、第一試薬Ｒ１と反応させたものであってもよい。また、試料液Ｓと第一試薬Ｒ１を、共に手作業で反応容器５０に導入してもよい。これらの場合、反応容器５０に試薬を保持させなくとも、二段反応が可能となる。 In the above embodiment, the first reagent R1 is held in the reaction vessel 50 and the two-stage reaction is performed. However, it is not essential to hold the reagent in the reaction vessel 50. When the reagent is not held in the reaction vessel 50, a one-step reaction between the sample solution S and the second reagent R2 can be performed. Further, the sample solution S may be previously reacted with the first reagent R1. Alternatively, both the sample solution S and the first reagent R1 may be manually introduced into the reaction vessel 50. In these cases, the two-stage reaction can be performed without holding the reagent in the reaction vessel 50.
［エンドトキシンの測定］
　本発明の試薬容器、試薬入り試薬容器、反応ユニット、および分析システムを、エンドトキシンの測定に適用する場合、反応容器に保持する第一試薬Ｒ１としては、エンドトキシンとの結合により活性化されるＣ因子と、ペプチドに発光基質が結合してなる発光合成基質を含有する試薬を用いる。また、試薬容器に保持する第二試薬Ｒ２としては、発光酵素および発光反応に必要な他の化合物を含有する試薬を用いる。
　あるいは、エンドトキシンとの結合により活性化されるＣ因子を、反応容器に保持する第一試薬Ｒ１として用い、ペプチドに発光基質が結合してなる発光合成基質を含有する試薬と発光酵素および発光反応に必要な他の化合物を含有する試薬とを、試薬容器に保持する第二試薬Ｒ２として用いてもよい。 [Measurement of endotoxin]
When the reagent container, reagent-containing reagent container, reaction unit, and analysis system of the present invention are applied to the measurement of endotoxin, the first reagent R1 retained in the reaction container is a factor C activated by binding with endotoxin And a reagent containing a luminescent synthetic substrate formed by binding a luminescent substrate to a peptide. In addition, as the second reagent R2 held in the reagent container, a reagent containing a luminescent enzyme and other compounds necessary for the luminescent reaction is used.
Alternatively, factor C activated by binding to endotoxin is used as the first reagent R1 held in the reaction vessel, and a reagent containing a luminescent synthetic substrate formed by binding a luminescent substrate to a peptide, a luminescent enzyme, and a luminescent reaction. A reagent containing other necessary compounds may be used as the second reagent R2 held in the reagent container.
　エンドトキシンとの結合により活性化されるＣ因子を含有する試薬としては、カブトガニ血球抽出成分（リムルス試薬）を好適に使用できる。
　ペプチドに発光基質が結合してなる発光合成基質は、活性型Ｃ因子、活性型Ｂ因子、および凝固酵素の少なくともいずれか１種の作用（プロテアーゼ活性）により、発光基質とペプチドとの結合が切断される構造を有するものである。活性型Ｃ因子は、Ｃ因子が活性化されることにより生成される。活性型Ｂ因子は、活性型Ｃ因子の作用によりＢ因子が活性化されて生成される。凝固酵素は、活性型Ｂ因子や活性型Ｃ因子の作用により前凝固酵素が活性化されて生成される。
　発光基質としては、アミノルシフェリンが好適に使用できる。発光基質と結合するペプチドとしては、該ペプチドのＣ末端におけるアミノルシフェリンとのアミド結合が、活性型Ｃ因子、活性型Ｂ因子および凝固酵素の少なくともいずれか１種のプロテアーゼ活性により切断されるアミノ酸配列からなるものであればよい。
　なお、試料液中の塩分濃度に起因する誤差を解消するため、第一試薬Ｒ１中に、ＮａＣｌを添加しておいてもよい。
　発光酵素は、発光合成基質から遊離される発光基質の生物発光を触媒とし、光を発生させる酵素である。発光基質がアミノルシフェリンである場合の発光酵素はルシフェラーゼであり、発光反応に必要な他の化合物は、ＡＴＰおよび２価金属イオンである。 As a reagent containing Factor C activated by binding to endotoxin, a horseshoe crab blood cell extract component (Limulus reagent) can be suitably used.
A luminescent synthetic substrate formed by binding a luminescent substrate to a peptide cleaves the bond between the luminescent substrate and the peptide by the action (protease activity) of at least one of active factor C, active factor B, and clotting enzyme. It has a structure. The activated factor C is generated by activating factor C. The activated factor B is generated by activating factor B by the action of activated factor C. The clotting enzyme is produced by the activation of the procoagulase by the action of active factor B or active factor C.
As the luminescent substrate, aminoluciferin can be preferably used. As the peptide that binds to the luminescent substrate, an amino acid sequence in which an amide bond with aminoluciferin at the C-terminus of the peptide is cleaved by at least one protease activity of active factor C, active factor B and coagulation enzyme Anything consisting of can be used.
In order to eliminate an error caused by the salt concentration in the sample solution, NaCl may be added to the first reagent R1.
A luminescent enzyme is an enzyme that generates light by using bioluminescence of a luminescent substrate released from a luminescent synthetic substrate as a catalyst. When the luminescent substrate is aminoluciferin, the luminescent enzyme is luciferase, and other compounds required for the luminescent reaction are ATP and divalent metal ions.
　具体的な試薬容器及び反応ユニットの構造、および分析システムの具体的手順は、上記で説明したものを、好適に適用できる。
　エンドトキシンの測定のように、発光を利用する測定では、第二試薬Ｒ２を保持した試薬保持部を試料液に浸漬してから、発光反応を光学的に検出するまでの時間は短い方が好ましい。そのため、第二反応工程でプランジャ３０を押し下げてから、押し下げを解除して測定工程に移行する動作は、できるだけ速やかに行うことが好ましい。また、光学測定手段により発光反応の検出が可能な位置に容器ユニットを配置してから、プランジャ３０の押し下げを行うことが好ましい。 The specific structure of the reagent container and reaction unit and the specific procedure of the analysis system can be suitably applied as described above.
In the measurement using luminescence as in the measurement of endotoxin, it is preferable that the time from when the reagent holding part holding the second reagent R2 is immersed in the sample solution until the luminescence reaction is optically detected is shorter. For this reason, it is preferable that the operation of depressing the plunger 30 in the second reaction step and releasing the depressing to move to the measurement step is performed as quickly as possible. In addition, it is preferable that the plunger 30 is pushed down after the container unit is arranged at a position where the light measurement reaction can be detected by the optical measuring means.
　第一試薬Ｒ１がカブトガニ血球抽出成分（リムルス試薬）とペプチドにアミノルシフェリンが結合している合成基質との混合試薬であり、第二試薬Ｒ２がルシフェラーゼとＡＴＰおよび２価金属イオンを含む混合試薬である場合、第一試薬Ｒ１を保持した反応容器に試料液を最初に導入した後、３７℃で１５～３０分間程度インキュベートすることが好ましい。
　その後、プランジャ３０を押し下げて第二試薬Ｒ２を保持した試薬保持部を試料液に浸漬し、ルシフェリンとルシフェラーゼの発光反応（生物発光反応）を行わせる。発光反応の際の温度は、たとえば室温（２５℃）とすることができる。また、発光反応の反応時間、すなわち、プランジャ３０を押し下げてから、押し下げを解除して、発光反応を光学的に検出するまでの時間は、０秒から１０秒とすることが好ましい。 The first reagent R1 is a mixed reagent of a horseshoe crab blood cell extract component (Limulus reagent) and a synthetic substrate in which aminoluciferin is bound to a peptide, and the second reagent R2 is a mixed reagent containing luciferase, ATP and a divalent metal ion. In some cases, it is preferable to incubate at 37 ° C. for about 15 to 30 minutes after first introducing the sample solution into the reaction vessel holding the first reagent R1.
Thereafter, the plunger 30 is pushed down to immerse the reagent holding part holding the second reagent R2 in the sample solution to cause a luminescence reaction (bioluminescence reaction) between luciferin and luciferase. The temperature during the luminescence reaction can be, for example, room temperature (25 ° C.). Further, the reaction time of the luminescence reaction, that is, the time from when the plunger 30 is depressed to when the depression is released and the luminescence reaction is optically detected is preferably 0 to 10 seconds.
　本発明は、エンドトキシン、βグルカン等の微生物夾雑物の検出等、液状では不安定な試薬を用いる分析に好適に使用できる。 The present invention can be suitably used for analysis using a liquid unstable reagent such as detection of microbial contaminants such as endotoxin and β-glucan.
　１０…試薬容器、２０…シリンジ、３０…プランジャ、４０…バネ、５０…反応容器、３２Ａ…第一摺接部、３２Ｂ…第二摺接部、３２Ｃ…試薬保持部、５５…光学測定手段、Ｒ１…第一試薬、Ｒ２…第二試薬 DESCRIPTION OF SYMBOLS 10 ... Reagent container, 20 ... Syringe, 30 ... Plunger, 40 ... Spring, 50 ... Reaction container, 32A ... First sliding contact part, 32B ... Second sliding contact part, 32C ... Reagent holding part, 55 ... Optical measuring means, R1 ... first reagent, R2 ... second reagent

Claims (11)

  1. 　軸方向の一端が第一の開口端とされ、他端が第二の開口端とされているシリンジと、該シリンジ内を摺動可能なプランジャを備え、
    　前記プランジャは、前記シリンジの内面に摺接する摺接部と、前記シリンジの内面に摺接せず、かつ前記摺接部に囲まれている試薬保持部とを有し、
    　前記試薬保持部は、前記シリンジの内部から、前記シリンジの第一の開口端の外部まで移動可能とされていることを特徴とする試薬容器。     One end in the axial direction is a first opening end, and the other end is a second opening end, and includes a plunger that can slide in the syringe,
    The plunger has a sliding contact portion that is in sliding contact with the inner surface of the syringe, and a reagent holding portion that is not in sliding contact with the inner surface of the syringe and is surrounded by the sliding contact portion,
    The reagent container, wherein the reagent holding part is movable from the inside of the syringe to the outside of the first opening end of the syringe.
  2. 　前記シリンジの側面に試薬供給孔が形成され、該試薬供給孔は、前記試薬保持部が前記シリンジの内部に位置する状態で、前記試薬保持部と連通可能とされている請求項１に記載の試薬容器。 2. The reagent supply hole according to claim 1, wherein a reagent supply hole is formed in a side surface of the syringe, and the reagent supply hole can communicate with the reagent holding part in a state where the reagent holding part is located inside the syringe. Reagent container.
  3. 　前記シリンジは、軸方向の中間部に内側に突出する突出部を有する請求項１または２に記載の試薬容器。 The reagent container according to claim 1 or 2, wherein the syringe has a protruding portion that protrudes inward in an intermediate portion in the axial direction.
  4. 　さらに、弾性部材を備え、
    　前記試薬保持部を前記シリンジの第一の開口端の外部に移動させたとき、
    　前記弾性部材は、前記プランジャに、前記第二の開口端側に移動させる付勢力を与える請求項１～３のいずれか一項に記載の試薬容器。     Furthermore, an elastic member is provided,
    When the reagent holding part is moved outside the first open end of the syringe,
    The reagent container according to any one of claims 1 to 3, wherein the elastic member applies an urging force for moving the plunger toward the second opening end.
  5. 　前記シリンジは、第二の開口端またはその近傍に、外側に突出する鍔部を有する請求項１～４のいずれか一項に記載の試薬容器。 The reagent container according to any one of claims 1 to 4, wherein the syringe has a flange protruding outward at or near the second open end.
  6. 　請求項１～５のいずれか一項に記載の試薬容器と、前記試薬保持部に保持された試薬を備えることを特徴とする試薬入り試薬容器。 A reagent container containing a reagent container according to any one of claims 1 to 5 and a reagent held in the reagent holding part.
  7. 　請求項１～５のいずれか一項に記載の試薬容器と、上端が開口端とされている有底筒状の反応容器とを備え、
    　前記試薬容器は、前記シリンジの第一の開口端が前記反応容器の軸方向中間部に達するように、前記第一の開口端側から前記反応容器に挿入可能とされていることを特徴とする反応ユニット。     A reagent container according to any one of claims 1 to 5 and a bottomed cylindrical reaction container whose upper end is an open end,
    The reagent container can be inserted into the reaction container from the first opening end side so that the first opening end of the syringe reaches the intermediate portion in the axial direction of the reaction container. Reaction unit.
  8. 　前記試薬容器の前記試薬保持部に試薬が保持されている請求項７に記載の反応ユニット。 The reaction unit according to claim 7, wherein a reagent is held in the reagent holding part of the reagent container.
  9. 　前記反応容器内に第一試薬が、前記試薬容器の試薬保持部内に第二試薬が、各々保持されている請求項７に記載の反応ユニット。 The reaction unit according to claim 7, wherein the first reagent is held in the reaction container, and the second reagent is held in a reagent holding part of the reagent container.
  10. 　請求項８に記載の反応ユニットと、前記反応容器内の試料液を光学的に測定する光学測定手段を備え、
    　前記試薬容器を前記反応容器に挿入した状態で、前記試薬保持部を前記シリンジの第一の開口端の外部に移動させて予め前記反応容器に導入された試料液に接触させ、前記試薬を反応させる工程と、
    　前記試薬と反応後の試料液を、前記光学測定手段により測定する工程とを順次行うように構成されたことを特徴とする試料液の分析システム。     The reaction unit according to claim 8, and an optical measurement means for optically measuring the sample liquid in the reaction vessel,
    With the reagent container inserted into the reaction container, the reagent holding part is moved to the outside of the first opening end of the syringe and brought into contact with the sample solution previously introduced into the reaction container to react the reagent. A process of
    A sample solution analysis system configured to sequentially perform the step of measuring the sample solution after reaction with the reagent by the optical measuring means.
  11. 　請求項９に記載の反応ユニットと、前記反応容器内の試料液を光学的に測定する光学測定手段を備え、
    　前記反応容器に導入された試料液と、前記第一試薬を反応させる工程と、
    　前記試薬容器を前記反応容器に挿入した状態で、前記試薬保持部を前記シリンジの第一の開口端の外部に移動させて前記第一試薬と反応後の試料液に接触させ、前記第二試薬を反応させる工程と、
    　前記第二試薬と反応後の試料液を、前記光学測定手段により測定する工程とを順次行うように構成されたことを特徴とする試料液の分析システム。     The reaction unit according to claim 9, and an optical measurement means for optically measuring the sample liquid in the reaction vessel,
    Reacting the sample solution introduced into the reaction vessel with the first reagent;
    With the reagent container inserted into the reaction container, the reagent holding part is moved to the outside of the first open end of the syringe to contact the first reagent and the sample solution after the reaction, and the second reagent Reacting with
    A sample solution analysis system configured to sequentially perform the step of measuring the sample solution after reacting with the second reagent by the optical measuring means.
PCT/JP2014/084151 2013-12-24 2014-12-24 Reagent container, reagent-filled reagent container, reaction unit, and analysis system WO2015098967A1 (en)

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