WO2014148601A1 - Système de réaction à deux étages, système de mesure d'échantillon de fluide et procédé de mesure d'échantillon de fluide - Google Patents

Système de réaction à deux étages, système de mesure d'échantillon de fluide et procédé de mesure d'échantillon de fluide Download PDF

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Publication number
WO2014148601A1
WO2014148601A1 PCT/JP2014/057725 JP2014057725W WO2014148601A1 WO 2014148601 A1 WO2014148601 A1 WO 2014148601A1 JP 2014057725 W JP2014057725 W JP 2014057725W WO 2014148601 A1 WO2014148601 A1 WO 2014148601A1
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WIPO (PCT)
Prior art keywords
container
reagent
end side
sample liquid
sample
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PCT/JP2014/057725
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English (en)
Japanese (ja)
Inventor
悟史 八幡
秀和 長峯
荒川　智
Original Assignee
東亜ディーケーケー株式会社
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Publication of WO2014148601A1 publication Critical patent/WO2014148601A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • B01L3/5082—Test tubes per se
    • B01L3/50825—Closing or opening means, corks, bungs
    • 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
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/04—Closures and closing means
    • B01L2300/046—Function or devices integrated in the closure
    • B01L2300/047—Additional chamber, reservoir
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0848—Specific forms of parts of containers
    • B01L2300/0858—Side walls
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics
    • 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
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G01N21/03—Cuvette constructions
    • G01N2021/0325—Cells for testing reactions, e.g. containing reagents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor

Definitions

  • the present invention relates to a two-stage reaction system, a sample solution measurement system, and a sample solution measurement method.
  • a two-stage reaction system suitable for detection of microbial contaminants such as endotoxin and ⁇ -glucan a sample solution measurement system, and a sample solution measurement method are provided.
  • 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). In this technique, it is required to sequentially perform at least the steps of releasing the luminescent substrate and the step of adding the luminescent reagent, and at least 2 to 3 pipetting operations are required.
  • 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 diaphragm used in the container shown in FIG. 8 of Patent Document 3 has a property of easily adsorbing contaminants. In general, heat sterilization is also difficult. Therefore, the container shown in FIG. 8 of Patent Document 3 may be contaminated with microbial contaminants as detection targets in the production stage. In addition, when a small amount of sample solution of about several hundred ⁇ L is used, even if a hole is made in the diaphragm, the amount of the liquid that cannot be removed from the hole due to the sample liquid adhering to the diaphragm cannot be ignored, which hinders high-sensitivity detection. Met. Further, the container shown in FIG.
  • Patent Document 3 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 using a small amount of sample solution of about several hundred ⁇ L, the amount of solution that cannot be moved due to the sample solution adhering to the reagent-containing cup even if the container is turned over cannot be ignored, which hinders high-sensitivity detection. Met.
  • the present invention has been made in view of the above circumstances, and is a two-stage reaction system and sample that can perform detection that requires a two-step reaction with high sensitivity and simplicity, and can be automated as necessary. It is an object of the present invention to provide a liquid measurement system and a sample liquid measurement method.
  • the present invention employs the following configuration.
  • a first reagent to be reacted first with the sample solution is fixed to the inner lower end of the first container,
  • the sample after reacting with the first reagent at a position inside the second container and in contact with the aspirated sample liquid when a predetermined volume of the sample liquid is aspirated from the first container by the aspiration and discharge means A two-stage reaction system, wherein a second reagent to be reacted with a
  • the first container having an opening on the upper end side, into which the sample solution is first introduced, and the second container having openings on both the upper end side and the lower end side, the lower end side of the second container is A container unit inserted into the first container such that the opening on the lower end side is positioned in the vicinity of the lower end on the inner side of the first container; and the opening on the upper end side of the second container;
  • a suction means for sucking a predetermined volume of the sample liquid into the second container from A first reagent to be reacted first with the sample solution is fixed to the inner lower end of the first container,
  • the sample liquid after reacting with the first reagent at a position inside the second container and in contact with the aspirated sample liquid when a predetermined volume of the sample liquid is aspirated from the first container by the aspirating means A two-stage reaction system, wherein a second reagent to be reacted with is fixed.
  • the first container having an opening on the upper end side, into which the sample solution is first introduced, and the second container having openings on both the upper end side and the lower end side, the lower end side of the second container is A container unit inserted into the first container such that the opening on the lower end side is located near the lower end on the inside of the first container;
  • a first reagent to be reacted first with the sample solution is fixed to the inner lower end of the first container, The sample after reacting with the first reagent at a position inside the second container and in contact with the aspirated sample liquid when a predetermined volume of the sample liquid is aspirated from the first container by the aspiration and discharge means
  • the second reagent that reacts with the liquid is fixed, Reacting the sample introduced into the first container with
  • a system for measuring a sample liquid wherein the step of returning the sample liquid to the first container and measuring the sample liquid after reacting with the second reagent by the optical measuring means is sequentially performed.
  • the first container having an opening on the upper end side, into which the sample solution is first introduced, and the second container having openings on both the upper end side and the lower end side, the lower end side of the second container is A container unit inserted into the first container such that the opening on the lower end side is located near the lower end on the inside of the first container;
  • Optical measuring means A first reagent to be reacted first with the sample solution is fixed to the inner lower end of the first container, The sample liquid after reacting with the first reagent at a position inside the second container and in contact with the aspirated sample liquid when a predetermined volume of the sample liquid is aspirated from the first container by the aspirating means
  • the second reagent to be reacted with is fixed, Reacting the sample introduced into the first container with the first reagent;
  • a sample liquid measuring system configured to sequentially perform the step of measuring the sample liquid after reacting with the second reagent in the second container by the optical measuring means.
  • the first container having an opening on the upper end side, and a second container having an opening on both the upper end side and the lower end side, and the first container into which the sample solution is first introduced, Inserted into the first container so that the opening on the lower end side is located near the lower end inside the first container, A first reagent is fixed to the inner lower end of the first container, The inner side of the second container is in a position not in contact with the sample liquid when first introduced into the first container, and the first introduced sample liquid is placed on the lower end side of the second container.
  • the second reagent is fixed at a position where it comes into contact with the sucked sample liquid when sucked from the opening of
  • the first reagent comprises a factor C activated by binding to endotoxin, and a luminescent synthetic substrate that liberates the luminescent substrate when the factor C is activated by binding a luminescent substrate to the peptide.
  • detection that requires a two-stage reaction can be performed with high sensitivity and simplicity, and can be automated as necessary. Is possible.
  • FIG. 1 is an example of a container unit used in the two-stage reaction system and measurement system of the present invention.
  • the container unit 1 in FIG. 1 includes a first container 10 and a second container 20 having a lower end inserted into the first container 10.
  • the first container 10 is a container into which a sample solution is first introduced, and is a bottomed cylindrical container having an opening 10a at the upper end.
  • An air hole 11 is provided in the side surface of the first container 10.
  • a first reagent R1 that is first reacted with the sample solution is fixed to the inner lower end of the first container 10 in a dry state.
  • the first reagent R1 can be fixed in a dry state by lyophilization in a state where the solution of the first reagent R1 is placed in the first container 10.
  • the first container 10 is preferably made of a material that can be sterilized as necessary. Further, when used in the first embodiment described later, it is preferably made of a light transmissive material. Examples of the light transmissive material that can be sterilized include quartz, borosilicate glass, polystyrene, polypropylene, and acrylic resin. In addition, examples of materials that can be sterilized include fluororesins such as polytetrafluoroethylene and polyether ether ketone resins.
  • the second container 20 includes a second container body 21 having a circular cross section and a suction pipe 22 having an upper end inserted into the second container body 21.
  • the second container body 21 is provided with a large-diameter portion 21a having an outer diameter larger than the outer diameter of the first container 10 and a small-diameter portion having an outer diameter slightly smaller than the inner diameter of the first container 10. 21b.
  • the large diameter portion 21 a is provided with a columnar hole 24 from the upper end to the lower side, and the upper end of the columnar hole 24 is an opening 20 a on the upper end side of the second container 20.
  • an insertion hole 25 for inserting the suction pipe 22 is provided from the lower end side of the small diameter portion 21b, and a tapered intermediate hole 26 for connecting the two is provided between the columnar hole 24 and the insertion hole 25. ing.
  • a second reagent R2 to be reacted with the sample liquid after reacting with the first reagent R1 is fixed to the lower end side of the intermediate hole 26 in the second container 20 in a dry state.
  • the opening 20b on the lower end side of the second container 20 is sealed to form an air layer in the insertion tube 22, and the solution of the second reagent R2
  • freeze-drying is performed in a state in which is attached to the vicinity of the lower end side of the intermediate hole 26.
  • the presence of an air layer in the insertion tube 22 causes the second reagent R2 solution adhering to the vicinity of the lower end side of the intermediate hole 26 to be inserted into the insertion tube 22 before lyophilization. You can avoid falling in.
  • the solution of the second reagent R2 is attached to the vicinity of the lower end side of the intermediate hole 26, and after freeze-drying in this state, the insertion tube 22 is moved to the position shown in FIG. The method of returning is also mentioned.
  • the lower end side of the intermediate hole 26 to which the second reagent R2 is fixed is separated from the sample liquid when the sample liquid is first introduced into the first container 10, and a suction / discharge means (aspiration means), which will be described later.
  • a suction / discharge means aspiration means
  • the second container body 21 is preferably made of a material that can be easily processed because the columnar holes 24 and the like are provided. Moreover, it is preferable to consist of the material which can perform a sterilization process as needed. In addition, when used in the second embodiment described later, it is preferable to be made of a light transmissive material. Examples of materials that can be easily processed, can be sterilized, and are light transmissive include quartz, borosilicate glass, polystyrene, polypropylene, and acrylic resin. Other materials that can be easily processed and that can be sterilized include fluororesins such as polytetrafluoroethylene and polyether ether ketone resins.
  • the suction pipe 22 is inserted into the insertion hole 25 such that a part of the upper end side reaches the upper end of the insertion hole 25, and the remaining part is suspended from the lower end side of the small diameter part 21b.
  • the opening on the lower end side of the suction pipe 22 is an opening 20b on the lower end side of the second container 20, and the opening 20 a and the opening 20 b communicate with each other by a columnar hole 24, an intermediate hole 26, and a hole inside the suction pipe 22. is doing.
  • the suction pipe 22 is preferably made of a material having a strength necessary for the narrow pipe. Moreover, it is preferable to consist of the material which can perform a sterilization process as needed.
  • quartz, borosilicate glass, polystyrene, polypropylene, and acrylic resin can be used as the light-transmitting material that has the required strength and can be sterilized.
  • materials that have the necessary strength and can be sterilized include fluororesins such as polytetrafluoroethylene and polyether ether ketone resins.
  • the small diameter portion 21 b is inserted into the upper end of the first container 10 while the suction pipe 22 is suspended, and the lower end side of the large diameter portion 21 a is locked at the upper end of the first container 10. It can be placed on the container 10. In this way, when the second container 20 is placed on the first container 10, the lower end (lower end side opening 20 b) of the suction pipe 22 of the second container 20 is positioned near the lower end inside the first container 10. It is like that.
  • the sample liquid can be introduced to the lower end side of the suction pipe 22 up to a level equivalent to the liquid level of the sample liquid in the first container 10. At the stage where the suction discharge (suction) is not performed by the means (suction means), it does not reach the upper end side portion of the intermediate hole 26 from the lower end side of the columnar hole 24 to which the second reagent R2 is fixed.
  • the container unit 1 to which each reagent is fixed may be hermetically sealed using, for example, a shrink film in order to prevent contamination after manufacture.
  • the sample liquid is introduced into the first container by inserting an injection needle into the air hole 11 of the first container 10 through the shrink film, thereby contaminating the container unit 1 after the sample liquid is introduced. Can be prevented more reliably.
  • a gap through which air flows between the first container 10 and the second container 20 can be secured to the extent that suction discharge (suction) by a suction discharge means (suction means) described later is possible, the first The air hole 11 of the container 10 is not essential.
  • the sample solution may be introduced from the opening 10a with the second container 20 removed.
  • the second container 20 may be one in which the second container body 21 and the suction pipe 22 are integrally formed.
  • FIG. 2 is a schematic configuration diagram showing a first embodiment of the two-stage reaction system and measurement system for a sample solution of the present invention.
  • the system 100 in FIG. 2 is a two-stage reaction system of the present invention by using a plurality of the container units 1 in FIG. 1 and combining them with the suction / discharge means 30A. Further, in addition to the plurality of container units 1 and the suction / discharge means 30A, an optical measurement means 40A is further provided to provide the measurement system of the present invention.
  • the suction / discharge means 30A of the present embodiment includes a syringe pump 31, a plurality of suction cups 32 respectively corresponding to the plurality of container units 1, a branch pipe 33 connecting the syringe pump 31 and the plurality of suction cups 32, and a branch pipe 33. And an on-off valve 34 provided on the suction cup 32 side from each branch point. Moreover, the suction cup 32 corresponding to each container unit 1 can be moved up and down individually by lifting means not shown.
  • the suction cup 32 is sized to cover the opening 20a of the second container 20, and is pressed against the upper end of the second container 20 by the elevating means so as to be airtightly connected to the opening 20a.
  • a hole communicating with the branch pipe 33 is provided at the center of the suction cup 32.
  • a light (fluorescence) detection device that detects light (including fluorescence) emitted from the first container 10 of the container unit 1, a light source, and light reception that detects light transmitted through the first container 10
  • a light-absorbing device, a light source, and a scattered light detecting device including a light-receiving device that detects light scattered in the first container 10 can be appropriately employed.
  • the system 100 may include a heating means for appropriately heating the container unit 1 in order to promote the progress of the reaction.
  • first reaction step A A step of reacting the sample solution introduced into the first container 10 with the first reagent R1 (hereinafter referred to as “first reaction step A”).
  • first reaction step A A step of reacting the sample solution introduced into the first container 10 with the first reagent R1
  • second reaction step A A step of reacting the sample solution after the reaction with the first reagent R1 is sucked into the second container 20 and then returned to the first container 10, and the second reagent R2 is reacted with the first reagent R1.
  • second reaction step A A step of reacting with the sample solution
  • measurement step A A step of returning to the first container and optically measuring the sample solution after the reaction with the second reagent R2
  • the first reaction step A is a standby step after the container unit 1 introduced with the sample solution is set in the system 100 until the next second reaction step A is started.
  • the waiting time for the first reaction step A is appropriately set in consideration of the necessary reaction time in order to cause the sample introduced into the first container 10 to react with the first reagent R1.
  • the container unit 1 can be appropriately heated to promote the reaction.
  • the sample liquid can be introduced into the first container 10 from the air hole 11 or the opening 10a.
  • the introduction work is preferably performed manually, but may be automated by adding a sampling mechanism to the system 100 of FIG.
  • the first reagent R1 is dissolved by the introduced sample liquid, and the sample liquid and the first reagent R1 can react.
  • the suction means 32 is lowered by the lifting means and pressed against the upper end of the corresponding second container 20, and the suction / discharge means 30A is airtightly connected to the opening 20a.
  • the entire container unit 1 is sealed using a shrink film or the like, and the sample liquid is introduced by inserting a syringe needle into the air hole 11 of the first container 10 through the shrink film without removing the shrink film.
  • hook also descend
  • the on-off valve 34 only the on-off valve 34 between the suction cup 32 and the syringe pump 31 connected to the opening 20a is opened, and a piston of the syringe pump 31 is pulled to suck a certain amount of air.
  • the inside of the second container 20 is pulled through the branch pipe 33, and the sample liquid having a liquid volume corresponding to the air volume sucked by the syringe pump 31 is sucked up from the first container 10 to the second container 20.
  • the amount of the sample liquid sucked into the second container 20 is such that the liquid level of the sample liquid remaining in the first container falls below the lower end (lower end side opening 20b) of the suction pipe 22 of the second container 20.
  • the amount of liquid sucked into the second container 20 is excessive and the opening 20b is exposed to the gas phase on the sample liquid, the sample liquid moves rapidly, and the sample liquid causes the inside of the suction / discharge means 30A. May be contaminated. Further, the amount of the sample solution sucked into the second container 20 needs to be sufficient to reach the position where the second reagent R2 is fixed. When the aspirated sample liquid reaches the fixing position of the second reagent R2, the second reagent R2 is dissolved by the aspirated sample liquid, and the sample liquid and the second reagent R2 can react.
  • the sample is returned to the first container, and the sample solution after the reaction with the second reagent R2 is optically measured by the optical measurement means 40A.
  • the type of optical measurement is not particularly limited, and fluorescence detection, absorbance detection, scattered light detection, and the like can be appropriately employed.
  • finish of the measurement process A the connection with the container unit 1 of the suction cup 32 is cancelled
  • the optical measurement conditions can be easily uniformized by performing the suction and discharge in the second reaction step A and the measurement step A according to the predetermined time table for all the samples. Therefore, it is easy to eliminate detection errors due to variations in measurement conditions. In addition, since almost the entire amount of the sample solution is returned to the first container in the measurement step A, it can be detected with high accuracy even if a small amount of the sample solution is used.
  • FIG. 3 is a schematic configuration diagram showing a second embodiment of the two-stage reaction system and measurement system for a sample solution of the present invention. 3, the same components as those in FIG. 2 are denoted by the same reference numerals, and detailed description thereof is omitted.
  • the system 200 of FIG. 3 is a two-stage reaction system of the present invention by using a plurality of container units 1 of FIG. 1 and combining them with the suction means 30B. Further, in addition to the plurality of container units 1 and the suction means 30B, an optical measurement means 40B is further provided to provide the measurement system of the present invention.
  • the suction unit 30B of the present embodiment is composed of the same constituent members as the suction / discharge unit 30A of the first embodiment, but its operation is different from the suction / discharge unit 30A of the first embodiment as will be described later.
  • the optical measurement means 40B is the same as the optical measurement means 40A except that the object to be measured is not the sample liquid in the first container 10 but the sample liquid in the second container 20. That is, as the optical measurement means 40B, absorbance detection comprising a fluorescence detection device that detects fluorescence emitted from the second container 20 of the container unit 1, a light source, and a light receiving device that detects light transmitted through the second container 20.
  • a scattered light detection device including a device, a light source, and a light receiving device that detects light scattered in the second container 20 can be appropriately employed.
  • the system 200 may include a heating means for appropriately heating the container unit 1 in order to promote the progress of the reaction.
  • first reaction step B A step of reacting the sample solution introduced into the first container 10 with the first reagent R1 (hereinafter referred to as “first reaction step B”).
  • second reaction step B A step of sucking a certain amount of the sample solution after the reaction with the first reagent R1 into the second container 20 to cause the sample solution and the second reagent R2 to react. That said.
  • second reaction step B A step of optically measuring the sample solution after the reaction with the second reagent R2 (hereinafter referred to as “measurement step B”).
  • the first reaction step B is a standby step after the container unit 1 introduced with the sample solution is set in the system 200 until the next second reaction step B is started.
  • the waiting time for the first reaction step B is appropriately set in consideration of the necessary reaction time in order to cause the sample introduced into the first container 10 to react with the first reagent R1.
  • the container unit 1 can be appropriately heated to promote the reaction.
  • the sample liquid can be introduced into the first container 10 from the air hole 11 or the opening 10a.
  • the introduction work is preferably performed manually, but may be automated by adding a sampling mechanism to the system 200 of FIG.
  • the first reagent R1 is dissolved by the introduced sample liquid, and the sample liquid and the first reagent R1 can react.
  • the suction means 32 is lowered by the lifting means and pressed against the upper end of the corresponding second container 20, and the suction means 30B is airtightly connected to the opening 20a.
  • the entire container unit 1 is sealed using a shrink film or the like, and the sample liquid is introduced by inserting a syringe needle into the air hole 11 of the first container 10 through the shrink film without removing the shrink film.
  • hook also descend
  • the on-off valve 34 only the on-off valve 34 between the suction cup 32 and the syringe pump 31 connected to the opening 20a is opened, and a piston of the syringe pump 31 is pulled to suck a certain amount of air.
  • the inside of the second container 20 is pulled through the branch pipe 33, and a sample liquid having a liquid volume corresponding to the air volume sucked by the syringe pump 31 is sucked up from the first container 10 to the second container 20.
  • the amount of the sample liquid sucked into the second container 20 is such that the liquid level of the sample liquid remaining in the first container falls below the lower end (lower end side opening 20b) of the suction pipe 22 of the second container 20.
  • the amount of liquid sucked into the second container 20 is excessive and the opening 20b is exposed to the gas phase on the sample liquid, the sample liquid moves rapidly, and the sample liquid causes the inside of the suction / discharge means 30A. May be contaminated. Further, the amount of the sample solution sucked into the second container 20 needs to be sufficient to reach the position where the second reagent R2 is fixed. When the aspirated sample liquid reaches the fixing position of the second reagent R2, the second reagent R2 is dissolved by the aspirated sample liquid, and the sample liquid and the second reagent R2 can react.
  • the sample liquid is sucked from the first container 10 into the second container 20, and then the on-off valve 34 is closed. Thereby, the sample liquid after being sucked into the second container 20 can be held in the second container 20 as it is.
  • the time for holding the sample solution in the second container 20 is set to a time sufficient for the second reagent R2 to be dissolved by the sucked sample solution and to react with the sample solution.
  • the second reaction step B can be performed in a short time. It is preferable that the measurement process B is completed immediately after the completion.
  • the sample liquid held in the second container 20 is optically measured by the optical measurement means 40B while the on-off valve 34 is closed.
  • the type of optical measurement is not particularly limited, and luminescence detection, fluorescence detection, absorbance detection, scattered light detection, and the like can be appropriately employed.
  • finish of the measurement process B first, after opening the opening-and-closing valve 34, the piston of the syringe pump 31 is pushed back to the state before suction, so that the suction state in the second container 20 is eliminated. Next, the connection of the suction cup 32 to the container unit 1 is released, and then the suction cup 32 is raised by the elevating means to complete the measurement operation for the container unit 1.
  • the optical measurement conditions can be easily uniformized by performing the second reaction step B and the measurement step B according to a predetermined time table for all the samples. Therefore, it is easy to eliminate detection errors due to variations in measurement conditions. Further, since optical measurement can be performed immediately after the sample solution is brought into contact with the second reagent R2, the generation of light to be detected is completed in a short time after the sample solution and the second reagent R2 are brought into contact. Detection using a simple reaction system can also be performed with high accuracy.
  • FIG. 4 is a schematic configuration diagram showing a third embodiment of the two-stage sample liquid reaction system of the present invention.
  • 4 includes a pipette tip 51, a pipette body 52 capable of sucking a predetermined amount of sample liquid into the pipette tip 51, and a cap 53 that fits the pipette tip 51 and covers at least the tip thereof. It is configured.
  • pipettes such as Eppendorf (registered trademark) pipette manufactured by Eppendorf, Pipetteman (registered trademark) manufactured by Gilson, etc. can be used.
  • the cap 53 corresponds to the first container in the present invention
  • the pipette tip 51 corresponds to the second container in the present invention
  • the pipette main body 52 corresponds to the suction / discharge means or the suction means in the present invention.
  • the pipette tip 51 fitted with the cap 53 corresponds to the container unit in the present invention.
  • the cap 53 has a bottomed cylindrical shape with an upper end opened, and the first reagent R1 is fixed to the inner lower end in a dry state. It is preferable that the cap 53 is provided with irregularities so that a gap through which air can flow is formed between the cap 53 and the pipette tip 51 when the cap 53 is fitted. Thereby, it becomes easy to perform suction discharge (suction) by a suction discharge means (suction means) described later while the pipette tip 51 and the cap 53 are fitted.
  • the first reagent R1 can be fixed in a dry state, for example, by performing lyophilization with the solution of the first reagent R1 placed in the cap 53.
  • the cap 53 is preferably made of a material that can be sterilized as necessary.
  • the light transmissive material that can be sterilized include quartz, borosilicate glass, polystyrene, polypropylene, and acrylic resin.
  • materials that can be sterilized include fluororesins such as polytetrafluoroethylene and polyether ether ketone resins.
  • the pipette tip 51 has an opening at the upper end and the lower end, and is a tapered cylindrical body that decreases in diameter toward the lower end.
  • the second reagent R2 is fixed in a dry state in the vicinity of the inner middle.
  • the second reagent R2 can be fixed in a dry state, for example, by performing lyophilization with the solution of the second reagent R2 attached in the vicinity of the inner middle.
  • the vicinity of the inner middle of the pipette tip 51 to which the second reagent R2 is fixed is separated from the sample solution when it is fitted with the cap 53 into which the sample solution has been introduced. When the sample liquid in the cap 53 is sucked by a certain amount, the position can come into contact with the sucked sample liquid.
  • the tip of the pipette tip 51 is positioned near the lower end inside the cap 53.
  • the sample liquid can be introduced to the lower end side of the pipette tip 51 up to a level equivalent to the liquid level of the sample liquid in the cap 53.
  • the pipette tip 51 and the cap 53 to which each reagent is fixed may be sealed as a whole using, for example, a shrink film or the like in a state where both are fitted.
  • the system 300 can be used to sequentially perform the steps shown in the following first usage method or second usage method, for example.
  • First usage A step of introducing the sample solution into the cap 53 and reacting the sample solution with the first reagent R1 (hereinafter referred to as “first reaction step C”).
  • first reaction step C A predetermined amount of the sample solution reacted with the first reagent R1 is sucked into the pipette tip 51 and then returned to the cap 53, and the second reagent R2 is reacted with the first reagent R1.
  • second reaction step C A step of returning to the cap 53 and optically measuring the sample solution after the reaction with the second reagent R2 (hereinafter referred to as “measurement step C”).
  • the time from the introduction of the sample solution into the cap 53 in the first reaction step C to the start of the next second reaction step C is to allow the sample introduced into the cap 53 to react with the first reagent R1. In consideration of the required reaction time, it is set appropriately. When the reaction between the sample and the first reagent R1 is fast, the process may be promptly shifted to the next second reaction step C. In the first reaction step C, the cap 53 can be appropriately heated to promote the reaction. When the pipette tip 51 and the cap 53 are fitted, the sample liquid is introduced into the cap 53 after the pipette tip 51 is removed.
  • the pipette tip 51 is attached to the pipette body 52. Thereafter, the cap 53 into which the sample solution has been introduced is fitted into the pipette tip 51. Next, by performing a suction operation with the pipette main body 52, the set amount of sample liquid is sucked up from the cap 53 to the pipette tip 51. At this time, the liquid volume of the sample liquid sucked into the pipette tip 51 is set to a liquid volume in which the liquid surface of the sample liquid remaining in the cap 53 does not fall below the lower end (lower end side opening) of the pipette tip 51. is required.
  • the amount of the sample liquid sucked into the pipette tip 51 is excessive and the opening at the lower end of the pipette tip 51 is exposed to the gas phase on the sample liquid, the sample liquid suddenly moves, and the pipette body There is a possibility that the inside of 52 may be contaminated. Further, the amount of the sample solution sucked up by the pipette tip 51 needs to be sufficient to reach the position where the second reagent R2 is fixed. When the aspirated sample liquid reaches the fixing position of the second reagent R2, the second reagent R2 is dissolved by the aspirated sample liquid, and the sample liquid and the second reagent R2 can react.
  • a discharge operation is performed by the pipette main body 52, whereby the sample liquid of the sucked liquid amount (the liquid amount set by the pipette main body 52) is returned to the cap 53.
  • the time until the discharge is performed after the suction is sufficient if the second reagent R2 is dissolved by the sucked sample solution. If the second reagent R2 is highly soluble, the discharge is performed immediately after the suction. It can be carried out. 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 and the second reagent R2 as in fluorescence measurement, it is preferable to discharge immediately after aspiration.
  • optical measurement can be performed immediately. If the reaction between the sample solution and the second reagent R2 is not sufficient at the end of discharge, the process proceeds to the measurement step C after an appropriate reaction time has elapsed.
  • the sample liquid returned to the cap 53 and optically measured after the reaction with the second reagent R2 is measured.
  • the cap 53 removed from the pipette tip 51 may be placed in the measuring part of the optical measuring means for measurement. If the pipette tip 51 is light transmissive, the cap 53 that is still fitted to the pipette tip 51 attached to the pipette main body 52 may be placed in the measuring part of the optical measuring means and measured. In that case, since the cap 53 can be moved while holding the pipette main body 52, the cap 53 can be easily moved to the optical measuring means.
  • the type of optical measurement is not particularly limited, and fluorescence detection, absorbance detection, scattered light detection, and the like can be appropriately employed.
  • first reaction step D A step of introducing the sample solution into the cap 53 and reacting the sample solution with the first reagent R1 (hereinafter referred to as “first reaction step D”).
  • first reaction step D A step of introducing the sample solution into the cap 53 and reacting the sample solution with the first reagent R1 (hereinafter referred to as “first reaction step D”).
  • second reaction step D A step of aspirating the sample solution and reacting the sample solution with the second reagent R2 (hereinafter referred to as “second reaction step D”).
  • second reaction step D A step of optically measuring the sample solution after the reaction with the second reagent R2 (hereinafter referred to as “measurement step D”).
  • the first reaction step D is the same as the first reaction step C.
  • the sample solution is sucked from the cap 53 into the pipette tip 51 and then held in the pipette tip 51 as it is.
  • the time for holding the sample solution in the pipette tip 51 is set to a time sufficient for the second reagent R2 to be dissolved by the aspirated sample solution and to react with the sample solution.
  • the second reaction step D can be performed in a short time. It is preferable that the measurement process D is completed immediately after the completion.
  • the sample liquid that is held in the pipette tip 51 and reacted with the second reagent R2 is optically measured.
  • the pipette tip 51 that is still attached to the pipette main body 52 may be removed from the cap 53 and placed in the measuring section of the optical measuring means. If the cap 53 is light transmissive, the pipette tip 51 may be placed in the measuring part of the optical measuring means while the cap 53 is fitted to the pipette tip 51 for measurement. In this case, it is possible to prevent the sample liquid from dripping from the pipette tip 51 and contaminating the optical measuring means. In either case, since the pipette tip 51 can be moved while holding the pipette body 52, the movement of the pipette tip 51 to the optical measuring means is easy.
  • both the first usage method and the second usage method can perform all reactions by a single pipette operation. Moreover, the member which interferes with a high precision detection like the diaphragm is not used. Therefore, highly sensitive detection can be easily performed.
  • the syringe pump is used as the suction / discharge means (suction means).
  • the suction / discharge means is a syringe as long as it can feed a constant volume of sample liquid.
  • the suction / discharge means is a syringe as long as it can feed a constant volume of sample liquid.
  • pumps for example, a piezoelectric pump, a diaphragm pump, a tube pump, etc. can be used.
  • the liquid feeding amount of these pumps can be set to a predetermined liquid feeding amount by controlling the operation time.
  • the liquid level of the sample liquid in the first container is detected by a liquid level sensor, and when the liquid level of the sample liquid in the first container is lowered to a predetermined height, liquid feeding of the pump is stopped. Also good.
  • the suction / discharge means of the first embodiment it is necessary to reverse the direction of liquid feeding between the suction process and the discharge process.
  • a tube pump the direction of squeezing the tube may be reversed.
  • the on-off valve 34 is changed to a three-way valve whose one port can be released to the atmosphere, and the three-way valve By the operation, the sample liquid sucked into the second container 20 can be returned to the first container 10 by releasing the suction state in the second container 20 to atmospheric pressure.
  • the suction discharge means 30A suction means 30B
  • the second container 20 are connected by the suction cup 32, but this connection method is limited to the suction cup. Absent.
  • a connection plug that fits airtightly inside the opening 20 a may be provided at the tip of the branch pipe 33.
  • the first reagent R1 immobilized on the container unit is activated by binding to endotoxin.
  • a reagent containing a luminescent synthetic substrate formed by binding a luminescent substrate to a peptide.
  • the second reagent R2 a reagent containing a luminescent enzyme and other compounds necessary for the luminescent reaction is used.
  • 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 has the above luminescence by the action of any one of activated factor C, activated factor B and coagulase produced by activating factor C. It has a structure in which the bond between the substrate and the peptide is cleaved.
  • aminoluciferin can be preferably used as the luminescent substrate.
  • the amide bond with aminoluciferin at the C-terminus of the peptide is any of active factor C, active factor B, and coagulation enzyme. Or any amino acid sequence that is cleaved by one protease activity.
  • NaCl may be added to the first reagent R1.
  • the luminescent enzyme is an enzyme that generates light by catalyzing the bioluminescence of the luminescent substrate released from the luminescent synthetic substrate.
  • 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 container unit structure, the two-stage reaction system, the configuration of the measurement system, and the specific procedure of the measurement method, those described in the first to third embodiments can be suitably applied.
  • luminescence such as measurement of endotoxin
  • the suction operation for introducing the sample solution into the second container to which the second reagent R2 is fixed is performed as quickly as possible within a range where stable suction can be performed.
  • the operation immediately proceeds to the discharge operation, and the discharge operation can be performed as quickly as possible within a range where stable discharge can be performed. Preferably it is done.
  • 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 first container in which the first reagent R1 is fixed.
  • the sample solution is sucked into the second container in which the second reagent R2 is fixed from the opening on the lower end side, and the sample solution is introduced to cause a luminescence 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 sample solution is introduced into the second container in which the second reagent R2 is fixed to when the luminescence reaction is optically detected should be 0 to 10 seconds. Is preferred.
  • the present invention can be suitably used for detecting microbial contaminants such as endotoxin and ⁇ -glucan.
  • SYMBOLS 1 Container unit, 10 ... 1st container, 20 ... 2nd container, 30A ... Suction discharge means, 30B ... Suction means, 40A ... Optical measurement means, 40B ... Optical measurement means, 51 ... Pipette tip, 52 ... Pipette main body, 53 ... Cap, 100 ... System, 200 ... System, 300 ... System, R1 ... First reagent, R2 ... Second reagent

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Abstract

La présente invention concerne un système de réaction à deux étapes, capable de réaliser, de façon simple et avec une grande sensibilité, des détections qui exigent des réactions à deux étapes et, si nécessaire, qui peut également être automatisé ; un système de mesure d'échantillon de fluide ; et un procédé de mesure d'échantillon de fluide. L'invention concerne un système de mesure, qui effectue, à la suite, les étapes suivantes : l'introduction d'un échantillon de fluide dans un premier contenant (10), dans lequel est fixé un premier réactif et sa réaction avec le premier réactif ; l'aspiration d'une quantité fixe d'une partie de l'échantillon de fluide, après sa réaction avec le premier réactif, par un moyen (30a) d'aspiration et d'évacuation, dans un second contenant (20), dans lequel est fixé un second réactif, puis son renvoi au premier contenant (10) et la réaction de l'échantillon de fluide avec le second réactif ; et la mesure optique de l'échantillon de fluide qui a été renvoyé au premier contenant (10) et a réagi avec le second réactif, à l'aide d'un moyen de mesure optique.
PCT/JP2014/057725 2013-03-22 2014-03-20 Système de réaction à deux étages, système de mesure d'échantillon de fluide et procédé de mesure d'échantillon de fluide WO2014148601A1 (fr)

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JP2013-060405 2013-03-22
JP2013060405A JP5783200B2 (ja) 2013-03-22 2013-03-22 二段反応システム、試料液の測定システム、および試料液の測定方法

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6117394A (en) * 1996-04-10 2000-09-12 Smith; James C. Membrane filtered pipette tip
JP2000304754A (ja) * 1999-02-12 2000-11-02 Ortho Clinical Diagnostics Inc 液体を混合するための方法及び装置
JP2001337039A (ja) * 2000-05-26 2001-12-07 Dkk Toa Corp 発光検出装置
WO2007029616A1 (fr) * 2005-09-05 2007-03-15 Universal Bio Research Co., Ltd. Support et appareil de traitement de support de susbstances diverses, et procédé de traitement mentionné
WO2009063840A1 (fr) * 2007-11-12 2009-05-22 Hiroshima University Procédé et coffret de mesure de niveau d'endotoxine
JP2012132878A (ja) * 2010-12-24 2012-07-12 Sekisui Chem Co Ltd 微生物夾雑物検出容器、微生物夾雑物検出システム、並びに、微生物夾雑物の検出方法

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP5686308B2 (ja) * 2009-02-20 2015-03-18 国立大学法人広島大学 βグルカンの濃度測定方法および濃度測定用キット

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6117394A (en) * 1996-04-10 2000-09-12 Smith; James C. Membrane filtered pipette tip
JP2000304754A (ja) * 1999-02-12 2000-11-02 Ortho Clinical Diagnostics Inc 液体を混合するための方法及び装置
JP2001337039A (ja) * 2000-05-26 2001-12-07 Dkk Toa Corp 発光検出装置
WO2007029616A1 (fr) * 2005-09-05 2007-03-15 Universal Bio Research Co., Ltd. Support et appareil de traitement de support de susbstances diverses, et procédé de traitement mentionné
WO2009063840A1 (fr) * 2007-11-12 2009-05-22 Hiroshima University Procédé et coffret de mesure de niveau d'endotoxine
JP2012132878A (ja) * 2010-12-24 2012-07-12 Sekisui Chem Co Ltd 微生物夾雑物検出容器、微生物夾雑物検出システム、並びに、微生物夾雑物の検出方法

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