WO2023166784A1 - Procédé d'extraction d'un échantillon biologique contenu dans une puce de la puce, et dispositif de mise en œuvre dudit procédé - Google Patents

Procédé d'extraction d'un échantillon biologique contenu dans une puce de la puce, et dispositif de mise en œuvre dudit procédé Download PDF

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Publication number
WO2023166784A1
WO2023166784A1 PCT/JP2022/039451 JP2022039451W WO2023166784A1 WO 2023166784 A1 WO2023166784 A1 WO 2023166784A1 JP 2022039451 W JP2022039451 W JP 2022039451W WO 2023166784 A1 WO2023166784 A1 WO 2023166784A1
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Prior art keywords
container
chip
biological sample
shaking
sample
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PCT/JP2022/039451
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English (en)
Japanese (ja)
Inventor
大輔 川上
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株式会社島津製作所
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Publication of WO2023166784A1 publication Critical patent/WO2023166784A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • 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
    • G01N35/02—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N37/00—Details not covered by any other group of this subclass

Definitions

  • the present invention relates to a method for extracting a biological sample held in a chip from the chip and an apparatus for executing the method.
  • a microsampling device for collecting a very small amount of biological sample has been proposed and implemented (see Patent Document 1).
  • a microsampling device draws a biological sample from an inlet using capillary force and holds it in a microchannel provided inside.
  • a piece hereinafter referred to as a chip
  • the biological sample in the chip cut out from the microsampling device is taken out from the chip into another container, subjected to pretreatment such as deproteinization, and then subjected to analysis by HPLC, LC-MS/MS, or the like.
  • Patent Document 2 proposes to use a stirring function of a pretreatment device in order to shake a container containing chips.
  • an object of the present invention is to enable efficient extraction of a biological sample held in a chip.
  • a method according to the present invention is a method for extracting a biological sample held in a microchannel of a chip having therein a microchannel whose both ends are open, wherein the microchannel of the chip is placed in a container. an immersion step of immersing an end of a microchannel in an extraction solvent; and an extraction step of extracting the biological sample from the chip into the extraction solvent by shaking the container after the immersion step.
  • the device includes a container arrangement place where a container is arranged, a nozzle for dispensing an extraction solvent into the container at a predetermined dispensing position, and a shaking process for shaking the container.
  • a transport mechanism for transporting the filtration container; and a control unit for controlling operations of the nozzle, the shaking treatment unit, the pretreatment unit, and the transport mechanism, wherein the control unit comprises When recognizing from pre-registered information about a biological sample to be pretreated that the biological sample is held in a chip and housed in the container arranged at the container placement location, a dispensing step of transporting the container containing the chip to the dispensing position by the transport mechanism and dispensing a predetermined amount of extraction solvent into the container by the nozzle; After the container into which the solvent has been dispensed is transported to the shaking processing unit by the transport mechanism, the biological sample is extracted from the chip into the extraction solvent by shaking the container in the shaking processing unit. and an extraction step.
  • biological samples include blood samples such as whole blood, serum, and plasma.
  • the end of the microchannel of the chip is immersed in the extraction solvent so that the biological sample is in contact with the extraction solvent.
  • the surface tension acting on the biological sample at the end is reduced, and the biological sample can be efficiently extracted from the tip.
  • the chip when the target biological sample is held in the chip and accommodated in the container arranged at the container arrangement place, the chip contains a predetermined amount of the extraction solvent. Since the filtering container is shaken to take out the biological sample from the chip after dispensing into the container, the biological sample held in the chip can be efficiently taken out from the chip.
  • FIG. 4 is a flow chart for explaining one embodiment of a method for extracting a biological sample from a chip;
  • FIG. 1 shows an example of a micro-sampling device;
  • FIG. 4 is a cross-sectional view showing an example of a filtration container that can be used in the method of the same embodiment; It is a figure which shows the state which the chip
  • FIG. 4 is a conceptual diagram for explaining an example of shaking processing of the filtration container;
  • FIG. 4 is a cross-sectional view showing an example of a collection container that can be used in the method of the same embodiment.
  • FIG. 4 is a cross-sectional view showing a state in which the recovery container is arranged below the filtration container; 1 is a schematic configuration diagram showing an example of a pretreatment device; FIG. 5 is a flow chart showing an example of pretreatment operation in the same pretreatment apparatus.
  • the method described below is a method of extracting a biological sample such as whole blood, plasma, or serum retained in the chip from the chip, and is used as a pretreatment of the biological sample to be subjected to HPLC, LC-MS/MS. It is to be implemented.
  • a chip has inside a microchannel with both ends open.
  • An example of a chip is a piece cut from a microsampling device such as that shown in FIG.
  • the microsampling device 100 is provided with an inlet 102 for sucking a biological sample at the tip, and a microchannel 104 for holding the biological sample sucked from the inlet 102 is provided inside.
  • Microchannel 104 is a U-shaped channel that includes two straight portions extending along the longitudinal direction of microsampling device 100 .
  • the micro-sampling device 100 has two grooves 116, 118 on its surface so that it can be divided into three parts: a distal portion 108, a proximal portion 110, and a central portion 114. At each groove 116, 118, a micro-sampling device is formed. By breaking the sampling device 100, the central portion 114 can be taken out. The interior of the central portion 114 includes partial sections of the two straight portions of the microchannel 104 , and by cutting the central portion 114 from the microsampling device 100 , the living body held in the microchannel 104 can be extracted. A certain amount of the sample can be removed. In this example, a central portion 114 cut from the microsampling device 100 is used as a tip to hold the biological sample inside. The central portion 114 cut from the microsampling device 100 is hereinafter referred to as the "tip 114".
  • the chip 114 and the extraction solvent are placed in a top-open container, and the end of the chip 114 is immersed in the extraction solvent (Fig. 1: step 101).
  • the container is the filtration container 200 shown in FIG.
  • the filter container 200 is provided with a protein-removing filter 204 at the bottom of an internal space 202 for containing the chips 114 and an extraction solvent, and an extraction chamber below the protein-removing filter 204 for downwardly extracting the filtrate that has passed through the protein-removing filter.
  • a mouth 206 is provided.
  • FIG. 4 shows the end of tip 114 submerged in the extraction solvent in filtration vessel 200 .
  • the extraction solvent is primarily water.
  • An organic solvent for example, methanol
  • for activation of the protein removal filter 204 may be mixed with the extraction solvent.
  • the filtration container 200 is shaken while the end of the chip 114 is immersed in the extraction solvent (FIG. 1: step 102).
  • the shaking process is to periodically move the filtering container 200 so that the inside of the filtering container 200 is stirred.
  • the shaking process as shown in FIG. 5, the bottom portion of the filtering vessel 200 is swirled in a horizontal plane with the filtering vessel 200 tilted.
  • protein removal treatment for removing proteins in the biological sample taken out from the chip 114 can be performed.
  • a predetermined amount of protein-removing liquid for example, methanol
  • the protein-removing filter is used.
  • Deproteinization of the biological sample can be performed by passing the liquid through.
  • a collection vessel 300 as shown in FIG. 6 can be used when performing the protein removal process.
  • the collection container 300 has an internal space 302 for collecting the filtrate dropped from the extraction port 206 of the filtration container 200 .
  • the collection container 300 can accommodate the lower portion of the filtration container 200, as shown in FIG.
  • steps 1 and 2 of FIG. 1 when the biological sample is extracted from the tip 114 in a container such as a test tube not equipped with a protein removal filter, the sample is placed in the filtration container 200 or a container having an equivalent function. Either by transferring the solution and performing filtration, or by centrifuging the container containing the sample solution in a centrifuge (for example, 15000 rpm for 10 minutes) and then collecting the supernatant in the container. Deproteinization can be performed.
  • the pretreatment apparatus 400 is provided with a container table 412 on which the separation device 200 and the collection container 300 are arranged in a pair as a container installation place. Multiple sets of separation devices 200 and collection vessels 300 are arranged on the vessel table 412 .
  • the separation device 200 and collection container 300 are transported by a transport arm 424 (transport mechanism).
  • the transport arm 424 can hold the separation device 200 and the collection container 300 at its tip, and transports the held separation device 200 and collection container 300 in the circumferential direction.
  • a rotary sample table 402 for setting a sample container 406 containing a sample is provided at a position different from the container table 412 .
  • a sample rack 404 holding a plurality of sample containers 406 is arranged in an annular shape on the sample table 402 .
  • a reagent table 408 that rotates independently of the sample table 402 is provided inside the sample table 402 .
  • a reagent container 410 containing various reagents such as a protein-free solution is arranged on the reagent table 408 .
  • a sample arm 420 is provided near the sample table 412 for collecting a sample from a sample container placed on the sample table 402 .
  • the sample arm 420 rotates in the horizontal plane and moves up and down in the vertical direction.
  • a sample nozzle 420a is provided at the tip of the sample arm 420 to aspirate and dispense the sample.
  • a reagent arm 426 is provided at a position separate from the sample arm 420 .
  • a reagent nozzle 427 for aspirating and dispensing the reagent is provided at the tip of the reagent arm 426 .
  • a cleaning port 445 for cleaning the sample nozzle 420a is provided in the vicinity of the sample arm 420 .
  • a dispensing position is set between the sample table 402 and the container table 412, and a dispensing port 432 is provided at the dispensing position.
  • the dispensing position is a position where the transport arm 424 can transport the separation device 200, the sample nozzle 420a can dispense the sample, and the reagent nozzle 427 can dispense the reagent.
  • Dispense port 432 can receive a separation device 200 transported by transport arm 424 .
  • the container table 412 rotates in the horizontal plane to move the separation device 200 and the collection container 300 in the circumferential direction so that the separation device 200 and the collection container 300 used for pretreatment are aligned with the track of the holding portion 425 of the transfer arm 424 .
  • the transport arm 424 can hold an unused separation device 200 or collection container 300 positioned along the track of the holding portion 425 .
  • Filtration processing unit 430 is configured to be able to depressurize internal space 302 of collection container 300 when disposed with collection container 300 attached to the bottom of filtration container 200 (state shown in FIG. 7).
  • the structure of the filtration processing unit 430 may be equivalent to that disclosed in FIGS. 4A to 4C of Patent Document 2 (Japanese Patent Application Laid-Open No. 2019-132749).
  • the shaking processor 436 has a function of receiving the separation device 200 and shaking the separation device 200 .
  • the structure of the shaking processor 436 may be equivalent to that disclosed in FIGS. 6A and 6B of Patent Document 2.
  • a biological sample is contained in a sample container 406 set on a sample table 402 , or is contained in a separation device 200 set on a container table 412 while being held by a chip 114 . Whether the biological sample is contained in the sample container 406 or held in the chip 114 in the separation device 200 is registered in advance in the apparatus as sample information.
  • the control unit 450 recognizes from the registered sample information whether the target sample is contained in the sample container 406 or is held by the chip 114 in the separation device 200, and performs perform an action.
  • the control unit 450 confirms sample information registered for the target biological sample (step 201), and recognizes whether or not the biological sample is held in the chip 114 (step 202). If the target biological sample is held by the tip 114 (step 202: Yes), the control unit 450 moves the filtration container 200 containing the tip 114 from the container table 412 to the dispensing port 432 by the transfer arm. 424 (step 203), and the reagent nozzle 427 dispenses a predetermined amount (eg, 100 ⁇ L) of extraction solvent into the filtration container 200 (step 204). As a result, the ends of the chips 114 are immersed in the extraction solvent in the filtration container 200 .
  • a predetermined amount eg, 100 ⁇ L
  • control unit 450 transports the filtration container 200 into which the extraction solvent has been dispensed to the shaking processing unit 436 by the transportation arm 424, and shakes the filtration container 200 in the shaking processing unit 436 (step 205). ).
  • the biological sample extracted from the chip 114 is mixed with the extraction solvent in the filtration container 200 to form a sample solution.
  • the control unit 450 transports the filter container 200 containing the sample solution to the pipetting port 432 by the transport arm 424 (step 206). of deproteinized solution is added (step 207). After that, the control unit 450 transports the filtering container 200 to the shaking processing unit 436 by the transporting arm 424, and starts the shaking processing of the filtering container 200 in the shaking processing unit 436 (step 208). This shaking treatment agitates the inside of the filtration container 200, and mixes the sample solution and the deproteinized solution.
  • the control unit 450 transports and places the empty collection container 300 placed on the container table 412 to the filtration processing unit 430 by the transport arm 424 (step 209). .
  • the control section 450 transports the filtration container 200 of the shaking processing section 436 to the filtration processing section 430 by the transport arm 424, and onto the collection container 300 of the filtration processing section 430.
  • a filtration container 200 is placed and a filtration process is performed (step 210). As a result, the filtrate from which the protein has been removed is recovered in the recovery container 300 .
  • the control unit 450 if the target biological sample is not held on the chip 114, that is, if the target biological sample is stored in the sample container 406 of the sample table 402 (step 202: No), the control unit 450 The empty filtration container 200 is transported from the container table 412 to the dispensing port 432 by the transport arm 424 (step 211), and the target biological sample is dispensed from the sample container 406 to the filtration container 200 by the sample nozzle 420a (step 212). . Further, the controller 450 dispenses a predetermined amount of protein-removed liquid into the filtration container 200 through the reagent nozzle 427 (step 213).
  • control unit 450 transports the filtration container 200 into which the biological sample and the deproteinized liquid have been dispensed to the shaking processing unit 436 by the transportation arm 424, and starts the shaking processing of the filtration container 200 in the shaking processing unit 436. (step 214).
  • the inside of the filtration container 200 is agitated and the biological sample and the deproteinized liquid are mixed.
  • Subsequent steps 215 and 216 are the same as steps 209 and 210, respectively, and through these steps, the filtrate from which proteins have been removed is recovered in the recovery container 300.
  • a method for extracting a biological sample held in a microchannel of a chip having therein a microchannel open at both ends comprising: an immersion step of immersing the ends of the microchannels of the chip in an extraction solvent; and an extraction step of extracting the biological sample from the chip into the extraction solvent by shaking the container after the immersion step.
  • the container is provided with the protein-removing filter at the bottom of the internal space that accommodates the chip and the extraction solvent, and an extraction port below the protein-removing filter for downwardly extracting the filtrate that has passed through the protein-removing filter.
  • It may be a filtration container provided with. According to this aspect, there is no need to transfer the biological sample to another container in order to deproteinize the biological sample taken out from the chip, so the deproteinization can be efficiently performed without causing loss of the biological sample. becomes possible.
  • a container placement location where the container is placed, a nozzle for dispensing the extraction solvent into the container at a predetermined dispensing position, and a nozzle for shaking the container.
  • a shaking processing unit, a transport mechanism for transporting the container, and a control unit for controlling the operation of the nozzle, the shaking processing unit, and the transport mechanism When the control unit recognizes from the information registered in advance about the biological sample that the biological sample is held in the chip and accommodated in the container arranged at the container placement location, a dispensing step of transporting the container containing the chip to the dispensing position by the transport mechanism and dispensing a predetermined amount of the extraction solvent into the container by the nozzle; After the container into which the extraction solvent has been dispensed in the dispensing step is transported to the shaking processing unit by the transportation mechanism, the biological sample is shaken in the shaking processing unit by shaking the container. and extracting from the chip.

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  • Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Sampling And Sample Adjustment (AREA)

Abstract

La présente invention concerne un procédé d'extraction d'un échantillon biologique contenu dans un micro-passage d'écoulement d'une puce (114) qui comprend intérieurement le micro-passage d'écoulement, dont les deux extrémités sont ouvertes, le procédé comprenant: une étape d'immersion consistant à immerger une partie d'extrémité de le micro-passage d'écoulement de la puce (114) dans un solvant d'extraction, dans un récipient (200); et, après l'étape d'immersion, une étape d'extraction consistant à extraire l'échantillon biologique de la puce (114) dans le solvant d'extraction en secouant le récipient (200).
PCT/JP2022/039451 2022-03-03 2022-10-24 Procédé d'extraction d'un échantillon biologique contenu dans une puce de la puce, et dispositif de mise en œuvre dudit procédé WO2023166784A1 (fr)

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JP2022-032446 2022-03-03
JP2022032446 2022-03-03

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WO2023166784A1 true WO2023166784A1 (fr) 2023-09-07

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017122372A1 (fr) * 2016-01-14 2017-07-20 株式会社島津製作所 Dispositif de prélèvement d'échantillon, support de dispositif de prélèvement d'échantillon et procédé de prétraitement d'échantillon qui utilise le dispositif de prélèvement d'échantillon
JP2019132749A (ja) * 2018-02-01 2019-08-08 株式会社島津製作所 マイクロ流路内に保持された検体の前処理方法、その前処理方法を実行するための前処理装置及びその前処理装置を備えた分析システム
JP2021536564A (ja) * 2018-08-22 2021-12-27 クエスト ダイアグノスティックス インヴェストメンツ エルエルシー サンプリング装置のプレートおよびマイクロサンプリング装置のマイクロ遠心バイアル

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017122372A1 (fr) * 2016-01-14 2017-07-20 株式会社島津製作所 Dispositif de prélèvement d'échantillon, support de dispositif de prélèvement d'échantillon et procédé de prétraitement d'échantillon qui utilise le dispositif de prélèvement d'échantillon
JP2019132749A (ja) * 2018-02-01 2019-08-08 株式会社島津製作所 マイクロ流路内に保持された検体の前処理方法、その前処理方法を実行するための前処理装置及びその前処理装置を備えた分析システム
JP2021536564A (ja) * 2018-08-22 2021-12-27 クエスト ダイアグノスティックス インヴェストメンツ エルエルシー サンプリング装置のプレートおよびマイクロサンプリング装置のマイクロ遠心バイアル

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