WO2020021784A1 - Biological component collecting method and biological component analysis method - Google Patents

Biological component collecting method and biological component analysis method Download PDF

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Publication number
WO2020021784A1
WO2020021784A1 PCT/JP2019/015888 JP2019015888W WO2020021784A1 WO 2020021784 A1 WO2020021784 A1 WO 2020021784A1 JP 2019015888 W JP2019015888 W JP 2019015888W WO 2020021784 A1 WO2020021784 A1 WO 2020021784A1
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component
collecting
sample
solvent
biological component
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French (fr)
Japanese (ja)
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岩本　慎一
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株式会社島津製作所
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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
    • G01N1/02—Devices for withdrawing samples
    • G01N1/04—Devices for withdrawing samples in the solid state, e.g. by cutting

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  • the present invention relates to a method for collecting a biological component for collecting a component derived from a living body and a method for analyzing the biological component.
  • Non-Patent Document 1 an iron oxide nanoparticle processing plate is pressed as a sample plate on a human fingertip. Thereby, the components on the skin surface are transferred to the sample plate. The transferred components are analyzed by an analyzer using SALDI (surface assisted laser desorption / ionization).
  • SALDI surface assisted laser desorption / ionization
  • the component can be collected by pressing the sample plate against the living body as described above.
  • the living body is covered with body hair and is not exposed, it is difficult to collect components from the living body.
  • An object of the present invention is to provide a method for collecting a biological component and a method for analyzing a biological component, which can easily collect a component derived from a living body.
  • the inventor of the present invention conducted a preliminary experiment to collect components from human scalp. In this preliminary experiment, it was found that by rubbing the chip-shaped member against the scalp while avoiding the hair, an analyzable amount of the component was collected on the chip-shaped member. In addition, the inventors have studied a member capable of directly contacting a living body without substantially damaging the living body even when the living body is covered with body hair, as a collecting member for collecting components from the living body. As a result, it has been found that the sampling member has flexibility and a rod-shaped member is appropriate. Based on these findings, the present invention has been made.
  • a flexible, rod-shaped collection member is rubbed against the surface of a living body to obtain a component on the surface of the living body as a sample component. And extracting the sample component attached to the collection member from the collection member using a solvent.
  • the tip of a flexible and rod-shaped collection member is rubbed against the surface of a living body.
  • the collection member has a rod shape, even when the living body is covered with body hair, the tip of the collection member can be easily rubbed against the surface of the living body while avoiding the body hair.
  • the collection member has flexibility, the tip of the collection member can be easily rubbed against the surface of the living body without substantially damaging the living body of a small animal such as a mouse or a rat.
  • the sampling member has a rod-shaped part and an annular part formed at one end of the rod-shaped part, and may be formed of resin. In this case, the worker can easily attach a sufficient amount of the sample component to the annular portion by gripping the rod-shaped portion and rubbing the annular portion against the surface of the living body.
  • the sampling member may include a disposable loop. In this case, the sampling member can be easily realized.
  • the step of extracting the sample component attached to the sampling member from the sampling member includes separating the portion of the sampling member having the sample component attached from the other portion of the sampling member, and placing the separated portion of the sampling member in a container. It may include storing, injecting the solvent into the container, and dissolving the sample component attached to the separated portion of the collection member in the container in the container. In this case, sample components can be efficiently extracted from the sampling member.
  • Dissolving the sample components in the solvent may include centrifuging the sample components by stirring the solvent. In this case, the sample components can be more efficiently extracted from the sampling member.
  • the step of extracting the sample component attached to the collection member from the collection member includes extracting the first component contained in the sample component from the collection member using the first solvent as a solvent; Extracting the second component contained in the sample component from the collection member using a second solvent having a lower polarity than the first solvent as a solvent after the extraction of the second solvent.
  • a method for analyzing a biological component according to another aspect of the present invention includes a step of collecting a sample component by the method for collecting a biological component according to one aspect of the present invention, and a step of analyzing the collected sample component.
  • the sample component collected by the above-described method of collecting a biological component is analyzed.
  • a component derived from a living body is easily collected. Thereby, the component derived from the living body can be easily analyzed.
  • components derived from living organisms can be easily collected.
  • FIG. 1 is a diagram showing a collecting member used in a method for collecting a biological component according to one embodiment of the present invention.
  • FIG. 2 is a flowchart showing a method of analyzing a biological component using the sampling member of FIG.
  • FIG. 1 is a diagram showing a collecting member used in a method for collecting a biological component according to one embodiment of the present invention.
  • the sampling member 10 is sterilized, has a rod shape, and has flexibility.
  • the sampling member 10 is preferably formed of a resin.
  • sampling member 10 may be a disposable loop made of resin, which is sterilized by gamma rays, for example, or a pipette tip.
  • sampling member 10 is a disposable loop formed of a resin such as polypropylene or polytetrafluoroethylene, and includes rod-shaped portion 1 and annular portion 2.
  • the annular part 2 is formed at one end of the rod part 1.
  • the total length of the sampling member 10 is, for example, 220 mm, and the outer diameter of the annular portion 2 is, for example, about 2 mm.
  • the annular portion 2 may have an annular shape, an elliptical shape, or an oblong shape.
  • the other end of the rod portion 1 is used as a grip portion 3.
  • FIG. 2 is a flowchart showing a method of analyzing a biological component using the sampling member 10 of FIG. Steps S1 to S7 in FIG. 2 correspond to a method of collecting a biological component.
  • an operator who collects a component grips the grip portion 3 of the collection member 10 and rubs the annular portion 2 against the surface of the living body (step S1).
  • sample components components on the surface of the living body (hereinafter, referred to as sample components) adhere to the annular portion 2.
  • Step S2 the operator cuts the tip portion (the portion shown by the dotted line in FIG. 1) of the sampling member 10 including the annular portion 2 to which the sample component has adhered in Step S1 with scissors or the like (Step S2).
  • the tip portion of the sampling member 10 to which the sample component has adhered is cut off from other portions of the sampling member 10.
  • the length of the tip of the separated sampling member 10 is, for example, about 25 mm to 35 mm.
  • the tip portion of the sampling member 10 can be stored in the container.
  • the container is, for example, a microtube.
  • the operator stores the tip portion of the sampling member 10 cut off in step S2 in a microtube with a cap (step S3). Further, the operator injects a predetermined amount of a predetermined solvent into the microtube (step S4).
  • a predetermined solvent into the microtube.
  • the plurality of solvents having different polarities may be, for example, solvents of different types such as water, ethanol, methanol, acetonitrile, and hexane, or may be solvents of the same type but different concentrations.
  • step S5 the operator stirs the solvent injected into the microtube in step S4 (step S5).
  • the stirring may be performed using, for example, tweezers, or may be performed using a stirring device such as a vortex mixer. This causes the sample components to be centrifuged and eluted in the solvent.
  • the worker extracts the sample components eluted in the solvent in step S5 from the microtube (step S6).
  • step S7 the operator determines whether all the desired solvents prepared in advance have been used. If all the solvents have not been used, the operator returns to step S4. Steps S4 to S7 are repeated until all the solvent is used. In this case, each time step S4 is repeated, a solvent having a larger polarity is injected into the microtube. This makes it possible to sequentially extract and analyze components such as lipids having different solubility in a solvent while separating the components.
  • step S8 the operator analyzes the sample components extracted in step S6 using the analyzer (step S8). Specifically, an operator drops an appropriate amount of the extracted sample component on a sample support plate using a micropipette or the like, and sets the sample support plate on a mass spectrometer. Thereafter, the mass of the sample component is analyzed by MALDI-MS (matrix-assisted laser desorption / ionization mass spectrometry) or LDI-MS (laser desorption / ionization mass spectrometry).
  • MALDI-MS matrix-assisted laser desorption / ionization mass spectrometry
  • LDI-MS laser desorption / ionization mass spectrometry
  • step S9 the worker determines whether all the sample components extracted in step S6 have been analyzed. If not all sample components have been analyzed, the operator returns to step S8. Steps S8 and S9 are repeated until all sample components have been analyzed. When all the sample components have been analyzed, the operator ends the biological component analysis method.
  • the annular portion 2 at the tip of the flexible and rod-shaped collection member 10 is rubbed against the surface of the living body.
  • the collection member 10 has a rod shape, even when the living body is covered with body hair, the annular portion 2 can be easily rubbed against the surface of the living body while avoiding the body hair.
  • the sampling member 10 has flexibility, the annular portion 2 can be easily rubbed against the surface of a small animal such as a mouse or a rat without substantially damaging the living body.
  • Steps S1 to S3 and steps S4 to S9 in FIG. 2 may be executed by different operators.
  • the microtube obtained by one worker performing steps S1 to S3 may be stored or transported and then handed over to another worker. In this case, another worker can execute steps S4 to S9 using the handed microtube.
  • step S8 may be performed between step S6 and step S7.
  • the sample component is analyzed in step S8.
  • the sample components are analyzed by the mass spectrometer, but the present invention is not limited to this.
  • the sample components may be analyzed by other analyzers such as GC (gas chromatography) or LC (liquid chromatography).

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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

This biological component collection method includes: a step for attaching, to a flexible and rod-shaped collection member, a component of the surface of a living body as a sample component, by rubbing a tip portion of the collection member on the surface of the living body; and a step for extracting, from the collection member, the sample component attached to the collection member by using a solvent. This biological component analysis method includes: a step for collecting a sample component by means of the biological component collection method; and a step for analyzing the collected sample component.

Description

生体成分の採取方法および生体成分の分析方法Biological component collection method and biological component analysis method
　本発明は、生体に由来する成分を採取するための生体成分の採取方法および生体成分の分析方法に関する。 The present invention relates to a method for collecting a biological component for collecting a component derived from a living body and a method for analyzing the biological component.
　生体に由来する成分を分析することにより、生体の診断または生体への薬効の評価等の医学または薬学のための分析を行うことができる。生体から成分を採取する方法として、例えば、非特許文献１においては、ヒトの指先に酸化鉄ナノ粒子加工プレートがサンプルプレートとして押圧される。これにより、サンプルプレートに皮膚表面の成分が転写される。転写された成分がＳＡＬＤＩ（表面支援レーザ脱離イオン化）法を用いた分析装置により分析される。 分析 By analyzing components derived from the living body, analysis for medicine or pharmacy such as diagnosis of the living body or evaluation of drug efficacy on the living body can be performed. As a method for collecting components from a living body, for example, in Non-Patent Document 1, an iron oxide nanoparticle processing plate is pressed as a sample plate on a human fingertip. Thereby, the components on the skin surface are transferred to the sample plate. The transferred components are analyzed by an analyzer using SALDI (surface assisted laser desorption / ionization).
　また、上記の酸化鉄ナノ粒子加工プレートではなく、標準的なステンレス製のサンプルプレートをヒトの指先に押圧した場合でも、サンプルプレートに皮膚表面の成分を転写可能であることが報告されている。このように転写された成分は、例えばＭＡＬＤＩ（マトリックス支援レーザ脱離イオン化）法を用いた分析装置により分析される。
Maiko Kusano et al., "Laser Desorption/Ionization Mass Spectrometry (LDI-MS) of Lipids with Iron Oxide Nanoparticle-Coated Targets," Mass Spectrometry, 2014, Vol. 3, No. 1, A0026 In addition, it has been reported that even if a standard stainless steel sample plate is pressed against a human fingertip instead of the iron oxide nanoparticle-processed plate, the components on the skin surface can be transferred to the sample plate. The components transferred in this manner are analyzed by an analyzer using, for example, MALDI (matrix assisted laser desorption / ionization).
Maiko Kusano et al., "Laser Desorption / Ionization Mass Spectrometry (LDI-MS) of Lipids with Iron Oxide Nanoparticle-Coated Targets," Mass Spectrometry, 2014, Vol. 3, No. 1, A0026
　成分を採取すべき生体の部分が露出しかつ当該部分に荷重を加えることが可能な場合には、上記のようにサンプルプレートを生体に押圧することにより成分を採取することが可能である。しかしながら、生体が体毛に覆われており、露出していない場合には生体から成分を採取することは困難である。また、荷重を加えることができない小動物のような生体から成分を採取することは困難である。そのため、このような生体に由来する成分を分析することは困難である。 (4) When a portion of the living body from which the component is to be collected is exposed and a load can be applied to the portion, the component can be collected by pressing the sample plate against the living body as described above. However, if the living body is covered with body hair and is not exposed, it is difficult to collect components from the living body. Further, it is difficult to collect components from a living body such as a small animal to which a load cannot be applied. Therefore, it is difficult to analyze such a component derived from a living body.
　本発明の目的は、生体に由来する成分を容易に採取することが可能な生体成分の採取方法および生体成分の分析方法を提供することである。 目的 An object of the present invention is to provide a method for collecting a biological component and a method for analyzing a biological component, which can easily collect a component derived from a living body.
　本発明の発明者は、ヒトの頭皮から成分を採取する予備実験を行った。この予備実験において、毛髪を回避しつつチップ状の部材を頭皮に擦りつけることにより、分析可能な量の成分がチップ状の部材に採取されるという知見を得た。また、発明者は、生体から成分を採取するための採取部材として、生体が体毛に覆われている場合でも、生体をほとんど損傷させることなく生体に直接接触することが可能な部材について検討した。その結果、採取部材は可撓性を有しかつ棒状の部材が適切であるという知見を得た。これらの知見に基づいて、以下の本発明に想到した。 発 明 The inventor of the present invention conducted a preliminary experiment to collect components from human scalp. In this preliminary experiment, it was found that by rubbing the chip-shaped member against the scalp while avoiding the hair, an analyzable amount of the component was collected on the chip-shaped member. In addition, the inventors have studied a member capable of directly contacting a living body without substantially damaging the living body even when the living body is covered with body hair, as a collecting member for collecting components from the living body. As a result, it has been found that the sampling member has flexibility and a rod-shaped member is appropriate. Based on these findings, the present invention has been made.
　（１）本発明の一局面に従う生体成分の採取方法は、可撓性を有しかつ棒状の採取部材の先端部分を生体の表面に擦りつけることにより生体の表面の成分を試料成分として採取部材に付着させるステップと、採取部材に付着した試料成分を溶媒を用いて採取部材から抽出するステップとを含む。 (1) In a method for collecting a biological component according to one aspect of the present invention, a flexible, rod-shaped collection member is rubbed against the surface of a living body to obtain a component on the surface of the living body as a sample component. And extracting the sample component attached to the collection member from the collection member using a solvent.
　この生体成分の採取方法においては、可撓性を有しかつ棒状の採取部材の先端部分が生体の表面に擦りつけられる。ここで、採取部材は棒状を有するため、生体が体毛に覆われている場合でも、体毛を回避しつつ採取部材の先端部分を生体の表面に容易に擦りつけることが可能である。また、採取部材は可撓性を有するため、マウスまたはラット等の小動物の生体をほとんど損傷させることなく採取部材の先端部分を当該生体の表面に容易に擦りつけることが可能である。 In this method of collecting biological components, the tip of a flexible and rod-shaped collection member is rubbed against the surface of a living body. Here, since the collection member has a rod shape, even when the living body is covered with body hair, the tip of the collection member can be easily rubbed against the surface of the living body while avoiding the body hair. In addition, since the collection member has flexibility, the tip of the collection member can be easily rubbed against the surface of the living body without substantially damaging the living body of a small animal such as a mouse or a rat.
　上記の手順によれば、分析を行うために十分な量の生体の表面の成分が試料成分として採取部材の先端部分に付着する。そのため、採取部材に付着した試料成分を溶媒を用いて採取部材から容易に抽出することが可能である。その結果、生体に由来する成分を容易に採取することができる。 According to the above procedure, a sufficient amount of components on the surface of the living body to perform the analysis adhere to the distal end portion of the sampling member as sample components. Therefore, it is possible to easily extract the sample component attached to the collection member from the collection member using the solvent. As a result, a component derived from a living body can be easily collected.
　（２）採取部材は、棒状部と、棒状部の一端に形成された環状部とを有し、樹脂により形成されてもよい。この場合、作業者は、棒状部を把持して環状部を生体の表面に擦りつけることにより、環状部に十分な量の試料成分を容易に付着させることができる。 (2) The sampling member has a rod-shaped part and an annular part formed at one end of the rod-shaped part, and may be formed of resin. In this case, the worker can easily attach a sufficient amount of the sample component to the annular portion by gripping the rod-shaped portion and rubbing the annular portion against the surface of the living body.
　（３）採取部材は、ディスポザブルループを含んでもよい。この場合、採取部材を容易に実現することができる。 (3) The sampling member may include a disposable loop. In this case, the sampling member can be easily realized.
　（４）採取部材に付着した試料成分を採取部材から抽出するステップは、試料成分が付着した採取部材の部分を採取部材の他の部分から切り離すことと、採取部材の切り離された部分を容器に収納することと、溶媒を容器に注入することと、容器内で採取部材の切り離された部分に付着した試料成分を溶媒に溶解させることとを含んでもよい。この場合、採取部材から試料成分を効率よく抽出することができる。 (4) The step of extracting the sample component attached to the sampling member from the sampling member includes separating the portion of the sampling member having the sample component attached from the other portion of the sampling member, and placing the separated portion of the sampling member in a container. It may include storing, injecting the solvent into the container, and dissolving the sample component attached to the separated portion of the collection member in the container in the container. In this case, sample components can be efficiently extracted from the sampling member.
　（５）試料成分を溶媒に溶解させることは、溶媒を撹拌することにより試料成分を遠心分離させることを含んでもよい。この場合、採取部材から試料成分をより効率よく抽出することができる。 (5) Dissolving the sample components in the solvent may include centrifuging the sample components by stirring the solvent. In this case, the sample components can be more efficiently extracted from the sampling member.
　（６）採取部材に付着した試料成分を採取部材から抽出するステップは、第１の溶媒を溶媒として用いて試料成分に含まれる第１の成分を採取部材から抽出することと、第１の成分が抽出された後、第１の溶媒よりも低い極性を有する第２の溶媒を溶媒として用いて試料成分に含まれる第２の成分を採取部材から抽出することとを含んでもよい。この場合、脂質等の溶媒への溶解性が異なる成分を分離しつつ、順次抽出することができる。 (6) The step of extracting the sample component attached to the collection member from the collection member includes extracting the first component contained in the sample component from the collection member using the first solvent as a solvent; Extracting the second component contained in the sample component from the collection member using a second solvent having a lower polarity than the first solvent as a solvent after the extraction of the second solvent. In this case, it is possible to sequentially extract components while separating components having different solubility in a solvent such as lipids.
　（７）本発明の他の局面に従う生体成分の分析方法は、本発明の一局面に従う生体成分の採取方法により試料成分を採取するステップと、採取された試料成分を分析するステップとを含む。 (7) A method for analyzing a biological component according to another aspect of the present invention includes a step of collecting a sample component by the method for collecting a biological component according to one aspect of the present invention, and a step of analyzing the collected sample component.
　この生体成分の分析方法においては、上記の生体成分の採取方法により採取された試料成分が分析される。生体成分の採取方法においては、生体に由来する成分が容易に採取される。これにより、生体に由来する成分を容易に分析することができる。 In this method of analyzing a biological component, the sample component collected by the above-described method of collecting a biological component is analyzed. In the method of collecting a biological component, a component derived from a living body is easily collected. Thereby, the component derived from the living body can be easily analyzed.
　本発明によれば、生体に由来する成分を容易に採取することができる。 According to the present invention, components derived from living organisms can be easily collected.
図１は本発明の一実施の形態に係る生体成分の採取方法において用いられる採取部材を示す図である。FIG. 1 is a diagram showing a collecting member used in a method for collecting a biological component according to one embodiment of the present invention. 図２は図１の採取部材を用いた生体成分の分析方法を示すフローチャートである。FIG. 2 is a flowchart showing a method of analyzing a biological component using the sampling member of FIG.
　（１）生体成分の採取方法および生体成分の分析方法
　以下、図面を参照しながら本発明の実施の形態に係る生体成分の採取方法および生体成分の分析方法について説明する。図１は、本発明の一実施の形態に係る生体成分の採取方法において用いられる採取部材を示す図である。図１に示すように、採取部材１０は、滅菌されており、棒状を有しかつ可撓性を有する。採取部材１０は、樹脂により形成されることが好ましい。 (1) Biological Component Collection Method and Biological Component Analysis Method Hereinafter, a biological component collection method and a biological component analysis method according to an embodiment of the present invention will be described with reference to the drawings. FIG. 1 is a diagram showing a collecting member used in a method for collecting a biological component according to one embodiment of the present invention. As shown in FIG. 1, the sampling member 10 is sterilized, has a rod shape, and has flexibility. The sampling member 10 is preferably formed of a resin.
　採取部材１０は、例えばガンマ線により滅菌された樹脂製の使い捨て白金耳（ディスポザブルループ）であってもよいし、ピペットチップであってもよい。本実施の形態においては、採取部材１０は、ポリプロピレンまたはポリテトラフルオロエチレン等の樹脂により形成されたディスポザブルループであり、棒状部１および環状部２を含む。環状部２は、棒状部１の一端に形成される。採取部材１０の全長は例えば２２０ｍｍであり、環状部２の外径は例えば約２ｍｍである。環状部２は、円環状であってもよいし、楕円環状であってもよいし、長楕円環状であってもよい。棒状部１の他端部は、把持部３として用いられる。 The sampling member 10 may be a disposable loop made of resin, which is sterilized by gamma rays, for example, or a pipette tip. In the present embodiment, sampling member 10 is a disposable loop formed of a resin such as polypropylene or polytetrafluoroethylene, and includes rod-shaped portion 1 and annular portion 2. The annular part 2 is formed at one end of the rod part 1. The total length of the sampling member 10 is, for example, 220 mm, and the outer diameter of the annular portion 2 is, for example, about 2 mm. The annular portion 2 may have an annular shape, an elliptical shape, or an oblong shape. The other end of the rod portion 1 is used as a grip portion 3.
　図２は、図１の採取部材１０を用いた生体成分の分析方法を示すフローチャートである。図２のステップＳ１～Ｓ７は、生体成分の採取方法に対応する。まず、成分の採取を行う作業者は、採取部材１０の把持部３を把持して環状部２を生体の表面に試料に擦りつける（ステップＳ１）。これにより、生体の表面の成分（以下、試料成分と呼ぶ。）が環状部２に付着する。 FIG. 2 is a flowchart showing a method of analyzing a biological component using the sampling member 10 of FIG. Steps S1 to S7 in FIG. 2 correspond to a method of collecting a biological component. First, an operator who collects a component grips the grip portion 3 of the collection member 10 and rubs the annular portion 2 against the surface of the living body (step S1). As a result, components on the surface of the living body (hereinafter, referred to as sample components) adhere to the annular portion 2.
　次に、作業者は、ステップＳ１で試料成分が付着した環状部２を含む採取部材１０の先端部分（図１の点線で示される部分）をハサミ等により切断する（ステップＳ２）。これにより、試料成分が付着した採取部材１０の先端部分が採取部材１０の他の部分から切り離される。なお、切り離された採取部材１０の先端部分の長さは、例えば２５ｍｍ～３５ｍｍ程度である。これにより、採取部材１０の当該先端部分を容器に収納可能となる。本実施の形態においては、容器は例えばマイクロチューブである。 Next, the operator cuts the tip portion (the portion shown by the dotted line in FIG. 1) of the sampling member 10 including the annular portion 2 to which the sample component has adhered in Step S1 with scissors or the like (Step S2). As a result, the tip portion of the sampling member 10 to which the sample component has adhered is cut off from other portions of the sampling member 10. The length of the tip of the separated sampling member 10 is, for example, about 25 mm to 35 mm. Thereby, the tip portion of the sampling member 10 can be stored in the container. In the present embodiment, the container is, for example, a microtube.
　続いて、作業者は、ステップＳ２で切り離された採取部材１０の先端部分をキャップ付きのマイクロチューブに収納する（ステップＳ３）。また、作業者は、所定の溶媒をマイクロチューブに適量注入する（ステップＳ４）。ここで、極性が異なる複数の溶媒が予め準備されている場合には、最も極性が大きい溶媒がマイクロチューブに注入される。極性が異なる複数の溶媒とは、例えば、水、エタノール、メタノール、アセトニトリルおよびヘキサンのように種類が異なる溶媒であってもよいし、同一の種類でかつ濃度が異なる溶媒であってもよい。 Next, the operator stores the tip portion of the sampling member 10 cut off in step S2 in a microtube with a cap (step S3). Further, the operator injects a predetermined amount of a predetermined solvent into the microtube (step S4). Here, when a plurality of solvents having different polarities are prepared in advance, the solvent having the highest polarity is injected into the microtube. The plurality of solvents having different polarities may be, for example, solvents of different types such as water, ethanol, methanol, acetonitrile, and hexane, or may be solvents of the same type but different concentrations.
　その後、作業者は、ステップＳ４でマイクロチューブに注入された溶媒を撹拌する（ステップＳ５）。撹拌は、例えばピンセットを用いて行われてもよいし、ボルテックスミキサ等の撹拌装置により行われてもよい。これにより、試料成分が遠心分離され、溶媒中に溶出される。次に、作業者は、ステップＳ５で溶媒に溶出した試料成分をマイクロチューブから抽出する（ステップＳ６）。 Thereafter, the operator stirs the solvent injected into the microtube in step S4 (step S5). The stirring may be performed using, for example, tweezers, or may be performed using a stirring device such as a vortex mixer. This causes the sample components to be centrifuged and eluted in the solvent. Next, the worker extracts the sample components eluted in the solvent in step S5 from the microtube (step S6).
　続いて、作業者は、予め準備された所望の全部の溶媒が使用されたか否かを判定する（ステップＳ７）。全部の溶媒が使用されていない場合、作業者はステップＳ４に戻る。全部の溶媒が使用されるまで、ステップＳ４～Ｓ７が繰り返される。この場合、ステップＳ４が繰り返されるごとに、より大きい極性を有する溶媒がマイクロチューブに注入される。これにより、脂質等の溶媒への溶解性が異なる成分を分離しつつ順次抽出し、分析することが可能になる。 Next, the operator determines whether all the desired solvents prepared in advance have been used (step S7). If all the solvents have not been used, the operator returns to step S4. Steps S4 to S7 are repeated until all the solvent is used. In this case, each time step S4 is repeated, a solvent having a larger polarity is injected into the microtube. This makes it possible to sequentially extract and analyze components such as lipids having different solubility in a solvent while separating the components.
　ステップＳ７で全部の溶媒が使用された場合、作業者はステップＳ６で抽出された試料成分を分析装置により分析する（ステップＳ８）。具体的には、作業者は、抽出された試料成分の適量をマイクロピペット等により試料支持プレートに滴下し、その試料支持プレートを質量分析装置にセットする。その後、ＭＡＬＤＩ－ＭＳ（マトリックス支援レーザ脱離イオン化質量分析法）またはＬＤＩ－ＭＳ（レーザ脱離イオン化質量分析法）等により試料成分の質量が分析される。 (4) When all the solvents have been used in step S7, the operator analyzes the sample components extracted in step S6 using the analyzer (step S8). Specifically, an operator drops an appropriate amount of the extracted sample component on a sample support plate using a micropipette or the like, and sets the sample support plate on a mass spectrometer. Thereafter, the mass of the sample component is analyzed by MALDI-MS (matrix-assisted laser desorption / ionization mass spectrometry) or LDI-MS (laser desorption / ionization mass spectrometry).
　次に、作業者は、ステップＳ６で抽出された全部の試料成分が分析されたか否かを判定する（ステップＳ９）。全部の試料成分が分析されていない場合、作業者はステップＳ８に戻る。全部の試料成分が分析されるまで、ステップＳ８，Ｓ９が繰り返される。全部の試料成分が分析された場合、作業者は、生体成分の分析方法を終了する。 Next, the worker determines whether all the sample components extracted in step S6 have been analyzed (step S9). If not all sample components have been analyzed, the operator returns to step S8. Steps S8 and S9 are repeated until all sample components have been analyzed. When all the sample components have been analyzed, the operator ends the biological component analysis method.
　（２）効果
　本実施の形態に係る生体成分の採取方法においては、可撓性を有しかつ棒状の採取部材１０の先端の環状部２が生体の表面に擦りつけられる。ここで、採取部材１０は棒状を有するため、生体が体毛に覆われている場合でも、体毛を回避しつつ環状部２を生体の表面に容易に擦りつけることが可能である。また、採取部材１０は可撓性を有するため、マウスまたはラット等の小動物の生体をほとんど損傷させることなく環状部２を当該生体の表面に容易に擦りつけることが可能である。 (2) Effect In the method for collecting a biological component according to the present embodiment, the annular portion 2 at the tip of the flexible and rod-shaped collection member 10 is rubbed against the surface of the living body. Here, since the collection member 10 has a rod shape, even when the living body is covered with body hair, the annular portion 2 can be easily rubbed against the surface of the living body while avoiding the body hair. Moreover, since the sampling member 10 has flexibility, the annular portion 2 can be easily rubbed against the surface of a small animal such as a mouse or a rat without substantially damaging the living body.
　この手順によれば、分析を行うために十分な量の生体の表面の成分が試料成分として環状部２に付着する。そのため、環状部２に付着した試料成分を溶媒を用いて環状部２から容易に抽出することが可能である。その結果、生体に由来する成分を容易に採取することができる。また、採取された試料成分が分析装置により分析される。これにより、生体に由来する成分を容易に分析することができる。 According to this procedure, a sufficient amount of components on the surface of the living body for performing analysis adhere to the annular portion 2 as sample components. Therefore, the sample component attached to the annular portion 2 can be easily extracted from the annular portion 2 using a solvent. As a result, a component derived from a living body can be easily collected. The collected sample components are analyzed by the analyzer. Thereby, the component derived from the living body can be easily analyzed.
　（３）他の実施の形態
　上記実施の形態においては、生体成分の採取方法および生体成分の分析方法が一の作業者により実行されるが、本発明はこれに限定されない。図２のステップＳ１～Ｓ３とステップＳ４～Ｓ９とが異なる作業者により実行されてもよい。例えば、一の作業者がステップＳ１～Ｓ３を実行することにより取得されたマイクロチューブが、保管または輸送された後、他の作業者に手渡されてもよい。この場合、他の作業者は、手渡されたマイクロチューブを用いてステップＳ４～Ｓ９を実行することができる。 (3) Other Embodiments In the above embodiment, a method of collecting a biological component and a method of analyzing a biological component are executed by one operator, but the present invention is not limited to this. Steps S1 to S3 and steps S4 to S9 in FIG. 2 may be executed by different operators. For example, the microtube obtained by one worker performing steps S1 to S3 may be stored or transported and then handed over to another worker. In this case, another worker can execute steps S4 to S9 using the handed microtube.
　また、生体成分の分析方法の一部の手順が異なる順序で実行されてもよい。例えば、ステップＳ８がステップＳ６とステップＳ７との間に実行されてもよい。この場合、ステップＳ４～Ｓ６において一の溶媒を用いて試料成分が抽出されるごとに、その試料成分がステップＳ８において分析される。さらに、ステップＳ８において、質量分析装置により試料成分が分析されるが、本発明はこれに限定されない。ＧＣ（ガスクロマトグラフィー）またはＬＣ（液体クロマトグラフィー）等の他の分析装置により試料成分が分析されてもよい。 Some procedures of the biological component analysis method may be performed in a different order. For example, step S8 may be performed between step S6 and step S7. In this case, each time a sample component is extracted using one solvent in steps S4 to S6, the sample component is analyzed in step S8. Further, in step S8, the sample components are analyzed by the mass spectrometer, but the present invention is not limited to this. The sample components may be analyzed by other analyzers such as GC (gas chromatography) or LC (liquid chromatography).

Claims (7)

  1. 可撓性を有しかつ棒状の採取部材の先端部分を生体の表面に擦りつけることにより生体の表面の成分を試料成分として前記採取部材に付着させるステップと、
    　前記採取部材に付着した試料成分を溶媒を用いて前記採取部材から抽出するステップとを含む、生体成分の採取方法。     A flexible and rod-shaped collecting member is attached to the collecting member as a sample component by rubbing the tip of the collecting member against the surface of the living body as a sample component,
    Extracting a sample component attached to the collection member from the collection member using a solvent.
  2. 前記採取部材は、棒状部と、前記棒状部の一端に形成された環状部とを有し、樹脂により形成された、請求項１記載の生体成分の採取方法。 The method for collecting a biological component according to claim 1, wherein the collection member has a rod-shaped portion and an annular portion formed at one end of the rod-shaped portion, and is formed of a resin.
  3. 前記採取部材は、ディスポザブルループを含む、請求項２記載の生体成分の採取方法。 The method for collecting a biological component according to claim 2, wherein the collecting member includes a disposable loop.
  4. 前記採取部材に付着した試料成分を前記採取部材から抽出するステップは、
    　前記試料成分が付着した前記採取部材の部分を前記採取部材の他の部分から切り離すことと、
    　前記採取部材の切り離された部分を容器に収納することと、
    　前記溶媒を前記容器に注入することと、
    　前記容器内で前記採取部材の前記切り離された部分に付着した試料成分を前記溶媒に溶解させることとを含む、請求項１～３のいずれか一項に記載の生体成分の採取方法。     Extracting the sample component attached to the collection member from the collection member,
    Separating the portion of the sampling member to which the sample component is attached from the other portion of the sampling member;
    Storing the separated portion of the sampling member in a container,
    Injecting the solvent into the container;
    The method for collecting a biological component according to any one of claims 1 to 3, further comprising: dissolving a sample component attached to the separated portion of the collection member in the container in the solvent.
  5. 前記試料成分を前記溶媒に溶解させることは、前記溶媒を撹拌することにより前記試料成分を遠心分離させることを含む、請求項４記載の生体成分の採取方法。 The method for collecting a biological component according to claim 4, wherein dissolving the sample component in the solvent includes centrifuging the sample component by stirring the solvent.
  6. 前記採取部材に付着した試料成分を前記採取部材から抽出するステップは、
    　第１の溶媒を前記溶媒として用いて前記試料成分に含まれる第１の成分を前記採取部材から抽出することと、
    　前記第１の成分が抽出された後、前記第１の溶媒よりも低い極性を有する第２の溶媒を前記溶媒として用いて前記試料成分に含まれる第２の成分を前記採取部材から抽出することとを含む、請求項１～３のいずれか一項に記載の生体成分の採取方法。     Extracting the sample component attached to the collection member from the collection member,
    Extracting a first component contained in the sample component from the collection member using a first solvent as the solvent;
    After the first component is extracted, extracting a second component contained in the sample component from the collection member using a second solvent having a lower polarity than the first solvent as the solvent. The method for collecting a biological component according to any one of claims 1 to 3, comprising:
  7. 請求項１～３のいずれか一項に記載の生体成分の採取方法により試料成分を採取するステップと、
    　採取された前記試料成分を分析するステップとを含む、生体成分の分析方法。      Collecting a sample component by the method for collecting a biological component according to any one of claims 1 to 3,
    Analyzing the collected sample components.
PCT/JP2019/015888 2018-07-25 2019-04-12 Biological component collecting method and biological component analysis method WO2020021784A1 (en)

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