JPH0723796A - Intracellular sampling and analyzing method therefor - Google Patents
Intracellular sampling and analyzing method thereforInfo
- Publication number
- JPH0723796A JPH0723796A JP16885593A JP16885593A JPH0723796A JP H0723796 A JPH0723796 A JP H0723796A JP 16885593 A JP16885593 A JP 16885593A JP 16885593 A JP16885593 A JP 16885593A JP H0723796 A JPH0723796 A JP H0723796A
- Authority
- JP
- Japan
- Prior art keywords
- sampling
- needle
- sample
- tip
- adsorbed
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は、生体組織及び細胞内か
らサンプリングした核酸及びタンパク質等を直接観察試
料とする高感度分析方法に関するものである。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a highly sensitive analytical method in which nucleic acids, proteins and the like sampled from living tissues and cells are used as direct observation samples.
【０００２】[0002]
【従来の技術】従来の技術では、ガラスキャピラリー等
を用いて生体組織及び細胞内から核酸及びタンパク質等
をサンプリングしていた。従って、細胞壁にキャピラリ
ーの断面積分の穴をあけることになり破壊的サンプリン
グしか可能ではなかったし、高感度な分析法とサンプリ
ング法がうまく組み合っていないために、複数の生体組
織及び細胞内からサンプリングし、サンプル量を増やす
必要があり局部的分析が不可能であった。2. Description of the Related Art In the prior art, nucleic acids, proteins and the like were sampled from living tissues and cells using glass capillaries and the like. Therefore, a hole for the cross-sectional integration of the capillary was made in the cell wall, and only destructive sampling was possible.Since the sensitive analysis method and sampling method were not well combined, sampling was performed from multiple living tissues and cells. However, it was necessary to increase the sample size and local analysis was impossible.
【０００３】また、その採取した核酸及びタンパク質等
の一部しか有効に分析されていなかった。つまり、測定
装置の検出限界は、分析に必要な最低サンプル量とは等
しくなく、かなり余分なサンプル量が必要であった。Further, only a part of the collected nucleic acids, proteins and the like have been effectively analyzed. That is, the detection limit of the measuring device was not equal to the minimum sample amount required for the analysis, and a considerably extra sample amount was required.
【０００４】[0004]
【発明が解決しようとする課題】サンプリング棒をガラ
スキャピラリー等より断面積を小さくすることにより細
胞壁の破壊を極力小さくする。また、測定装置の検出限
界と、分析に必要な最低サンプル量とをほぼ等しい分析
法と組み合わせて採取した核酸及びタンパク質等を分析
する。従って、生体組織及び細胞内の局部的分析が可能
になる。The cross-sectional area of the sampling rod is made smaller than that of a glass capillary or the like to minimize the destruction of the cell wall. In addition, the nucleic acid, protein, etc. collected are analyzed by combining the detection limit of the measuring device and the minimum sample amount required for the analysis with substantially the same analysis method. Therefore, local analysis in living tissues and cells becomes possible.
【０００５】[0005]
【課題を解決するための手段】本発明は、試料採取針の
先端部に生体組織（細胞）内において核酸及びタンパク
質等を電気的または化学的に吸着（反応）させ、その先
端部に吸着された試料を直接観察試料とする。そのた
め、測定装置の検出限界が、分析に必要な最低サンプル
量とほぼ等しくなり、高感度な分析法が可能となる。ま
た、分析したい生体組織（細胞）部分に試料採取針を刺
しこむので局部的分析を可能とする。According to the present invention, nucleic acid and protein are electrically or chemically adsorbed (reacted) in a biological tissue (cell) to the tip of a sampling needle, and the tip is adsorbed. The sample is used as the direct observation sample. Therefore, the detection limit of the measuring device becomes almost equal to the minimum sample amount required for analysis, and a highly sensitive analysis method becomes possible. In addition, since a sampling needle is inserted into a living tissue (cell) portion to be analyzed, local analysis is possible.
【０００６】[0006]
【作用】本発明は、試料採取針を生体組織（細胞）内に
刺しこむため、生きた生体組織からサンプリングが可能
で、サンプリングによる破壊も最小限に抑えることがで
きる。従って、サンプリング後の生体組織（細胞）の活
性にほとんど影響を与えない。あるパラメーターに対す
る１つの生体組織（細胞）の経時変化を計測することも
可能になる。In the present invention, since the sample collecting needle is inserted into the living tissue (cell), it is possible to sample from living living tissue, and the destruction due to sampling can be minimized. Therefore, the activity of the living tissue (cell) after sampling is hardly affected. It is also possible to measure the time-dependent change of one living tissue (cell) with respect to a certain parameter.
【０００７】[0007]
実施例１ 図１に、先端部分を親水性にし、核酸及びタンパク質を
先端部分にのみ吸着する試料採取針１を示す。この例で
は、試料採取針１自体は表面は疎水性とされ、界面活性
剤２により表面が親水性とされる。親水性とされた表面
がサンプリング部３として使用される。Example 1 FIG. 1 shows a sampling needle 1 in which the tip portion is made hydrophilic and nucleic acids and proteins are adsorbed only to the tip portion. In this example, the surface of the sampling needle 1 itself is hydrophobic, and the surface of the sampling needle 1 is hydrophilic by the surfactant 2. The hydrophilic surface is used as the sampling unit 3.
【０００８】試料採取針１は、針スキャナー７に内臓さ
れている圧電素子により粗動及び微動可能とされる。分
析目的の試料９はサンプルホルダーである光学顕微鏡の
試料台１０に固定されている。針スキャナー７により、
試料採取針１は試料細胞片９の目的の個所に移動された
後、試料を刺し、数分間そのままに保持された後抜き取
られる。The sampling needle 1 can be coarsely and finely moved by a piezoelectric element incorporated in the needle scanner 7. A sample 9 for analysis is fixed on a sample stage 10 of an optical microscope which is a sample holder. With the needle scanner 7,
The sample collection needle 1 is moved to a desired position on the sample cell piece 9, punctured with the sample, held for a few minutes, and then withdrawn.
【０００９】本実施例においては、生体組織（細胞）内
の伝令リボ核酸(m-RNA)を試料採取針１のサンプリング
部３に吸着させ、その存在の有無を原子間力顕微鏡AFM
（Atomic Force Microprobe)で観察する。試料採取針１
は、例えば、タングステン針（直径100μｍ）とされ、
この先端部を親水性にするために両親媒性の界面活性剤
２（例えばステアリン酸カドミニウム）を吸着させて、
核酸が吸着しやすいようにする。試料採取針１の先端部
は、nmオーダーで言えばそれほど尖っているものではな
いので、この試料採取針の形状のためにAFM観察が不可
能になることはない。In this embodiment, messenger ribonucleic acid (m-RNA) in living tissue (cells) is adsorbed to the sampling unit 3 of the sampling needle 1, and the presence or absence thereof is checked by the atomic force microscope AFM.
Observe with (Atomic Force Microprobe). Sampling needle 1
Is, for example, a tungsten needle (diameter 100 μm),
To make this tip hydrophilic, adsorb amphiphilic surfactant 2 (eg cadmium stearate),
Make it easier for nucleic acids to be adsorbed. Since the tip of the sampling needle 1 is not so sharp in nm order, the shape of the sampling needle does not make AFM observation impossible.
【００１０】実施例２ 実施例１と同じ試料採取針１のサンプリング部３に分析
目的のm-RNAの相補的な塩基配列（例えば、化学合成す
る）を吸着させ、リファレンスとしてAFMにて観察す
る。Example 2 A complementary nucleotide sequence (for example, chemically synthesized) of a target mRNA for analysis is adsorbed to the sampling section 3 of the same sampling needle 1 as in Example 1 and observed by AFM as a reference. .
【００１１】次に、分析目的のm-RNAの相補的な塩基配
列が吸着された試料採取針１を実施例１と同様に試料に
差し込み、サンプリング部３に吸着された分析目的のm-
RNAを観察する。この場合、m-RNAの存在を判り易くする
ために、m-RNAに特有な結合能力のある分子を反応さ
せ、その分子をAFMにて観察する。例えば、m-RNAには必
ず存在するメチオニン(塩基配列:AUG)をビオチン化した
分子の含まれ水溶液にサンプリング部３を浸け、次に、
アビジンを含んだ水溶液に、再度、サンプリング部３を
浸けて、AFMで一辺数十nmあるサイコロ状のアビジンの
存在の有無を確認することで局部的な生体組織（細胞）
内のm-RNAの存在を測定することができる。Next, the sampling needle 1 to which the complementary nucleotide sequence of the m-RNA to be analyzed is adsorbed is inserted into the sample in the same manner as in Example 1, and the m-RNA to be adsorbed to the sampling unit 3 is adsorbed.
Observe RNA. In this case, in order to make it easy to recognize the presence of m-RNA, a molecule having a binding ability specific to m-RNA is reacted and the molecule is observed by AFM. For example, the sampling unit 3 is dipped in an aqueous solution containing a biotinylated molecule of methionine (base sequence: AUG) that is always present in m-RNA, and then
By immersing the sampling unit 3 again in an aqueous solution containing avidin and confirming the presence or absence of dice-shaped avidin with a side length of several tens of nm by AFM, local biological tissue (cells)
The presence of m-RNA within can be measured.
【００１２】勿論、目的のm-RNAの相補的な塩基配列を
吸着させた試料採取針１にm-RNAを吸着させただけでもm
-RNAの存在が確認出来る可能性はあるが、基本的にAFM
は表面の形状のみの情報しか与えないので上記のような
m-RNA確認のための過程をトルコとが、詳細な分析のた
めには有用である。Of course, even if m-RNA is adsorbed only on the sampling needle 1 on which the complementary nucleotide sequence of the target m-RNA is adsorbed,
-It is possible to confirm the presence of RNA, but basically AFM
Since only gives information on the surface shape,
Turkey has a process for confirmation of m-RNA, but is useful for detailed analysis.
【００１３】実施例３ 本実施例では、生体組織（細胞）内のタンパク質を試料
採取針１の表面に電気的に吸着し、その存在の有無を電
界蒸発型質量分析法で分析する。Example 3 In this example, proteins in living tissues (cells) are electrically adsorbed on the surface of the sampling needle 1, and the presence or absence thereof is analyzed by field evaporation mass spectrometry.
【００１４】本実施例では試料採取針１は金属針（例え
ば、タングステン（直径100μｍ））とされ、サンプリ
ング部３となる部分以外は絶縁層１２で覆われる。In this embodiment, the sampling needle 1 is a metal needle (for example, tungsten (diameter 100 μm)), and the portion other than the portion to be the sampling portion 3 is covered with the insulating layer 12.
【００１５】実施例１と同様に試料採取針１を目的の個
所に移動して試料を試料採取針１で刺し、試料採取針１
にプラスの電位をかけ、数分間そのままに保持した後抜
き取る。生体組織（細胞）内に存在しているタンパク質
は通常マイナスの電荷を持っているので、存在している
ものは、ほとんどがサンプリング部３に吸着される。Similar to the first embodiment, the sample-collecting needle 1 is moved to a desired position, and the sample is pierced with the sample-collecting needle 1, and the sample-collecting needle 1 is inserted.
Apply a positive potential to, hold it for a few minutes, and then remove. Since proteins existing in living tissues (cells) usually have a negative charge, most of the existing proteins are adsorbed by the sampling unit 3.
【００１６】この場合、複数のタンパク質がサンプリン
グされるが、そのタンパク質の同定は、電界蒸発型質量
分析により求められた質量スペクトルより既存のタンパ
ク質の質量スペクトルと比較することにより行う。ま
た、試料採取針１は先端部分の吸着物を非常に効率的に
蒸発させるので、もし先端部以外に吸着されたタンパク
質が存在した場合でもほとんど電界蒸発されない。従っ
て、先端部分以外にもタンパク質の吸着が起こった場合
でも試料採取針１による局部的分析が可能となる。In this case, a plurality of proteins are sampled, and the proteins are identified by comparing the existing mass spectra of the proteins with the mass spectra obtained by the field evaporation mass spectrometry. Further, since the sample collecting needle 1 evaporates the adsorbate at the tip portion very efficiently, even if there is adsorbed protein other than the tip portion, almost no electric field evaporation occurs. Therefore, even if protein adsorption occurs in other than the tip portion, local analysis by the sampling needle 1 is possible.
【００１７】[0017]
【発明の効果】試料採取針の先端部をサンプリング部と
することにより、生体組織（細胞）の局部的分析が可能
になり、また、原子間力顕微鏡や質量分析法などのサン
プルとして、採取したサンプルのほぼすべてを利用でき
るので、非常に高感度な分析が可能となる。EFFECTS OF THE INVENTION By using the tip of the sample collecting needle as a sampling part, it becomes possible to perform local analysis of living tissue (cells), and the sample is collected as a sample for an atomic force microscope or mass spectrometry. Almost all of the samples are available, allowing for very sensitive analysis.
【図１】先端部分を親水性にし、サンプリング部とした
試料採取針。FIG. 1 is a sampling needle that has a hydrophilic tip portion and is used as a sampling portion.
【図２】試料採取針を用いた生体組織及び細胞サンプリ
ング装置。FIG. 2 shows a biological tissue and cell sampling device using a sampling needle.
【図３】先端部分を導電性にし、サンプリング部とした
試料採取針。FIG. 3 is a sampling needle that has a conductive tip portion and is used as a sampling portion.
１…試料採取針、２…界面活性剤、３…サンプリング
部、６…光学顕微鏡、７…試料採取針スキャナー、９…
試料細胞片、１０…サンプルホルダー、１２…絶縁層。DESCRIPTION OF SYMBOLS 1 ... Sampling needle, 2 ... Surfactant, 3 ... Sampling part, 6 ... Optical microscope, 7 ... Sampling needle scanner, 9 ...
Sample cell piece, 10 ... Sample holder, 12 ... Insulating layer.
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.6 識別記号 庁内整理番号 ＦＩ 技術表示箇所 Ｇ０１Ｎ 1/10 Ｐ // Ｃ１２Ｎ 15/09 Ｃ１２Ｑ 1/68 Ｚ 9453−4Ｂ ─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. 6 Identification code Internal reference number FI Technical display location G01N 1/10 P // C12N 15/09 C12Q 1/68 Z 9453-4B
Claims (5)
は化学的に針先端に核酸及びタンパク質等を吸着させる
ことを特徴とするサンプリング法。1. A sampling method, which comprises puncturing a cell with a sampling needle and electrostatically or chemically adsorbing a nucleic acid, a protein and the like to the tip of the needle.
とする請求項１記載のサンプリング法。2. The sampling method according to claim 1, wherein only the tip of the needle is made hydrophilic.
とする請求項１記載のサンプリング法。3. The sampling method according to claim 1, wherein only the tip of the needle is made conductive.
試料採取針先端部に吸着された核酸及びタンパク質等を
同定、定量化することを特徴とする分析方法。4. Utilizing biochemical methods and mass spectrometry,
An analysis method characterized by identifying and quantifying nucleic acids, proteins, etc. adsorbed on the tip of a sampling needle.
部に吸着された核酸及びタンパク質等を同定、定量化す
ることを特徴とする分析方法。5. An analysis method characterized by identifying and quantifying nucleic acids and proteins adsorbed on the tip of a sampling needle using an atomic force microscope.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP16885593A JPH0723796A (en) | 1993-07-08 | 1993-07-08 | Intracellular sampling and analyzing method therefor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP16885593A JPH0723796A (en) | 1993-07-08 | 1993-07-08 | Intracellular sampling and analyzing method therefor |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH0723796A true JPH0723796A (en) | 1995-01-27 |
Family
ID=15875810
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP16885593A Pending JPH0723796A (en) | 1993-07-08 | 1993-07-08 | Intracellular sampling and analyzing method therefor |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0723796A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5945678A (en) * | 1996-05-21 | 1999-08-31 | Hamamatsu Photonics K.K. | Ionizing analysis apparatus |
| JP2006246731A (en) * | 2005-03-09 | 2006-09-21 | National Institute Of Advanced Industrial & Technology | Method for detecting living cell protein by immunodynamic measurement |
| EP2017610A1 (en) * | 2006-04-28 | 2009-01-21 | University of Yamanashi | Ionizing method and device by electrospray |
| JP2009041931A (en) * | 2007-08-06 | 2009-02-26 | Nippon Telegr & Teleph Corp <Ntt> | Protein function analyzer |
| JP2011523064A (en) * | 2008-06-06 | 2011-08-04 | ユニバーシティー・オブ・ワシントン | Method and system for concentrating particles from solution |
| US8940092B1 (en) | 2008-10-27 | 2015-01-27 | University Of Washington Through Its Center For Commercialization | Hybrid fibers, devices using hybrid fibers, and methods for making hybrid fibers |
-
1993
- 1993-07-08 JP JP16885593A patent/JPH0723796A/en active Pending
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5945678A (en) * | 1996-05-21 | 1999-08-31 | Hamamatsu Photonics K.K. | Ionizing analysis apparatus |
| JP2006246731A (en) * | 2005-03-09 | 2006-09-21 | National Institute Of Advanced Industrial & Technology | Method for detecting living cell protein by immunodynamic measurement |
| JP4517074B2 (en) * | 2005-03-09 | 2010-08-04 | 独立行政法人産業技術総合研究所 | Detection method of live cell protein by immunodynamic measurement |
| EP2017610A1 (en) * | 2006-04-28 | 2009-01-21 | University of Yamanashi | Ionizing method and device by electrospray |
| EP2017610A4 (en) * | 2006-04-28 | 2010-02-03 | Univ Yamanashi | Ionizing method and device by electrospray |
| US7902499B2 (en) | 2006-04-28 | 2011-03-08 | University Of Yamanashi | Ionization method and apparatus using electrospray |
| JP2009041931A (en) * | 2007-08-06 | 2009-02-26 | Nippon Telegr & Teleph Corp <Ntt> | Protein function analyzer |
| JP2011523064A (en) * | 2008-06-06 | 2011-08-04 | ユニバーシティー・オブ・ワシントン | Method and system for concentrating particles from solution |
| US8632669B2 (en) | 2008-06-06 | 2014-01-21 | University Of Washington | Method and system for concentrating particles from a solution |
| US9097664B2 (en) | 2008-06-06 | 2015-08-04 | University Of Washington | Method and system for concentrating particles from a solution |
| US9518956B2 (en) | 2008-06-06 | 2016-12-13 | University Of Washington | Particle concentration system |
| US8940092B1 (en) | 2008-10-27 | 2015-01-27 | University Of Washington Through Its Center For Commercialization | Hybrid fibers, devices using hybrid fibers, and methods for making hybrid fibers |
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