JP3969361B2 - 固定化された物質の定量方法 - Google Patents
固定化された物質の定量方法 Download PDFInfo
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- JP3969361B2 JP3969361B2 JP2003271637A JP2003271637A JP3969361B2 JP 3969361 B2 JP3969361 B2 JP 3969361B2 JP 2003271637 A JP2003271637 A JP 2003271637A JP 2003271637 A JP2003271637 A JP 2003271637A JP 3969361 B2 JP3969361 B2 JP 3969361B2
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- fluorescence
- fluorescent
- substance
- measured
- molecules
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
-
- 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
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Optics & Photonics (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
a)前記被測定物質を基板に固定化する前又は固定化した後に、1分子当たり所定個数の蛍光物質で標識を行い、
b)前記基板全面又は該基板上の所定範囲にあって前記蛍光物質による蛍光が現れた画像を取得し、
c)前記画像に基づいて前記蛍光物質による蛍光の減少前の蛍光強度を測定し、
d)その後に前記蛍光物質による蛍光の減少を促進させ、蛍光減少の後に蛍光スポット数と蛍光強度とを測定し、その蛍光減少後における蛍光スポット数と蛍光強度との関係を参照して、前記蛍光減少前の蛍光強度から被測定物質の分子数又は分子密度を推算する、
ようにしたことを特徴とする。
まず、タンパク質、DNAなどの被測定物質を1分子当たり所定個数の蛍光物質で標識する(ステップS1)。通常は1分子当たり1個の蛍光標識でよい。このときの蛍光標識方法や蛍光物質の種類は、被測定物質の種類等に応じて適宜に選択すればよい。そうして蛍光標識した被測定物質を基板に固定化する(ステップS2)。基板への固定化の方法は従来知られている各種の方法を採用することができる。このようにして測定対象であるサンプルを作成する。
まず、被測定物質であるDNA(20mer)の5'末端をCy-5で標識し、3'末端をビオチン(Biotin)で標識する。基板12bとしては背景光の小さい石英スライドガラスを使用し、洗浄した石英スライドガラスの表面にBSAを添加したビオチン(Biotin-BSA)を非特異吸着させ、これにストレプトアビジン(Streptavidin)を反応させる。そして、これを介して上記のように蛍光標識したDNA(濃度は100pM)を基板12bに固定化する。その後、この被測定物質12aをカバーガラス12cと封止材12dとしてのマニュキュアによって封入し、気密性を確保する。また、蛍光測定には、プリズム式全反射蛍光顕微鏡を使用する。励起光の光源としては波長が635nmであるレーザを使用し、サンプル12への入射角度は69°とする。また、検出部15はイメージインテンシファイアと冷却したCCDセンサとの組み合わせであり、CCDセンサの画素数は200×200である。
11…レンズ
12…サンプル
12a…試料(被測定物質)
12b…基板
12c…カバーガラス
12d…封止材
13…分光器
14…対物レンズ
15…検出部
16…画像処理部
17…モニタ
18…データ処理部
Claims (2)
- 基板表面に固定された被測定物質の分子数又は分子密度を定量的に求める方法であって、
a)前記被測定物質を基板に固定化する前又は固定化した後に、1分子当たり所定個数の蛍光物質で標識を行い、
b)前記基板全面又は該基板上の所定範囲にあって前記蛍光物質による蛍光が現れた画像を取得し、
c)前記画像に基づいて前記蛍光物質による蛍光の減少前の蛍光強度を測定し、
d)その後に前記蛍光物質による蛍光の減少を促進させ、蛍光減少の後に蛍光スポット数と蛍光強度とを測定し、その蛍光減少後における蛍光スポット数と蛍光強度との関係を参照して、前記蛍光減少前の蛍光強度から被測定物質の分子数又は分子密度を推算する、
ようにしたことを特徴とする固定化された物質の定量方法。 - 固定化された被測定物質に対してレーザ光を照射することで蛍光の褪色を促進させることにより、又は、化学的なクエンチング手法によって蛍光の消失を促進させることにより、前記蛍光物質による蛍光を減少させることを特徴とする請求項1に記載の固定化された物質の定量方法。
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2003271637A JP3969361B2 (ja) | 2003-07-07 | 2003-07-07 | 固定化された物質の定量方法 |
US10/875,175 US20050009199A1 (en) | 2003-07-07 | 2004-06-25 | Method of measuring number of molecules or molecular density of a sample fixed on a substrate surface |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2003271637A JP3969361B2 (ja) | 2003-07-07 | 2003-07-07 | 固定化された物質の定量方法 |
Publications (3)
Publication Number | Publication Date |
---|---|
JP2005030950A JP2005030950A (ja) | 2005-02-03 |
JP2005030950A5 JP2005030950A5 (ja) | 2005-11-24 |
JP3969361B2 true JP3969361B2 (ja) | 2007-09-05 |
Family
ID=33562664
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2003271637A Expired - Fee Related JP3969361B2 (ja) | 2003-07-07 | 2003-07-07 | 固定化された物質の定量方法 |
Country Status (2)
Country | Link |
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US (1) | US20050009199A1 (ja) |
JP (1) | JP3969361B2 (ja) |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US10294519B2 (en) | 2011-07-19 | 2019-05-21 | Hitachi High-Technologies Corporation | Method and apparatus for nucleic acid analysis |
CN104165873A (zh) * | 2014-07-22 | 2014-11-26 | 中国科学院植物研究所 | 一种检测活体植物细胞中两种膜蛋白共定位程度的方法 |
US10465937B2 (en) * | 2017-08-08 | 2019-11-05 | Lennox Industries Inc. | Hybrid tandem compressor system and method of use |
WO2019131947A1 (ja) | 2017-12-27 | 2019-07-04 | 国立研究開発法人理化学研究所 | 分光分析装置、分光分析方法、プログラム、記録媒体及び顕微鏡 |
CN113671113B (zh) * | 2020-05-14 | 2024-02-23 | 山东中谷淀粉糖有限公司 | 一种玉米淀粉斑点的检查方法 |
Family Cites Families (7)
Publication number | Priority date | Publication date | Assignee | Title |
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US6207369B1 (en) * | 1995-03-10 | 2001-03-27 | Meso Scale Technologies, Llc | Multi-array, multi-specific electrochemiluminescence testing |
AU755913B2 (en) * | 1997-06-18 | 2003-01-02 | Masad Damha | Nucleic acid biosensor diagnostics |
US6716588B2 (en) * | 1999-12-09 | 2004-04-06 | Cellomics, Inc. | System for cell-based screening |
US6982147B2 (en) * | 2000-01-24 | 2006-01-03 | Ingeneus Corporation | Apparatus for assaying biopolymer binding by means of multiple measurements under varied conditions |
IL135884A (en) * | 2000-04-30 | 2005-08-31 | Yissum Res Dev Co | Method for measuring non-transferrin bound iron |
US6861224B2 (en) * | 2001-11-02 | 2005-03-01 | Fujitsu Limited | Protein detecting device |
US20040060987A1 (en) * | 2002-05-07 | 2004-04-01 | Green Larry R. | Digital image analysis method for enhanced and optimized signals in fluorophore detection |
-
2003
- 2003-07-07 JP JP2003271637A patent/JP3969361B2/ja not_active Expired - Fee Related
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2004
- 2004-06-25 US US10/875,175 patent/US20050009199A1/en not_active Abandoned
Also Published As
Publication number | Publication date |
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US20050009199A1 (en) | 2005-01-13 |
JP2005030950A (ja) | 2005-02-03 |
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