JP5871224B2 - 細胞放出物質検出装置、細胞放出物質検出方法および細胞放出物質検出用固定化酵素基板 - Google Patents
細胞放出物質検出装置、細胞放出物質検出方法および細胞放出物質検出用固定化酵素基板 Download PDFInfo
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Images
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- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analysing Biological Materials (AREA)
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Description
GAP+ATP→1,3−ビスホスホグリセリン酸+ADP
NAD+→NADH
受光する。このため、透明基板10内を全反射しつつ光路L1を伝播する励起光とは分離して、組織細胞Sからの光学情報である蛍光をCCD15に入力することができ、ダイクロイックミラー、カットフィルタ等を不要とすることができる。CCDカメラユニット13にて取得された画像信号は、測定装置1からケーブル18を介してPC20へと出力される。
プロピルカルボジイミド(EDC)などが例示される。尚、本実施形態においては、3-アミノプロピルトリメトキシシランが用いられている。
(実施例1)
横軸に時間(秒)、縦軸に蛍光強度を示している。尚、蛍光強度は、最大値を4096とする相対値である。図5、図6からも分かるように、サンプル片の位置a,b,c部分の蛍光強度が明確に上昇していることが示されている。
10 透明基板(基板、細胞放出物質検出用固定化酵素基板)
15 CCD(検出手段)
100 ATP検出システム(細胞放出物質検出装置)
S13,S14 (画像形成手段の一部)
Claims (7)
- 細胞組織が載置される載置面側に、基質をリン酸化するリン酸化反応とリン酸化された後の基質を酸化する酸化反応とを触媒するグリセルアルデヒド3−リン酸脱水素酵素を有する基板と、
その基板上での反応に伴って蛍光元物質から生成される蛍光物質を励起する励起光を、前記基板上の細胞組織に向けて照射する光照射手段と、
その光照射手段によって照射された励起光によって蛍光物質から放射される蛍光を検出する検出手段と、
その検出手段により検出された蛍光に基づいて画像を形成する画像形成手段とを備えており、
前記基板に載置された細胞組織にグリセルアルデヒド3−リン酸と蛍光元物質とを共存させた場合にグリセルアルデヒド3−リン酸脱水素酵素が触媒する反応によって生じた蛍光に基づいて前記画像形成手段により形成された画像により、前記基板に載置された細胞組織から放出されたヌクレオチドの濃度分布を検出することを特徴とする細胞放出物質検出装置。 - 前記基板は、前記酵素が載置面側表面に化学結合によって固定化されたものであることを特徴とする請求項1に記載の細胞放出物質検出装置。
- 前記光照射手段は、前記基板の側面へ励起光を入射する光源部を備えており、
その光源部から前記基板の側面に照射される励起光により、前記基板の載置面にエバネッセント光を発生させるよう構成されていることを特徴とする請求項1または2に記載の細胞放出物質検出装置。 - 前記検出手段は、前記光照射手段が照射する励起光の光路と重ならない位置に配設されていることを特徴とする請求項1から3のいずれかに記載の細胞放出物質検出装置。
- 細胞組織から放出されたヌクレオチドの濃度分布を検出する方法であって、
基質をリン酸化するリン酸化反応とリン酸化された後の基質を酸化する酸化反応とを触媒するグリセルアルデヒド3−リン酸脱水素酵素を有する基板に載置された細胞組織に向かって、グリセルアルデヒド3−リン酸と当該酵素反応に伴って蛍光物質に変換される蛍光元物質とが基板上に共存する状態で、蛍光物質の励起光を照射する光照射工程と、
その光照射工程により照射された励起光によって励起された蛍光物質から放射される蛍光を検出する蛍光検出工程と、
その蛍光検出工程により検出された蛍光に基づいて画像を形成する画像形成工程とを備え、
その画像形成工程により形成された画像により、細胞組織から放出されたヌクレオチドの濃度分布を検出することを特徴とする細胞放出物質検出方法。 - 前記酵素は基板表面に化学結合によって固定されており、細胞組織はその基板表面に載置されることを特徴とする請求項5に記載の細胞放出物質検出方法。
- 請求項1から4のいずれかに記載の細胞放出物質検出装置または請求項5若しくは6に記載の細胞放出物質検出方法に用いられる基板において、平滑面を有する透光性基板の表面上にグリセルアルデヒド3−リン酸脱水素酵素が化学的結合によって担持されていることを特徴とする細胞放出物質検出用固定化酵素基板。
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