JPWO2015097858A1 - 生体分子解析装置 - Google Patents
生体分子解析装置 Download PDFInfo
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Abstract
Description
本実施形態に係る生体分子解析装置は、レーザー蛍光顕微鏡を用いて接着系培養細胞からなるサンプルの光学イメージを得る手段と、レーザー光源を用いて細胞を破壊する手段とを備え、細胞中のmRNAをアレイデバイスに捕捉することで遺伝子発現解析を行うことができる装置である。図1に本実施形態に係る生体分子解析装置の構成図を示す。この生体分子解析装置は、光学イメージを取得し、光学イメージ上の所定の位置の細胞の一部又は全部を破壊する機能を有する顕微鏡システム1と、細胞から放出・拡散される生体分子(mRNA)を捕捉するための領域が配列したアレイデバイス、これを駆動する機構、及びサンプル細胞を移動させる機構を有する生体分子採取システム2と、これら2つのシステムの動きを制御する制御システム3とから構築される。
本実施形態では、PCR増幅の代わりにT7プロモーターを用いた場合の例を説明する。第1実施形態との相違点は、シーケンシングサンプルの調製方法にある。図5及び図6に対応するサンプル調製の手順を図13、図14及び図15に示す。細孔アレイシート51の内部に固定されたDNAプローブ80は、5’末端方向からT7プロモーター配列、emPCR増幅用共通配列(フォワード方向、配列番号24)、細胞識別用タグ配列、分子識別用タグ配列、及びオリゴ(dT)配列で構成される。T7プロモーター配列をDNAプローブ80へ導入することで、後続のIVT(In Vitro Transcription)によるcRNA63増幅工程(図6(e))におけるターゲット配列の増幅が可能となる。すなわち、T7プロモーター配列はT7RNAポリメラーゼにより認識され、その下流配列から転写(cRNA63増幅)反応が開始される。同様にPCR増幅用共通配列を導入することで、後続のemPCR増幅工程において共通プライマーとして利用することができる。また、細胞識別用タグ配列を例えば5塩基としてDNAプローブ80に導入することによって、45=1024個の単一細胞を識別することが可能となることは第1実施形態と同様である。さらに、分子識別用タグ配列(例えば15塩基)をDNAプローブ80へ導入することにより、415=1.1×109分子を認識することができるため、次世代シーケンサーで得られる膨大な解読データが、どの分子由来であるかを識別することが可能となることも第1実施形態と同様である。すわなち、IVT/emPCR等の増幅工程で生じた遺伝子間の増幅バイアスを修正することができるため、始めに細胞中に存在していたmRNA量を高い精度で定量することが可能となる。最も3’側に位置するオリゴ(dT)配列は、mRNAの3’側に付加されているポリAテールとハイブリダイズし、mRNAを捕捉するために利用される(図13(a))。
第1及び第2実施形態においては、細胞を破壊する手段としてレーザー光を用いたが、様々な材料からなるニードルを用いても良い。図16に本実施形態の装置構成図を示す。ニードル1001は半導体プロセスを用いて作製し、針の長さは例えば10μmであり、先端部分の直径は1μmとする。針部分の材料は、シリコン酸化膜を用いることができる。原子間力顕微鏡のカンチレバーと同様の作製方法を用いて作製することができる。ニードルの保持部材1002としてはシリコン基板を用いる。このシリコン基板はZステージ1003により駆動し、ニードル1001の先端部分を細胞膜に突き刺すことによって、細胞膜に穿孔し、漏れ出たmRNAを細孔アレイシート30まで電気泳動にて誘導する。ニードル1001の面内での位置は白金電極32の中心に配置して固定する。ニードル1001を中空のニードルに変えて、細胞中の生体分子が中空ニードル内を通って放出されるようにしても良い。
本実施形態では、生体分子としてタンパク質由来のペプチドを解析するための構成について説明する。細孔アレイシートの内部に、シランカップリング剤を用いて、DNAプローブではなく計測対象とするタンパク質やペプチドを抗原とする抗体を固定する。固定条件は同じで良い。タンパク質やペプチドは、マイナスの電荷を持つとは限らないので、電気泳動を用いて細孔アレイシートの内部までこれらを誘導することはできない。そこで、細孔アレイシートを対称面として、計測対象とする細胞の反対側に、溶液を吸引するためのノズルを配置する。ノズルにより吸引した溶液はシャーレの内部に戻して循環させることができる。ノズル内径は例えば0.1mmであり吸引速度は500μl/秒とすることができる。これによって、細孔アレイシートの内部及び細孔アレイシートと細胞の間の領域に溶液の流れが生じ、レーザーで細胞膜を破壊したときに放出されるタンパク質やペプチドを含む生体分子を細孔アレイシートに誘導することができる。なお、破壊する細胞は1細胞でも数細胞程度でも良い。目標とするサンプリング分解能に応じて選択する。
本実施形態では、アレイデバイスとして、多孔質メンブレンからなる細孔アレイシートの代わりに、表面にビーズがパッキングされたビーズアレイを用いる場合について説明する。生体分子解析装置の構成は図1の例と同じであり、細孔アレイシート30に代えて、図17に示すようなビーズアレイを用いた点のみが異なる。ビーズアレイ1701には、ビーズが保持された領域1702が配列している。領域1702が、図1における領域301に対応する。
2 生体分子採取システム
3 制御システム
4 蛍光顕微鏡用レーザー光源
5 細胞破壊用レーザー光源
6 ダイクロイックミラー
7 ダイクロイックミラー
9 ステージ
10 試料ステージ
11 バンドパスフィルタ
12 結像レンズ
13 受光器
14 ピンホール
20 シャーレ
21 接着系培養細胞
22 接着系培養細胞
23 接着系培養細胞
30 細孔アレイシート
31 分離壁
32 白金電極
33 シールド線
34 XYZステージ
35 電源
36 mRNA
39 参照電極
41 電源
42 石英基板
43 金電極
44 貫通孔
51 細孔アレイシート
54 mRNA
55 貫通孔
56 DNAプローブ
59 ファーストストランドcDNA鎖
60 ターゲット遺伝子特異的配列プライマー
61 セカンドストランドcDNA鎖
62 2本鎖cDNA
63 cRNA
71 ターゲット遺伝子特異的配列プライマー
72 cDNA
73 2本鎖DNA
80 DNAプローブ
301 領域
302 チップ
801 撮像素子
802 フォーカス調整レンズ
803 ダイクロイックミラー
1001 ニードル
1002 保持部材
1003 Zステージ
1401 光源
1402 偏光子
1403 ウォラストンフィルタ
1404 ウォラストンプリズム
1405 コンデンサレンズ
1406 対物レンズ
1501 光源
1502 ビームスプリッタ
1503 非線形ファイバ
1504 水浸対物レンズ
1505 ハイパスフィルタ
1506 分光器
1507 分光器用CCDカメラ
1508 結像レンズ
1701 ビーズアレイ
1702 領域
1703 ビーズ
Claims (13)
- 複数の細胞の光学イメージを得る手段と、
前記複数の細胞のうちの1つ以上の細胞の一部又は全部を破壊する手段と、
前記破壊する手段によって放出される細胞中の生体分子を捕捉するための領域が配列したアレイデバイスと、
前記光学イメージにおける、前記破壊する手段によって破壊された細胞に相当する部分に対し、前記アレイデバイスにおける生体分子を捕捉した領域を対応付ける手段と、
を備える生体分子解析装置。 - 破壊する手段によって細胞を破壊する前に、アレイデバイスの領域と、破壊する細胞とを接近させる手段をさらに備える請求項1に記載の生体分子解析装置。
- 放出される細胞中の生体分子を、アレイデバイスの領域に吸引し又は泳動させる手段をさらに備える請求項1又は2に記載の生体分子解析装置。
- アレイデバイスが、多孔質メンブレン、又は表面にビーズがパッキングされたビーズアレイである請求項1〜3のいずれかに記載の生体分子解析装置。
- アレイデバイスの表面又は内部に、細胞中の生体分子を選択的に捕捉するためのプローブ分子が固定されている請求項1〜4のいずれかに記載の生体分子解析装置。
- 細胞中の生体分子がmRNAであり、プローブ分子がDNAプローブである請求項5に記載の生体分子解析装置。
- DNAプローブが、アレイデバイスの位置ごとに異なる配列を有する請求項6に記載の生体分子解析装置。
- 細胞中の生体分子がタンパク質又はペプチドであり、プローブ分子が抗体である請求項5に記載の生体分子解析装置。
- 破壊する手段が、レーザーである請求項1〜8のいずれかに記載の生体分子解析装置。
- 破壊する手段が、ニードルである請求項1〜8のいずれかに記載の生体分子解析装置。
- 破壊する手段が、中空ニードルであり、細胞中の生体分子は前記中空ニードル内を通って放出される請求項1〜8のいずれかに記載の生体分子解析装置。
- 破壊する手段が、電子線又は荷電粒子線である請求項1〜8のいずれかに記載の生体分子解析装置。
- 複数の細胞が、ゲル中に3次元的に配置されている請求項1〜12のいずれかに記載の生体分子解析装置。
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