JP2010008378A - バイオチップ用基板及びその製造方法 - Google Patents
バイオチップ用基板及びその製造方法 Download PDFInfo
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- JP2010008378A JP2010008378A JP2008171322A JP2008171322A JP2010008378A JP 2010008378 A JP2010008378 A JP 2010008378A JP 2008171322 A JP2008171322 A JP 2008171322A JP 2008171322 A JP2008171322 A JP 2008171322A JP 2010008378 A JP2010008378 A JP 2010008378A
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Abstract
【解決手段】基板の表面にアミノ基含有ポリマーを共有結合で固定化することにより、基板表面上にアミノ基を均一に、高密度に、かつ安定に結合することができ、このアミノ基を利用してタンパク質や核酸等の生体関連物質から成るプローブを固定化することにより、プローブ固定化率が高く、且つ固定化密度が均一であり、さらに、タンパク質の非特異吸着を防止することにより検出感度が高く且つ再現性が高くなる。
【選択図】図2
Description
基板表面のアミノ基の均一性は、基板表面全面をアミノ基と反応性を有するように活性化した蛍光分子(TAMRA)溶液でアミノ基に蛍光分子を結合させ、蛍光スキャナーでスキャンすることで評価した。具体的には10mM N,N,N',N'-テトラメチル-(5又は6)-カルボキシローダミン(TAMRA)、10mM HBTU(2−1H[ベンゾトリアゾルー1−イル]―1,1,3,3,テトラメチルウロニウムーヘキサフルオロホスフェート)、20mM DIEA(ジイソプロピルエチルアミン)/DMF(ジメチルホルムアミド)溶液に基板を浸漬し室温で1時間振とうさせてアミノ基にTAMRAを結合させた。続いてメタノール洗浄で過剰なTAMRAを洗い流した後、基板を減圧乾燥した。これを蛍光スキャナー(日立ソフトウェア、CRBIO IIe)でスキャン(Power 40%、Resolution 30μm、PMT 65%、励起光532 nm、発光573 nm)し、得られた画像および輝度の分布ヒストグラムで均一性を評価した。
基板表面のアミノ基量は基板表面をブロモアセチル化したのち、メルカプトエタノールで処理することで遊離した臭化物イオンをイオンクロマトグラフィー(島津製作所)で定量する方法で測定した。具体的には、10mM無水ブロモ酢酸(ブロモ酢酸とDCC[ジシクロヘキシルカルボジイミド]をジオキサン中で混合することで調製)、10mMピリジンのジオキサン溶液中に基板を浸漬し1時間振とうさせアミノ基をブロモ化アセチルした。続いて過剰なブロモ酢酸をメタノールで洗浄後、減圧乾燥させた。さらに、10mMメルカプトエタノール水溶液に基板を浸漬し1時間振とうさせ臭化物イオンを遊離させた。この臭化物イオンを含有する溶液を10 μL分取しイオンクロマトグラフィーに供して臭化物イオン濃度を定量し、基板の表面積から単位面積当たりのアミノ基量を算出した。
プローブと基板とのリンカーとしてEMCA(6-マレイミドカプロン酸)を導入した。EMCAをDCCにより対称無水物としたのち、ピリジンを加え10mMに調整した溶液にPAA固定化したアモルファスカーボン基板を1時間浸漬した。これにプローブとして、α−ヘリックス構造の合成ペプチド(アミノ酸配列GLQQLARALRRLAQAGC)に蛍光分子TAMRAを標識したペプチドを1%酢酸水溶液に濃度10μMになるように調整した溶液を上記基板表面にスポット(アレイ化)した。さらに、過剰なEMCAをキャッピングするため、10mMメルカプトエタノール液に当該基板を浸漬し、メタノールで洗浄後、スピン乾燥させたのち、蛍光スキャナーで蛍光を測定しプローブの存在を確認した。さらに、50%イソプロパノール水溶液で洗浄後、真空乾燥し再度蛍光スキャナーで蛍光を測定し、プローブの残存(すなわち固定化されている)を確認した。
タンパク質として牛血清アルブミン(BSA)を蛍光分子TAMRAで標識したものを、リン酸バッファーに溶解して濃度0.01%に調整した溶液を基板表面にスポットして1時間静置した。これを、50%イソプロパノール水溶液で洗浄後、スピン乾燥した基板を蛍光スキャナーで測定した。発光画像によりタンパク質吸着状態を評価した。
1. バイオチップ用基板の作製
(1) 基板及び紫外線処理
表面粗さRaが1nmになるように研磨したアモルファスカーボン板(25.0×75.0mm 公差±0.1mm 板厚1.000mm 公差±0.025mm)を基板材料とし、紫外線照射装置(セン特殊光源株式会社,フォト・サーフェイス・プロセッサーPL16-110)で15分間紫外線照射(18.5 mW/cm2、254nm)を行った。
PAA(平均分子量3,000)をエタノールで1質量%となるように濃度調整したものをコーティング液とした。PAAコーティング液を適量ピペットで分取し、基板材料表面に滴下した後、PAA塗布量が20μg/cm2になるように塗工厚さを3mil(約0.076mm)に設定したベーカー式アプリケーターにより基板材料表面全体にコーティング液を塗り広げた。溶媒が揮発してからさらに1時間真空乾燥した(真空度-0.098MPa)のち、そのまま真空下で3分間紫外線を照射(18.5mW/cm2,254nm)しPAAを固定化した。さらに超純水で1時間振とう洗浄して未反応のPAAを除去してからスピン乾燥させ本発明のバイオチップ用基板を得た。
作製された基板の2枚について上記方法によりアミノ基の均一性評価した(図1a,b)。また、画像データのドット強度のヒストグラムによりアミノ基の分布を評価した(図2a,b)。PAAコーティングカーボン基板は市販のアミノ化ガラス基板(後述の比較例2)に比べて均一にアミノ基が分布している。さらに、アミノ基量を遊離臭化物イオン濃度から算出したところ、PAAコーティングしたカーボン基板は基板表面に5.6±0.4nmol/cm2のアミノ基量を有しており、後述する比較例2のアミノ化ガラス基板よりも高密度であった。また、上記方法により固定化したプローブは洗浄2回繰り返しても固定化されたままであった(図5)。さらに、上記方法によりタンパク質の吸着性を評価したところ、タンパク質の吸着(図7)は比較例2のガラス基板(図8)に比べて少なかった。さらに、
XPSによる解析を行なったところ、表面アミノ基をブロモアセチル化した後XPSでC-Br結合が観察された(図9)。このことによりPAAが共有結合で基板表面に結合していることが確認された。
アルミニウム合金(5000系合金)の圧延板(75×25mm厚さ1mm)をPVA砥石で厚さ0.98mmまで研削し、表面粗さをRa30nmとした後、全面をアルカリ脱脂、硝酸デスマット、亜鉛ジンケート処理を行い、ニッケル−リン無電解めっきを片面あたり厚さ13μmつけた。さらに、両面をアルミナスラリーにより片面あたり3μm研磨し表面粗さRaを1nmとした。続いて、表面にイオン化蒸着装置を用いて、ヘキサメチルシロキサンガス中SiCを20nm成膜した後、メタンガス中でDLCを200nm成膜して表面にカーボンをコーティングしたものを基板材料に供した。以後は、実施例1と同様にしてポリマーを固定化し、バイオチップ用基板を得た。
信越化学工業製の合成石英スライドガラス(75×25mm板厚1mm)をフッ化水素酸で洗浄後、真空乾燥した。続いて、表面にカーボンを実施例2と同じ方法で200nmコーティングしたものを基板材料に供した。以後は、実施例1と同様にしてポリマーを固定化し、バイオチップ用基板を得た。
ポリメタクリル酸メチル板(75×25mm板厚1mm)をメタノールで洗浄し基板材料に供した。以後は、実施例1と同様にしてポリマーを固定化し、バイオチップ用基板を得た。
ポリマー溶液としてPAA1%エタノール溶液に親水性ポリマーとしてPEGが濃度0.3%になるように調整した溶液をコーティング液とし、モルファスカーボン表面に塗布方法に従って塗布した。以後は、実施例1と同様にしてポリマーを固定化し、バイオチップ用基板を得た。
アモルファスカーボンを実施例1と同様に紫外線処理(PAA溶液塗布前の紫外線処理)した後、アミノ基の均一性を評価した(図1c)。また、画像データのドット強度のヒストグラムによりアミノ基の分布を評価した(図2c)。アミノ基含有ポリマーを固定化していないため、アミノ基は検出されなかった。
2種類の市販のアミノ化ガラス基板についてアミノ基の均一性評価した(図3)。また、画像データのドット強度のヒストグラムによりアミノ基の分布を評価した(図4)。実施例に比べてアミノ基の量は不均一であり、画像の輝度のムラおよびヒストグラムの幅が大きくなっている。さらに、アミノ基量を遊離臭化物イオン濃度から算出したところ、臭化物イオンは検出されなかった。これはガラス表面には共有結合での固定化が難しいことを意味する。プローブは図6のように残るが色素の吸着が激しくかつ共有結合できないためにじむ。タンパク質の吸着は、(図8)のように激しい。
Claims (19)
- 基板表面に、アミノ基含有ポリマーが共有結合により固定化されたバイオチップ用基板。
- 前記基板表面に固定化される前の状態で、前記アミノ基含有ポリマーを構成する構成単位の50%以上が、それぞれアミノ基を少なくとも1個有する請求項1記載の基板。
- 前記アミノ基含有ポリマーを構成する構成単位の90%以上が、それぞれアミノ基を少なくとも1個有する請求項2記載の基板。
- 前記アミノ基含有ポリマーがポリアリルアミンである請求項3記載の基板。
- 前記アミノ基含有ポリマーの固定化量が、ポリアリルアミン換算で4μg/cm2以上である請求項1〜4のいずれか1項に記載の基板。
- 前記アミノ基含有ポリマーの固定化量が、40μg/cm2以下である請求項5記載の基板。
- 前記アミノ基含有ポリマーの重量平均分子量が、ポリアリルアミン換算で1000以上である請求項1〜6のいずれか1項に記載の基板。
- 前記アミノ基含有ポリマーの重量平均分子量が6万以下である請求項7記載の基板。
- 基板表面に、前記アミノ基含有ポリマーと均一に混合された親水性ポリマーが共有結合によりさらに固定化された請求項1ないし8のいずれか1項に記載の基板。
- 前記親水性ポリマーが、ポリエチレングリコール、ポリアクリルアミド及びアガロースから成る群より選ばれる少なくとも1種である請求項9記載の基板。
- 前記アミノ基含有ポリマーの固定化量が、ポリアリルアミン換算で4μg/cm2以上であり、前記アミノ基含有ポリマー及び前記親水性ポリマーの固定化量の合計が40μg/cm2以下である請求項9又は10記載の基板。
- 前記基板表面が、カーボン又はプラスチックから成る請求項1ないし11のいずれか1項に記載の基板。
- 前記基板表面が、カーボンから成る請求項12記載の基板。
- 請求項1ないし13のいずれか1項に記載のバイオチップ用基板の前記アミノ基に、直接又はリンカーを介して生体関連物質が共有結合されたバイオチップ。
- 前記基板表面に、前記アミノ基含有ポリマーを塗布した後、減圧雰囲気下又は不活性ガス雰囲気下で該基板表面を光照射することを含む請求項1記載のバイオチップ用基板の製造方法。
- 前記基板表面を、プラズマ照射又は光照射し、次いで該基板を前記アミノ基含有ポリマーと接触させることを含む請求項1記載のバイオチップ用基板の製造方法。
- アミノ基含有ポリマーと接触させた後、さらに減圧雰囲気下又は不活性ガス雰囲気下で該基板表面を光照射することを含む請求項16記載の方法。
- 前記基板表面が、カーボン又はプラスチックから成る請求項16又は17記載の方法。
- 前記基板表面が、カーボンから成る請求項18記載の方法。
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WO2014109312A1 (ja) | 2013-01-09 | 2014-07-17 | 日本軽金属株式会社 | バイオチップ用基板及びその製造方法 |
WO2015025634A1 (ja) * | 2013-08-21 | 2015-02-26 | 日本軽金属株式会社 | バイオチップ用基板及びその製造方法 |
WO2015033668A1 (ja) | 2013-09-06 | 2015-03-12 | 日本軽金属株式会社 | バイオチップ用基板 |
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EP2306197A4 (en) | 2011-08-03 |
US9857369B2 (en) | 2018-01-02 |
JP5459990B2 (ja) | 2014-04-02 |
WO2010001876A1 (ja) | 2010-01-07 |
EP2306197B1 (en) | 2014-11-26 |
US20110152409A1 (en) | 2011-06-23 |
EP2306197A1 (en) | 2011-04-06 |
US20140336081A1 (en) | 2014-11-13 |
US8822607B2 (en) | 2014-09-02 |
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