JP2004085557A - Probe-coupling substrate, its manufacturing method, and its analytical method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a probe coupling substrate and its manufacturing method, capable of easily analyzing probes, as well as minimizing loss in sampling inspection, to enable quick and inexpensively detection and quantitative analysis of a target matter, when using various surface analyses, and to provide a method for quantitatively analyzing the target matter in a specimen quickly and inexpensively. <P>SOLUTION: The probe-coupling substrate and its manufacturing method are probes which are specifically combinable with the target matter located on the substrate surface of at least insulative face, and a conductive membrane is located on at least the same level as the probe. This method is for analyzing probe arrays having those probes specifically combined with the target matter which may be contained in the measuring specimen, as a plurality of mutually-independent spots on the substrate. In the method, the probes are analyzed, after the conductive membrane is located on the substrate surface to be electrically connected with a ground. <P>COPYRIGHT: (C)2004,JPO

Description

【０１３６】
（６）ＢＪプリンターによるＤＮＡ吐出、および基板への結合
上記２５種類のＤＮＡを最終濃度が約４００ｍｇ／ｍｌになるようにＴＥ溶液（１０ｍｍｏｌ／Ｌ　Ｔｒｉｓ−ＨＣｌ（ｐＨ８）／１ｍｍｏｌ／Ｌ　ＥＤＴＡ水溶液）に溶解し、それぞれ一本鎖ＤＮＡ溶液を調製した（正確な濃度は吸収強度から算出）。
【０１３７】
グリセリン７．５ｗｔ％、尿素７．５ｗｔ％、チオジグリコール７．５ｗｔ％、及びアセチレンアルコール（商品名：アセチレノールＥＨ；川研ファインケミカル（株）社製）１ｗｔ％を含む水溶液を用意し、上記ＤＮＡ溶液に加え、一本鎖ＤＮＡの最終濃度が８μｍｏｌ／Ｌとなるように調整した。この液体の表面張力は３０〜５０ｍＮ／ｍ（３０〜５０ｄｙｎｅ／ｃｍ）の範囲内であり、また粘度は１．８ｍＰａ・ｓ（＝１．８ｃｐｓ）（Ｅ型粘度計：東京計器（株）社製）であった。この液体をバブルジェットプリンター（商品名：ＢＪＣ６２０；キヤノン（株）社製）用インクタンクに充填しバブルジェットヘッドに装着した。なおここで用いたバブルジェットプリンター（商品名：ＢＪＣ６２０；キヤノン（株）社製）は平板への印刷が可能で、２５種類のＤＮＡのそれぞれを導入可能な様に改造を施したものである。またこのプリンターは３６０×７２０ｄｐｉの解像度で印字可能である。次いでこのプリンターに上記（２）で処理した石英基板を装着し、プローブ核酸を含む液体を石英基板上にスポッティングした。吐出した液滴の数は、１５×１５＝２２５個とし、５×５＝２５個を１ユニットとして、１ユニット中にはそれぞれ種類の異なるＤＮＡプローブを有し、各ユニットは同じＤＮＡ配列を有する構造とした。この際、上記（４）で形成した酸化パラジウムのドットとの位置とＤＮＡプローブの位置がずれないよう酸化パラジウムドットで位置合わせを行なうようにした。スポッティング終了後、石英基板を３０分間加湿チャンバー内に静置し、石英基板表面のマレイミド基と核酸プローブ末端のチオール基とを反応させた。なお上記プリンターの１吐出動作あたりのＤＮＡプローブ溶液の吐出量は約２４ｐＬであった。
【０１３８】
（７）スポット位置検査
上記（６）で作製したＤＮＡプローブスポットの吐出位置を、酸化パラジウムドットの位置を参考に光学顕微鏡で確認した。各ＤＮＡプローブスポットとも、位置ずれすることなく、所望の位置に吐出されていることを確認した。また、各スポットとも、スポットが不必要に広がる事なく、スポット径は約７０μｍと均一な形状が得られていた。
【０１３９】
（８）ブロッキング反応
マレイミド基とチオール基との反応終了後、石英基板を１ｍｏｌ／Ｌ　ＮａＣｌ／５０ｍｍｏｌ／Ｌリン酸緩衝液（ｐＨ７．０）溶液で洗浄し、石英基板と結合しなかった余分なＤＮＡを含む液体を洗い流した。次いで石英基板を２％ウシ血清アルブミン水溶液中に浸して２時間放置し、ブロッキング反応を行なった。
【０１４０】
（９）スポット製膜検査
ブロッキング反応後のプローブ結合基板を純水で洗浄し、低真空走査電子顕微鏡で観察を行なった。低真空走査電子顕微鏡はＦＥＩ社製　ＸＬ３０ＥＳＥＭを用い、加速電圧は１５ｋＶ、真空度は８０Ｐａ（＝０．６Ｔｏｒｒ）として観察を行なった。
【０１４１】
観察の結果、各ＤＮＡプローブスポットとも、洗浄・及びブロッキング反応を行なった後でも、ＤＮＡスポットが吐出時と変わらず所望の位置に結合されていることが確認された。また、洗浄やブロッキング反応を行なうことにより、スポットの一部が欠けることにより変形したり、所望の場所以外の領域にＤＮＡが再結合したりといったトラブルもないことが確認できた。
【０１４２】
（１０）結果
従来のＤＮＡプローブスポットのみからなるプローブ結合基板では、ＤＮＡプローブが非常に薄く、かつ、２次電子放出効率の低い有機物からなるため、スポットの位置を探すことは非常に困難であった。また、蛍光色素をマーカーに用いたプローブ結合基板においても、同様にマーカーのスポットを探すことが非常に困難であった。
【０１４３】
しかし、本発明のプローブ結合基板においては、上記のように、ＤＮＡプローブスポットを特定できる位置、具体的には、Ｘ、Ｙ軸を同一にした酸化パラジウム化合物ドットを設けることにより、製造時検査でスポットの打ち損じがないかどうか、サテライトスポットがないかどうか、ゴミ等がスポットにかぶっていないか、の確認を、製造したＤＮＡプローブ基板の全てについて、早く確実にすることができた。
【０１４４】
また、ハイブリダイゼーション処理を行なわずに、製造時検査をすることができた。これにより、抜き取り検査により製品を無駄にする事なく、全ての検査合格品を出荷することができた。このことは品質保証上有効である。
【０１４５】
＜比較例１＞
酸化パラジウムからなる金属化合物ドットを設けない点以外は実施例１と全く同じプローブ結合基板を作製した。この際は、無色透明な石英基板上に無色透明のＤＮＡプローブを配置したため、プローブ位置を探すのが困難であったとともに、スポットの打ち損じ等が見つかった場合も、位置を特定するのが難しいため、バブルジェットプリンターの全てのヘッドを洗浄する必要があり、製造上のロスが大きくなってしまった。
【０１４６】
＜実施例２＞
金属化合物を酸化パラジウムから銀に変えた以外は実施例１と同じプローブ結合基板を作製した。このプローブ結合基板においても、実施例１と同様の効果が得られた。
【０１４７】
＜実施例３＞
本実施例では、図５のようにＸ，Ｙアドレス５０４を設け、ＤＮＡプローブスポットの吐出量を約１００ｐＬとした以外は実施例１と同じプローブ結合基板を作製した。
【０１４８】
Ｘ、Ｙアドレス５０４は、Ｘ方向の列番号と、Ｙ方向の行番号を、あらかじめ金属化合物ドットの外側に形成したものである。このアドレス５０４は金属化合物マーカー印字に使用した溶液を別のノズルに詰め、形成したものである。
このプローブ結合基板では、実施例１と同様の効果が得られただけでなく、あらかじめスポットのアドレスが低真空走査電子顕微鏡のＣＣＤ像、並びにＳＥＭ像で確認できるため、スポットの異常を発見した際に、どのプローブのノズルに異常があるかを早く発見することができた。
【０１４９】
＜実施例４＞
本実施例では、実施例１の方法を用いて、シリコン基板上に金属化合物ドットとＤＮＡプローブを作製し、Ｘ線光電子分光装置による分析を行なった。
【０１５０】
図４は本発明の一例を示す図であり、シリコン基板４０１上に複数個の互いに塩基配列の異なるプローブをスポット上に結合させたプローブ結合基板の平面図である。４０２はプローブのスポットである。４０３は銀のスポットであって、該プローブのスポット４０２の位置を特定可能な位置に配置されている。具体的にはマトリクス状に配置されているプローブのスポット４０２の各行及び各列に対応する位置に配置されており、それによってスポット４０２の位置の特定を可能としている。プローブ、及び銀のスポット径の設定値はそれぞれ１ｍｍとした。
【０１５１】
このプローブ結合基板を、金属スポットの材質を銀に変えた以外は実施例１と同じ製法で作製後、Ｘ線光電子分光法のチャンバー内に基板を入れ、各プローブのスポットについて、分析を行なった。Ｘ線光電子分光装置はＶＧ　Ｓｃｉｅｎｔｉｆｉｃ社製μ−ＥＳＣＡ　２５０ｉ−ＸＬを使用し、Ｘ線は単色化Ａｌ　Ｘ線を用いた。また、Ｘ線のフィラメント電流は１８ｍＡ、電圧は１７ｋＶとした。
【０１５２】
通常、ＤＮＡプローブスポットのみからなるプローブ結合基板では、ＤＮＡプローブが無色透明で、かつ、非常に薄い膜であるため、鏡筒内で位置を確認することができなかった。
【０１５３】
しかし、図４に示すように銀スポット４０３を、該プローブのスポット４０２の位置を特定可能な位置に配置した本発明のプローブ結合基板においては、銀スポットの位置により、分析位置を特定できるため、この特定位置に合わせて分析することにより、短時間に各スポットの分析を行なう事ができるようになった。
【０１５４】
この結果、基板上所望の位置に、各プローブがはがれや吐出不良を起こす事なく作製されていることを確認できた。即ち、検査用として十分信頼性あるプローブアレイが形成されていることが確認された。
【０１５５】
＜実施例５＞
本実施例では、インクジェット法を用いて、石英基板上に酸化パラジウムスポットとタンパク抗体を作製し、ＴｏＦ−ＳＩＭＳによる分析を行った。
【０１５６】
図１は本発明の一例を示す図であり、石英基板上にそれぞれ異なるタンパク質に対する抗体を配置した。この基板を実施例１と同じ製法で作製後、ＴｏＦ−ＳＩＭＳのチャンバー内に基板を入れ、各プローブのスポットについて分析を行った。ＴｏＦ−ＳＩＭＳはＩＯＮ−ＴＯＦ社製ＴｏＦ−ＳＩＭＳ　ＩＶ　を使用し、エミッターはＧａを用いた。また、加速電圧は２５ｋＶ、試料電流は２．４ｐＡとした。
【０１５７】
通常の抗体結合基板では、プローブが透明で薄膜のため、ＣＣＤカメラ等による位置の確認ができないため、プローブ位置を探すのが困難であると共に、スポットの位置を特定することが難しかった。しかし、本発明の基板においては、酸化パラジウムスポットの位置により分析位置を特定できるため、短時間に各スポットの分析を行うことができた。これにより、各プローブが抜けたりすることなく、設計通り形成されていることを確認した。
【０１５８】
＜実施例６＞
本実施例では、実施例１の方法を用いて、シリコン基板上に酸化パラジウムドットとＤＮＡプローブを作製し、通常の高真空タイプの走査電子顕微鏡による観察を行なった。
【０１５９】
図４は本発明の一例を示す図であり、シリコン基４０１上に複数個の互いに塩基配列の異なるプローブをスポット上に結合させたプローブ結合基板の平面図である４０２はプローブのスポットである４０３は酸化パラジウムのスポットであって、該プローブのスポット４０２の位置を特定可能な位置に配置されている。具体的にはマトリクス状に配置されているプローブのスポット４０２の各行及び各列に対応する位置に配置されており、それによってスポット４０２の位置の特定を可能としている。プローブ、及び銀のスポット径の設定値はそれぞれ７０μｍとした。走査電子顕微鏡は、日立製Ｓ−５０００Ｈを用い、加速電圧１ｋＶで観察した。
【０１６０】
このプローブ結合基板を実施例１と同じ製法で作製後、走査電子顕微鏡の鏡筒内に基板を入れ、各プローブのスポットについて、詳細な観察を行なった。通常、ＤＮＡプローブスポットは、無色透明で、かつ、非常に薄い膜であるため、鏡筒内で位置を確認することは非常に困難であった。
【０１６１】
しかし、図４に示すように酸化パラジウムスポット４０３を、該プローブのスポット４０２の位置を特定可能な位置に配置した本発明のプローブ結合基板においては、酸化パラジウムスポットの位置により、大体の観察位置を特定できるため、この特定位置を詳細に観察することにより、短時間に良好な像を得る事ができるようになった。
【０１６２】
この結果、各プローブが設定値通り約７０μｍの径で均一に作製されていることが確認された。更に、光学顕微鏡より微細な構造についても確認することができ、検査用として十分信頼性あるプローブアレイが形成されていることを確認できた。
【０１６３】
＜実施例７＞
図７は本発明の一例を示す図であり、石英基板７０１上にプローブをスポット上に結合させたプローブアレイの平面図である。７０２はプローブのスポットである。７０３は酸化パラジウムからなる導電性膜であって、外部アース電極と電気的に接続されている。
【０１６４】
このプローブ結合基板の作製方法を下記に示す。
【０１６５】
まず、特開平１１−１８７９００号公報（特許文献４）に記載の方法に準じて核酸プローブアレイを作製した。
【０１６６】
（１）基板洗浄
２５．４ｍｍ×２５．４ｍｍ×１ｍｍの合成石英基板をラックに入れ、純水で１０％に稀釈した超音波洗浄用洗剤（ブランソン：ＧＰＩＩＩ）に一晩浸した。その後、洗剤中で２０分間超音波洗浄を行い、その後、水洗により洗剤を除去した。純水ですすいだ後、純水の入った容器中でさらに超音波処理を２０分間行なった。次に、予め８０℃に加温した１ｍｏｌ／Ｌ（１Ｎ）水酸化ナトリウム水溶液に基板を１０分間浸した。引き続き水洗、純水洗浄を行なって、そのまま次工程に供した。
【０１６７】
（２）表面処理
アミノ基を結合したシランカップリング剤、Ｎ−β−（アミノエチル）−γ−アミノプロピルトリメトキシシラン　ＫＢＭ６０３（信越化学工業）の１ｗｔ％水溶液を室温下で２時間攪拌し、上記シラン化合物の分子内のメトキシ基を加水分解した。次いでこの溶液に上記（１）で得た基板を室温で１時間浸漬した後、純水で洗浄し、窒素ガスを基板の両面に吹き付けて乾燥させた。次に基板を１２０℃に加熱したオーブン中で１時間ベークして最終的に基板表面にアミノ基を導入した。
【０１６８】
次いでＮ−マレイミドカプロイロキシスクシンイミド（同仁化学研究所：以下ＥＭＣＳ）２．７ｍｇを、ジメチルスルホキシド（ＤＭＳＯ）／エタノールの１：１溶液に濃度が０．３ｍｇ／ｍｌとなる様に溶解した。シランカップリング処理を行なった石英基板をこのＥＭＣＳ溶液に室温で２時間浸漬して、シランカップリング処理によって基板表面に担持されているアミノ基とＥＭＣＳ溶液のスクシイミド基を反応させた。この段階で基板表面にはＥＭＣＳ由来のマレイミド基が存在することになる。ＥＭＣＳ溶液から引き上げた基板はＤＭＳＯ及びエタノールの混合溶媒及びエタノールで順次洗浄した後、窒素ガスを吹き付けて乾燥させた。
【０１６９】
（３）金属含有溶液の作製
次に金属含有溶液を作製した。０．８４ｇの酢酸パラジウムーモノエタノールアミン（以下ＰＡ−ＭＥと略す）を１２ｇの水に溶解し、さらにポリビニルアルコール（以下ＰＶＡと略す）を加え、溶液粘度を２０ｍＰａ・ｓ（＝２０センチポイズ）に調整したものをバブルジェット付与用水溶液とした。なお、ＰＡ−ＭＥは下記のようにして合成した。
【０１７０】
１０ｇの酢酸パラジウムを２００ｃｍ^３のイソプロピルアルコール（以下ＩＰＡと略す）に懸濁させ、更に１６．６ｇのモノエタノールアミンを加え室温で４時間撹拌させた。反応終了後、ＩＰＡをエバポレートより除き、固形物にエタノールを加え、溶解、ろ過し、ろ液からＰＡ−ＭＥを再結晶した。
【０１７１】
（４）ＢＪプリンターによる金属化合物吐出、および基板への結合
このようにして作製したバブルジェット付与溶液を、バブルジェットプリンター（商品名：ＢＪＣ６２０；キヤノン（株）社製）用インクタンクに充填しバブルジェットヘッドにセットした。なおここで用いたバブルジェットプリンター（商品名：ＢＪＣ６２０；キヤノン（株）社製）は平板への印刷が可能な様に改造を施したものである。その後、上記表面処理を施した石英基板上に図６のように金属含有溶液を付与し、乾燥させた。
【０１７２】
これを大気雰囲気のオーブン中で３００℃に加熱して前記ＰＡ−ＭＥ及びＰＶＡを基板上で分解堆積させ、酸化パラジウム微粒子からなる金属化合物スポットを形成した。
【０１７３】
（５）プローブＤＮＡの合成
ＤＮＡ合成業者（ベックス）に依頼して配列番号２６の一本鎖核酸（Ｔの４０量体）を合成した。なお配列番号２６の一本鎖ＤＮＡの５’末端には合成時にチオールモディファイア（グレンリサーチ）を用いる事によってチオール（ＳＨ）基を導入した。なお、脱保護、ＤＮＡの回収は定法により行い、また、精製にはＨＰＬＣを用いた。合成から精製までの一連の工程はすべて合成業者に依頼して行なった。
配列番号：２６
５’　ＨＳ−（ＣＨ_２）_６−Ｏ−ＰＯ_２−Ｏ−ＴＴＴＴＴＴＴＴＴＴ　ＴＴＴＴＴＴＴＴＴＴ　ＴＴＴＴＴＴＴＴＴＴ　ＴＴＴＴＴＴＴＴＴＴ　３’
（６）サーマルジェットプリンターによるＤＮＡ吐出、および基板への結合
上記配列番号２６の一本鎖ＤＮＡを８μｍｏｌ／Ｌの濃度でグリセリン７．５ｗｔ％、尿素７．５ｗｔ％、チオジグリコール７．５ｗｔ％、及び上記一般式（Ｉ）で示されるアセチレンアルコール（商品名：アセチレノールＥＨ；川研ファインケミカル（株）社製）１ｗｔ％を含む溶液を溶解した。サーマルジェット法の一種であるバブルジェット法を用いたバブルジェットプリンターＢＪＦ−８５０（キヤノン）用のプリンターヘッドＢＣ−５０（キヤノン）を数１００μＬの溶液を吐出可能とするべく改造し、このヘッドを上記石英基板上へ吐出可能となるよう改造した吐出描画機に搭載した。このヘッドの改造タンク部に上記ＤＮＡ溶液を数１００μＬ注入し、吐出描画機にＥＭＣＳ処理基板を装着して、ここにスポッティングした。なお、スポッティング時の吐出量は４ｐＬ／ｄｒｏｐｌｅｔで、スポッティングの範囲は基板の中央部に１０ｍｍ×１０ｍｍの範囲に２００ｄｐｉすなわち１２７μｍのピッチで吐出した。この条件ではスポッティングされたドットの直径は約５０μｍであった。
【０１７４】
スポッティング終了後、基板を３０分間加湿チャンバー内に静置し、ガラス板表面のマレイミド基と核酸プローブ末端のチオール基とを反応させた。次いで、基板を純水で洗浄し、純水中で保存した。
【０１７５】
（７）スポット製膜検査
上記保存したＤＮＡ結合基板（ＤＮＡチップ）は走査電子顕微鏡による観察直前に窒素ガスを吹き付けて乾燥し、真空デシケーター中でさらに乾燥した。乾燥後、試料をアルミ製の試料台上に、カーボンペーストで接着し、同様に試料表面の導電性膜６０３の端部と試料台とをカーボンペーストで接続することにより、導電性膜をアースと電気的に接続した。また、走査電子顕微鏡は日立製作所製Ｓ−５０００Ｈを用い、加速電圧は５ｋＶとして観察を行なった。
【０１７６】
（８）結果
観察の結果、加速電圧　５ｋＶにおいても、チャージアップを起こす事なく、観察を行なうことができた。また、各ＤＮＡプローブスポットとも、洗浄・及びブロッキング反応を行なった後でも、ＤＮＡスポットが吐出時と変わらず所望の位置に結合されていることが確認された。また、洗浄やブロッキング反応を行なうことにより、スポットの一部が欠けることにより変形したり、所望の場所以外の領域にＤＮＡが再結合したりといったトラブルもないことが確認できた。
【０１７７】
本発明のプローブ結合基板においては、上記のように、導電性膜を周囲に配置した形態とする事により、製造時検査でスポットの打ち損じがないかどうか、サテライトスポットがないかどうか、ゴミ等がスポットにかぶっていないか、の確認を、製造したＤＮＡプローブ基板の全てについて、早く確実にすることができた。
【０１７８】
また、ハイブリダイゼーション処理を行なわずに、製造時検査をすることができた。これにより、抜き取り検査により製品を無駄にする事なく、全ての検査合格品を出荷することができた。このことは品質保証上有効である。
【０１７９】
＜比較例２＞
酸化パラジウムからなる導電膜を設けない点以外は実施例７と全く同じプローブ結合基板を作製し、実施例７と同様に走査電子顕微鏡による観察を行なった。初めに、実施例７と同様に加速電圧５ｋＶで観察を行なったところ、絶縁性の石英基板上にＤＮＡプローブを配置したため、電子ビーム照射によりチャージアップが起こり、二次電子像が得られなかった。そこで、いったん鏡筒外にプローブ結合基板を取り出し、再度鏡筒内に導入した後、加速電圧１ｋＶまで加速電圧を下げて観察を行なったが、プローブの詳細な検査はできなかった。
【０１８０】
＜実施例８＞
金属化合物を酸化パラジウムから銀に変えた以外は実施例７と同じプローブ結合基板を作製した。このプローブ結合基板においても、実施例７と同様の効果が得られた。
【０１８１】
＜実施例９＞
導電性膜を図９のようにスパッタ法を用いて製膜した後で、プローブを作製した。図９において、９０１は青板基板、９０２はプローブアレイスポット、９０３は導電性膜である。導電性膜以外の製法は実施例７と同様に作製した。このプローブ結合基板を用いて、実施例７と同様に走査電子顕微鏡観察を行なったところ、実施例７と同様の効果が得られた。
【０１８２】
＜実施例１０＞
図８のように、導電性膜と電気的に接続したマーカーを設けてプローブを作製した。図８において、８０１は青板基板、８０２はプローブアレイスポット、８０３は導電性膜、８０４はマーカーである。導電性膜８０３はインクジェット法により、円形の酸化パラジウム膜を端部が重なるようにして、連続膜を成膜した。更に先端をアースと接続して、電気的にアースに落とした。マーカー８０４は酸化パラジウムのスポットであって、該プローブのスポット８０２の位置を特定可能な位置に配置されている。具体的にはマトリクス状に配置されているプローブのスポット８０２の各行、及び各列に対応する位置に配置されており、それによって、スポット８０２の位置の特定を可能としている。
【０１８３】
インクジェット法で酸化パラジウムを形成し、マーカー以外の製法は実施例７と同様に作製した。
【０１８４】
このプローブ結合基板を用いて、実施例７と同様に走査電子顕微鏡観察を行なったところ、実施例７と同様の効果が得られた。
【０１８５】
また、通常ＤＮＡプローブスポットは、無色透明で、非常に薄い膜であり、更に二次電子放出効率も低いために、鏡筒内で位置を確認することは非常に困難であった。しかし、図８に示すように酸化パラジウムスポット８０３を、該プローブのスポット８０２の位置を特定可能な位置に配置した本発明のプローブ結合基板においては、マーカーの位置を参考にプローブアレイスポット８０２の位置を探すことができたため、２次電子放出効率の低いプローブアレイスポットの位置を探すための時間を減らすことができ、この特定位置を詳細に観察することにより、短時間に良好な像を得ることができるようになり、分析効率を上げることができた。
【０１８６】
この結果、各プローブが約７０μｍの径で均一に作製されていることが確認された。更に、光学顕微鏡より微細な構造についても確認することができ、検査用として十分信頼性あるプローブアレイが形成されていることを確認できた。
【０１８７】
＜実施例１１＞
本実施例では、実施例７の方法を用いて、石英基板上に金属化合物ドットとＤＮＡプローブを作製し、Ｘ線光電子分光装置による分析を行なった。
【０１８８】
図１２は本発明の一例を示す図であり、石英基板１２０１上に複数個の互いに塩基配列の異なるプローブをスポット上に結合させたプローブ結合基板の平面図である。１２０２はプローブのスポットである。１２０３は銀のスポットであって、該プローブのスポット１２０２の位置を特定可能な位置に配置されている。具体的にはマトリクス状に配置されているプローブのスポット１２０２の各行及び各列に対応する位置に配置されており、それによってスポット１２０２の位置の特定を可能としている。プローブ、及び銀のスポット径の設定値はそれぞれ１ｍｍとした。
【０１８９】
このプローブ結合基板を、金属スポットの材質を銀に変えた以外は実施例７と同じ製法で作製後、Ｘ線光電子分光法のチャンバー内に基板を入れ、各プローブのスポットについて、分析を行なった。Ｘ線光電子分光装置はＶＧ　Ｓｃｉｅｎｔｉｆｉｃ社製μ−ＥＳＣＡ　２５０ｉ−ＸＬを使用し、Ｘ線は単色化Ａｌ　Ｘ線を用いた。また、Ｘ線のフィラメント電流は１８ｍＡ、電圧は１７ｋＶとした。
【０１９０】
通常、ＤＮＡプローブスポットのみからなるプローブ結合基板のＸ線光電子分光装置測定では、測定時のチャージアップを防ぐためにメタルマスクを試料の上に載せ、同時に中和電子銃を併用していた。
【０１９１】
その際、メタルマスクの穴があいている場所以外の場所を分析するためにはプローブアレイの位置に応じてメタルマスクを徐々にずらして行かなくてはならなかった。
【０１９２】
しかし、図１１に示す本発明のプローブ結合アレイにおいては、導電性膜がメタルマスクと同様の役割を果たすため、チャージアップを起こす事なく、測定することができた。
【０１９３】
この結果、基板上所望の位置に、各プローブがはがれや吐出不良を起こす事なく作製されていることを確認できた。即ち、検査用として十分信頼性あるプローブアレイが形成されていることが確認された。
【０１９４】
【発明の効果】
以上説明したように、本発明により、各種表面分析に有効なプローブ結合基板のマーキングが可能になった。また、本発明を利用すれば、標的物質との結合反応を行わなくても、製品検査用の分析が可能となった。
【０１９５】
【配列表】

【図面の簡単な説明】
【図１】本発明の実施態様の一例、実施例５を示す図である。
【図２】本発明の別の実施態様を示す図である。
【図３】本発明の別の実施態様を示す図である。
【図４】本発明の実施例を示す図である。
【図５】本発明の実施例３を示す図である。
【図６】本発明の別の実施態様を示す図である。
【図７】本発明の実施態様の一例と、実施例７，８を説明する図である。
【図８】本発明の実施例１０を説明する図である。
【図９】本発明の実施態様の一例と実施例９を説明する図である。
【図１０】本発明の実施態様の別の一例を説明する図である。
【図１１】本発明の別の実施態様を説明する図である。
【図１２】本発明の実施例１１を説明する図である。
【符号の説明】
１０１、２０１、３０１、４０１、５０１、６０１、７０１、８０１、９０１、１００１、１１０１、１２０１：基板
１０２、２０２、３０２、４０２、５０２、６０２、７０２、８０２、９０２、１００２、１１０２、１２０２：ＤＮＡプローブ
１０３、２０３、３０３、４０３、５０３、６０３、７０３、８０３、９０３、１００３、１１０３、１２０３：金属または金属化合物からなるプローブ
５０４：　Ｘ，Ｙアドレス
８０４：　マーカー[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a probe-bonded substrate, a method for manufacturing the same, and a method for analyzing the same.
[0002]
[Prior art]
So-called biochips such as DNA chips and protein chips have come to be used for the purpose of genomic analysis or gene expression analysis, and the results of those analyzes are used for cancer, hereditary diseases, and lifestyle. It is expected to provide important indices for diagnosis of disease, infectious disease, etc., prediction of prognosis, determination of treatment policy, and the like.
[0003]
Several methods are known for producing a biochip. In recent years, research and development on detection and quantification of a target substance using a solid-phase probe array have been vigorously conducted. For example, US Pat. No. 5,445,936 (Patent Literature 1) discloses a solid-phase oligonucleotide array manufactured by using photolithography. On the other hand, US Pat. No. 5,688,642 (Patent Document 2) discloses a method for producing a solid-phase DNA probe array using an inkjet method.
[0004]
Generally, biochips are made by either of these methods, but if these biochips are to be used for the applications described above, the reliability of the analysis, i.e., quantitative and reproducible, is guaranteed. In order to do so, it is important to know the amount of the probe present in each matrix, that is, in this case, the amount of the biological substance. It is also important to know what matrix shape (shape, size, state) actually exists. However, since probes on the chip exist in principle at the monolayer level, these analyzes require extremely sensitive surface analysis techniques.
[0005]
Although a method of isotopically labeling a probe is known as such a highly sensitive surface analysis technique, it is not general because the technique is complicated, dangerous, and requires special facilities and equipment.
[0006]
The same applies to other products, but especially when this probe-binding board that handles genetic information is manufactured, there is no defect in the manufacturing process, and testing before shipment is also to prevent medical accidents Deemed important.
[0007]
When detecting or quantifying a target substance using the probe-bound substrate, it is important to know which probe has reacted with the target substance among a plurality of different probes constituting the array.
[0008]
Therefore, a fluorescence method is generally used as these inspection methods. This is a method of detecting fluorescence by binding a probe array immobilized on a substrate to a target substance incorporating a fluorescent substance. For example, Cy3, Cy5, or the like is used as the fluorescent substance, and a confocal laser-type apparatus or the like is generally commercially available as a scanner for detecting fluorescence.
[0009]
In the case of using the above-mentioned fluorescence method in the manufacturing process or pre-shipment inspection, it is known in advance that a portion of the manufactured probe binding substrate is extracted and specifically bound to the probe array arranged on the substrate. A fluorescent substance is incorporated into a standard target substance, and the fluorescent substance is brought into contact with a probe array. After an appropriate reaction time, an extra target substance is removed, and fluorescence is detected by the scanner.
[0010]
When these inspection methods using the fluorescence method are applied to a solid-phase probe array manufactured by an ink-jet method, the position occupied by each probe with respect to a specific position on the substrate is determined by photolithography due to differences in manufacturing equipment. This can be difficult when compared to methods. For this reason, Japanese Patent Application Laid-Open No. 2000-270896 (Patent Document 3) discloses a probe array in which a dye or a fluorescent dye is arranged as a marker substance around a probe and the position of the probe can be specified from this position. The creation method is shown.
[0011]
Using this probe array, the emission position when the standard target substance binds to the probe can be determined by detecting the surrounding fluorescent dye by a fluorescence method.
[0012]
In addition, in the present invention, a technique described in JP-A-11-187900 (Patent Document 4) is used (described later).
[0013]
[Patent Document 1]
U.S. Pat. No. 5,445,936
[Patent Document 2]
U.S. Pat. No. 5,688,642
[Patent Document 3]
JP 2000-270896 A
[Patent Document 4]
JP-A-11-187900
[0014]
[Problems to be solved by the invention]
However, the present inventors have further studied techniques for detecting and quantifying a target substance using a probe array produced by an inkjet method, and as a result, have found the following problems.
[0015]
That is, when analysis methods other than the fluorescence method and the optical microscopy method are used, the dyes disclosed in JP-A-2000-270896 (Patent Document 3) and marker substances such as fluorescent dyes may not be distinguished in some cases. It is.
[0016]
It is important to confirm whether the probe array produced by the ink jet method is formed at a predetermined position or whether there is any defect, in a production process or in a post-production pre-shipment inspection. However, immediately after the probe droplet is dropped on the substrate, the position of the probe can be confirmed by confirming the droplet, but in the manufacturing process, the liquid containing the probe is allowed to flow after the completion of the bonding reaction between the probe and the substrate. After leaving, the relative position of the probe cannot be confirmed with a normal optical microscope or fluorescence microscope. Furthermore, it is not possible to confirm with an optical microscope whether or not the desired shape and quantity of probes are arranged on the substrate. That is, since the solution containing the probe is originally colorless and transparent, and the probe remaining on the substrate after the solution has been washed away is very fine, it cannot be seen with an optical microscope or a fluorescence microscope.
[0017]
Further, as described in JP-A-2000-270896 (Patent Document 3), a probe array is manufactured by arranging a fluorescent dye or a dye around, and a part of the manufactured probe array is extracted, and each probe is extracted. A target substance (with a fluorescent substance attached) that is known to bind to the array is bound to the probe array, and the position and shape of the probe can be checked by emitting light from each probe array. However, when performing the fluorescence method, the standard target substance is bound to the probe for detection, so that the probe-bound substrate after the inspection cannot be shipped as a product. Since the probe binding substrate is intended to detect various types of target substances with one binding substrate, a large number of probes exist on the binding substrate. It is a great loss for a manufacturer that the number of products that can be shipped is greatly reduced by performing a sampling inspection for each probe type for the inspection.
[0018]
For these reasons, inspection methods using different analytical methods are required for checking at the time of manufacture, and as a result of our intensive efforts, scanning electron microscopy, photoelectron spectroscopy, atomic force microscopy It has been found that surface analysis methods such as time-of-flight secondary ion mass spectrometry (hereinafter abbreviated as TOF-SIMS) are effective for probe inspection.
[0019]
However, in these analysis methods, when observing each flux such as an electron beam, an X-ray, and an ion beam on a probe-bonded substrate, not only the probe array but also the marker composed of the dye or the fluorescent dye is detected. We found that the probe position on the substrate could not be specified during analysis because the signal was weak.
[0020]
On the other hand, the substrate for the probe binding substrate is not particularly limited as long as the substance constituting the probe can bind thereto and does not hinder detection of the target substance, but one example is a glass substrate. . Other substrates such as silicon substrate, metal substrate, resin substrate, etc. may be used with appropriate surface treatment.However, when a glass substrate is used as a substrate, general cleaning methods and surface treatment methods are used. Various methods known in the art can be used, and there are advantages such as easy availability of the substrate itself.
[0021]
However, when the above-described analysis method is applied to the above-mentioned glass substrate, each beam such as an electron beam, an X-ray, and an ion beam is irradiated onto the insulating substrate. Found the problem of
[0022]
The present invention has been made in order to solve these technical problems, and its object is to perform surface analysis methods such as scanning electron microscopy, photoelectron spectroscopy, atomic force microscopy, and TOF-SIMS. It is another object of the present invention to provide a probe-bonded substrate and a method for manufacturing the same, which can quickly detect the position of a probe even when used.
[0023]
Further, the present invention has been made to solve the above technical problem, and an object of the present invention is to analyze an insulated substrate or a probe-bonded substrate manufactured on a substrate in which an insulating film is formed on a conductive substrate. Therefore, even when a surface analysis method such as scanning electron microscopy, X-ray photoelectron spectroscopy, TOF-SIMS, etc. is used, a probe-bonded substrate and a method of manufacturing the same that can perform probe analysis without any problem are provided. The point is to provide.
[0024]
The present invention also provides a probe-coupled board that can be shipped with a minimum loss in sampling inspection, and a method of manufacturing the same.
[0025]
Further, the present invention provides a probe-bound substrate capable of detecting and quantifying a target substance quickly and inexpensively, and a method for producing the same.
[0026]
The present invention also provides a method for quickly and inexpensively quantifying a target substance in a sample.
[0027]
Further, the present invention provides an analysis method capable of minimizing the loss due to the probe array inspection in the process of manufacturing a probe array capable of detecting or quantifying a target substance, or the inspection process after the manufacture, and performing the inspection quickly. I will provide a.
[0028]
[Means for Solving the Problems]
In order to achieve the above object, the present invention has the following features.
[0029]
That is, the present invention provides a probe binding substrate in which a probe capable of specifically binding to a target substance is bound to a surface of the substrate, wherein the probe is bound to a first position on the surface, and A probe-bonded substrate characterized in that a metal or a metal compound is arranged at a second position where the first position can be specified by having a specific positional relationship with the position.
[0030]
A method of manufacturing a probe-bound substrate that can achieve the above object, a first step of supplying a liquid having a probe capable of specifically binding to a target substance to a first position on the substrate surface,
A second step of disposing a metal or metal compound at a second position capable of specifying the first position by having a specific positional relationship with the first position,
The method is characterized in that the first step is performed after the second step.
[0031]
A first step of supplying a liquid having a probe capable of specifically binding to a target substance to a first position on the substrate surface;
A second step of supplying a metal or metal compound to a second position capable of specifying the first position by having a specific positional relationship with the first position,
The method is characterized in that the first step and the second step are performed simultaneously.
[0032]
Further, the present invention provides a method for analyzing a probe array having a plurality of independent spots on a substrate, each of which has a probe that specifically binds to a target substance that may be contained in a measurement sample, the method comprising: A method for analyzing a probe array, comprising the step of specifying the position of a spot based on the position of a marker made of a metal or a metal compound arranged so as to specify the position of the spot.
[0033]
Further, a step of preparing a probe array having probes that specifically bind to the target substance as a plurality of independent spots on a substrate,
Contacting a sample with each spot of the probe array,
Detecting the presence or absence of a reaction product of the probe and the target substance in any spot,
Specifying the position of the spot where the reactant is present based on the position of the metal or metal compound on the substrate surface when the reactant is detected. It is.
[0034]
Further, the present invention has the following features.
[0035]
That is, a probe capable of specifically binding to a target substance, at least a surface of which is bonded to a surface of an insulating substrate, in a probe-bonded substrate,
A probe-bonded substrate having a conductive film disposed on at least the same surface as the probe.
[0036]
A method of manufacturing a probe-bonded substrate that can achieve the above object, a first step of supplying a liquid having the probe to the substrate surface,
And a second step of disposing the conductive film on the substrate surface,
The method is characterized in that the first step and the second step are performed simultaneously.
[0037]
Further, the analysis method of the present invention is a method for analyzing a probe array having a plurality of independent spots on an insulative substrate having probes that specifically bind to a target substance, wherein the surface of the substrate is electrically conductive. The method is characterized in that the probe is analyzed after disposing the film and electrically connecting the conductive film to the ground.
[0038]
According to such an invention, even when analyzing a probe-bonded substrate in which probes are arranged on a flat substrate at high density without having a well or the like on the surface, the spot position of each probe is quickly and accurately specified. be able to.
[0039]
Furthermore, when a probe is placed on an insulating substrate or a substrate on which an insulating film is formed on a conductive substrate, charge-up may occur even when irradiated with X-rays, electron beams, ions, or the like. Therefore, the analysis of each probe can be performed accurately and without any problem.
[0040]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, each embodiment of the present invention will be described in detail with reference to the drawings.
[0041]
[First Embodiment]
FIG. 1 is a plan view of a probe array in which a plurality of probes having different base sequences from each other are spot-like bonded on a substrate 101. Reference numeral 102 denotes a probe spot, and reference numeral 103 denotes a metal or metal compound spot which is located at a position where the position of the probe spot 102 can be specified. Specifically, it is arranged at a position corresponding to each row and each column of the spots 102 of the probes 102 arranged in a matrix, thereby enabling the position of the spot 102 to be specified.
[0042]
The substrate 101 is not particularly limited as long as a substance constituting a probe can be bound thereto and does not prevent detection of a target substance, but one example is a glass substrate. In addition, a silicon substrate, a metal substrate, a resin substrate, or the like, or each substrate having an appropriate surface treatment may be used. When a glass substrate is used as a substrate, a cleaning method and a surface treatment method can be used by various generally known methods, and there are advantages such that the substrate itself is easily available. It is suitable.
[0043]
The probe 102 used in the present invention specifically binds to a target substance, and may have a label for detecting the binding to the target substance as needed. Typical examples of the probe include single-stranded nucleic acid and protein antibodies. Examples of the single-stranded nucleic acid include single-stranded DNA, single-stranded RNA, and single-stranded PNA ( Peptide nucleic acid). As such a probe, a conventionally known probe may be appropriately selected and used according to the type of the labeling substance.
[0044]
The metal or the metal compound used as the spot 103 of the metal or the metal compound is not particularly limited, and any metal or metal compound that is usually used in a semiconductor process or the like can be used.
[0045]
Specific examples of the metal or metal compound that can be used in the present invention include metals such as Au, Ag, Cu, Ni, Co, Cr, Al, Ta, Pt, Pd, Zn, and Sn, and compounds thereof. Although it is mentioned, it is not particularly limited.
[0046]
Further, for example, by being colored when irradiated with visible light, the spot made of the metal or the metal compound can be detected by an optical microscope or a stereoscopic microscope, and thus is suitable as a marker indicating the position of the probe array spot 102. is there.
[0047]
Further, the metal or metal compound is in the form of a fine particle film, specifically, a fine particle film is one in which the diameter of the particles forming the film is sufficiently smaller than 1 μm, and more specifically, the average particle diameter is 100 nm or less. This makes it possible to uniformly form a metal or a metal compound as a thin film, so that a metal film having an arbitrary shape can be produced when a probe array is evaluated, which is preferable.
[0048]
The shape of these spots may be a marker by providing a single or a plurality of circular spots, or may be another shape by combining a plurality of spots. For example, an address display representing a row or a column of spots may be used. It is also possible.
[0049]
The probe 102 used in the present invention, and a spot 103 of a metal or a metal compound, respectively, by applying a liquid containing a probe, and a liquid containing a metal or a metal compound on the substrate using, for example, an inkjet method, It can be formed.
[0050]
The ink jet method is suitable because it can discharge a small amount of droplets (several pL to several tens nL) and can accurately discharge a minute probe. At present, practically used ink jet methods include an ink jet method using a piezo element and a thermal jet method using a thermal element, and any method may be used in the present invention.
[0051]
By the way, when the spot 103 of the metal or the metal compound is provided on the substrate by the ink jet method as described above, the liquid composition is adjusted so that the droplet does not unnecessarily spread on the substrate and stays at a predetermined position. Is preferred. When the ink jet method is used, since the droplet supplied to the substrate surface is minute, it is also effective to include a humectant in the liquid to be discharged in order to prevent the liquid from being evaporated and dried after the supply. . In particular, in the thermal jet method, the temperature rises at the time of ejection, so the presence of a moisturizing component and a surface tension adjusting component is important. As a solvent for supplying such a probe to the substrate surface, a solvent containing 5 to 10 wt% of urea, 5 to 10 wt% of glycerin, 5 to 10 wt% of thiodiglycol, and 1 wt% of acetylene alcohol is preferable. Can be used.
[0052]
As a method for preparing a solution containing a metal or a metal compound when the spot 103 of the metal or the metal compound is applied by an ink-jet method, any method can be used as long as it can discharge minute droplets stably and continuously. Although such a method may be used, it is preferable to form the film as a fine particle film after the film is formed. Therefore, for example, it is preferable that the film is prepared by dissolving an organometallic complex or a metal colloid in an appropriate solvent.
[0053]
When an organometallic complex is used, examples of the central metal include metals such as Pt, Pd, Ru, Au, Ag, Cu, Cr, Ta, Fe, W, Zn, and Sn. As the solvent, water or an organic solvent is used depending on the solubility of the organometallic complex. However, water is more preferable in terms of cost reduction and environmental protection, and a method of dissolving the same in water in a solvent containing the moisturizing component and the surface tension adjusting component is preferable.
[0054]
When a metal colloid is used, for example, a metal such as Pd, Au, Ag, or Pt, SnO2, or the like is used. As the solvent, water or an organic solvent is used depending on the method of producing the colloid. Examples of the organic solvent include hexane and pentaethylene glycol decyl ether.
[0055]
The formation of the spot 103 of the metal or the metal compound by the ink-jet method may be performed in the same step as the formation of the spot 102 of the probe by the ink-jet method, may be formed before the probe, or may be performed either.
[0056]
When the spot 103 of a metal or a metal compound is formed by an ink jet method prior to the probe, the spot 103 of the metal or the metal compound is already formed. Even so, it is possible to use water as the main solvent for the solution containing the metal or metal compound.
[0057]
Specifically, an aqueous solution of a specific organometallic complex or a metal colloid, or a solution obtained by adding an appropriate humectant or surface tension retaining agent to these aqueous solutions can be used.
[0058]
As the formation method, after spots 103 of a metal or a metal compound are discharged in advance, the spots 103 are formed by air drying or by heating the substrate and evaporating the solvent. Further, depending on the material of the substrate and the type of the organometallic complex, the substrate on which the spots 103 are formed is dried at a high temperature of 200 to 400 ° C. to remove the organic components in the complex and to be composed of fine particles of a metal or a metal compound. The spot 103 can be formed.
[0059]
Thereafter, in the same manner as the above-described simultaneous spot formation, a probe-containing spot 102 is formed by discharging a solution containing a probe and reacting the solution with the substrate.
[0060]
By using this formation method, more kinds of metals or metal compound substances can be used as the spots 103 for markers.
[0061]
When the spot 103 of the metal or the metal compound and the spot 102 of the probe are formed in the same step, the probe array may be reacted with the target substance after forming both spots, and then the washing may be performed. It is necessary to devise a solution containing the metal or the metal compound so that the spot 103 of the metal or the metal compound is not removed.
[0062]
As one method, the solvent of the solution containing the metal or the metal compound contains the metal or the metal compound during the binding time between the solution containing the probe and the substrate by reducing the humectant from the solution containing the probe. It is also effective to dry only the solvent of the solution so that the spot 103 is not removed during the washing. The amount of the humectant is appropriately selected depending on the type of the main solvent other than the humectant.
[0063]
When the spot 103 of the metal or the metal compound and the spot 102 of the probe are formed in the same step, the alignment between the row or column of the spot of the probe and the spot 103 of the metal or the metal compound can be omitted, and more quickly. Can be made.
[0064]
Further, since the spots of these metal or metal compound substances can be easily formed at an arbitrary position by using the above-described ink jet method, in-line analysis at the time of manufacturing or shipping. Effective for pre-test analysis.
[0065]
Hereinafter, the method for analyzing a probe-bound substrate of the present invention will be described.
[0066]
FIG. 2 shows another embodiment of the probe binding substrate of the present invention. 201 is a substrate, 202 is a probe array, and 203 is a metal or metal compound.
[0067]
One example of the analysis method of the present invention is a method of analyzing the probe-bonded substrate of the present invention with an X-ray photoelectron spectrometer. First, the probe-bonded substrate of the present invention is introduced into a chamber.
[0068]
At this time, in order to determine the measurement position on the substrate introduced into the chamber, the substrate is observed with the attached CCD camera. However, since the probe array 202 is very small and is colorless and transparent, only the probe array is disposed. In the case of the conventional probe-bonded substrate described above, it was very difficult to confirm the position of each probe array with a CCD. However, in the probe-bonded substrate of the present invention, since the metal or metal compound 203 exists at the same position in the X and Y axes as the probe array, the measurement position is determined by following the position of the metal or metal compound. Can be.
[0069]
Further, for example, when the insulator is a glass substrate or the like and the measurement area is a minute area of 1 mm or less, a monochromatic X-ray source is used as the X-ray source. It is necessary to attach a cover, but in this case also, it becomes easy to cover the metal cover at a desired probe position.
[0070]
FIG. 3 shows another embodiment of the probe binding substrate of the present invention. 301 is a Si substrate, 302 is a probe array, and 303 is a metal or metal compound.
[0071]
One example of the analysis method of the present invention is a method of analyzing the probe-bonded substrate of the present invention with a scanning electron microscope. First, the probe-bonded substrate of the present invention is introduced into a measurement chamber.
[0072]
At this time, in order to determine the measurement position on the substrate introduced into the chamber, the entire image is confirmed in the low magnification mode. However, the probe array has a very small film thickness and low secondary electron emission efficiency. Since it is an organic substance, it is very difficult to confirm the position of each probe array in the low-magnification mode in the case of the conventional probe-bonded substrate in which only the probe array is arranged, and it takes a long time. However, in the probe-bonded substrate of the present invention, since the metal or metal compound 303 exists at the same position on the X and Y axes as the probe array, the position of the metal or metal compound can be traced quickly and easily. Can be determined. Further, fine structures can be observed by conventional optical microscopy and fluorescence methods.
[0073]
The analysis method using the probe-bonded substrate of the present invention has been described using an X-ray photoelectron spectrometer and a scanning electron microscope as examples. However, the present invention is not limited thereto. For example, an electron beam microanalyzer, Auger electron spectroscopy, It is effective for an analysis method of a probe-bonded substrate using various surface analysis methods such as SIMS and an electron microscope.
[0074]
Further, in the above embodiment, the analysis of only the probe array was described. However, the present invention is not limited to only the check of the production of the probe array. It is also effective for detection.
[0075]
[Second embodiment]
FIG. 9 is a plan view of a probe array in which a plurality of probes each having a different base sequence from each other are spot-like bonded on an insulating substrate 901. Reference numeral 902 denotes a probe array spot, and 903 denotes a conductive film, which is electrically connected to a ground potential as shown in FIG.
[0076]
The substrate 901 is not particularly limited as long as it is an insulating substrate to which a substance constituting a probe can be bonded and does not hinder detection of a target substance, but a glass substrate is one example. Other substrates such as a resin substrate and the like which have been subjected to appropriate surface treatment may be used. Further, on a conductive substrate such as a silicon substrate or a metal substrate, for example, SiO.sub.2 is formed by sputtering, printing, thermal oxidation, or the like. ₂ The present invention is also applied to a substrate having an insulating film formed thereon.
[0077]
Among them, when a glass substrate is used as a substrate, the cleaning method and the surface treatment method can be various commonly known methods, and also have advantages such as easy availability of the substrate itself. Yes, it is preferred.
[0078]
The probe array spot 902 used in the present invention specifically binds to a target substance, and may have a label for detecting the binding to the target substance, if necessary. Typical examples of the probe include single-stranded nucleic acid and protein antibodies. Examples of the single-stranded nucleic acid include single-stranded DNA, single-stranded RNA, and single-stranded PNA ( Peptide nucleic acid). As such a probe, a conventionally known probe may be appropriately selected and used according to the type of the labeling substance.
[0079]
Examples of a material used for the conductive film 903 include a metal, a metal compound, and carbon. Specifically, the metal or metal compound that can be used in the present invention includes a conductive film 903 having a volume resistivity of 1 × 10 ⁷ It is sufficient if the material is Ωcm or less. ⁵ It is more preferable that the resistance is Ωcm or less. Examples thereof include metals such as Au, Ag, Cu, Ni, Co, Cr, Al, Ta, Pt, Pd, Zn, and Sn, and compounds thereof, but are not particularly limited. As long as it satisfies the above, a material usually used in a semiconductor processing step or the like can be used.
[0080]
By using a material that satisfies the above conditions as the conductive film 903, charge-up can be prevented as described later when analyzing the probe-bonded substrate on the insulator substrate.
[0081]
Further, the conductive film 903 only needs to be at least on the same surface as the probe. However, if the conductive film 903 is formed at a position in contact with the probe, the wettability changes, and it is necessary to change the probe manufacturing conditions for each conductive film. Therefore, it is preferable that the film be located at a position different from the position of the probe. For example, as shown in FIG. 9, it is preferable to form a film around the probe.
[0082]
Further, since it is preferable to cover the insulating surface around the probe with a conductive film as much as possible, it is preferable to cover the surface to which the probe is not coupled with the conductive film as shown in FIGS. However, the present invention is not limited to this. If there is a conductive film connected to the ground around the probe, the insulating surface may be exposed as shown in FIGS.
[0083]
Further, the form of the conductive film 903 may be a continuous film as shown in FIGS. 9 and 11, or may be a continuous film as shown in FIGS. May be formed.
[0084]
The method for forming the conductive film 903 used in the present invention is not particularly limited as long as the conductive film can be provided at a desired position. Examples of the method include a vacuum deposition method, a sputtering method, a printing method, and an inkjet method. Law and the like.
[0085]
When a film is formed over the entire surface of the substrate by a vacuum evaporation method, a sputtering method, or the like, for example, a metal mask or the like is covered on the substrate at the time of film formation, whereby a desired shape can be patterned.
[0086]
Further, it can also be formed by applying a liquid containing a metal or a metal compound to the substrate by using, for example, an inkjet method.
[0087]
The probe array spot 902 used in the present invention can be formed by applying a liquid containing a probe to the substrate by using, for example, an inkjet method.
[0088]
The ink jet method is suitable because it can discharge a small amount of droplets (several pL to several tens nL) and can accurately discharge a minute probe. At present, practically used ink jet methods include an ink jet method using a piezo element and a thermal jet method using a thermal element, and any method may be used in the present invention.
[0089]
In the case of manufacturing by an ink-jet method, the metal or metal compound serving as the material of the conductive film is in the form of a fine particle film, specifically, the fine particle film means that the diameter of the particles forming the film is more than 1 μm. When the probe array is evaluated, it is possible to form a metal or a metal compound uniformly as a thin film by being small, more specifically, having an average particle diameter of 100 nm or less. A film can be produced, which is preferable.
[0090]
FIG. 6 shows an example of a probe-bonded substrate manufactured by applying a liquid containing a metal or a metal compound to the above-described substrate by an inkjet method.
[0091]
In FIG. 6, the conductive film is formed such that the ends of the liquid spots 603 containing a metal or a metal compound overlap with each other. In the case where a conductive film is provided, it is preferable to adjust the liquid composition so that the droplet does not unnecessarily spread on the substrate and stays at a predetermined position. When the ink jet method is used, since the droplet supplied to the substrate surface is minute, it is also effective to include a humectant in the liquid to be discharged in order to prevent the liquid from being evaporated and dried after the supply. . In particular, in the thermal jet method, the temperature rises at the time of ejection, so the presence of a moisturizing component and a surface tension adjusting component is important. As a solvent for supplying such a probe to the substrate surface, a solvent containing 5 to 10 wt% of urea, 5 to 10 wt% of glycerin, 5 to 10 wt% of thiodiglycol, and 1 wt% of acetylene alcohol is preferable. Can be used.
[0092]
In addition, as a method for preparing a solution containing a metal or a metal compound when applying the liquid spot 603 containing a metal or a metal compound by an ink-jet method, a method capable of stably and continuously ejecting fine droplets is used. Any method may be used as long as it is formed. However, since it is preferable to form a film as a fine particle film after film formation, it is preferable to prepare the film by dissolving an organometallic complex or a metal colloid in an appropriate solvent.
[0093]
When an organometallic complex is used, examples of the central metal include metals such as Pt, Pd, Ru, Au, Ag, Cu, Cr, Ta, Fe, W, Zn, and Sn. As the solvent, water or an organic solvent is used depending on the solubility of the organometallic complex. However, water is more preferable in terms of cost reduction and environmental protection, and a method of dissolving the same in water in a solvent containing the moisturizing component and the surface tension adjusting component is preferable.
[0094]
When a metal colloid is used, for example, a metal such as Pd, Au, Ag, or Pt, SnO2, or the like is used. As the solvent, water or an organic solvent is used depending on the method of producing the colloid. Examples of the organic solvent include hexane and pentaethylene glycol decyl ether.
[0095]
The formation of the spot 603 of the liquid containing a metal or a metal compound by the ink-jet method may be performed in the same step as the formation of the spot 602 of the probe by the ink-jet method, may be formed before the probe, or may be either. It is.
[0096]
In the case where the formation of the liquid spot 603 containing a metal or a metal compound by the ink jet method is performed before the probe, the spot 603 of the liquid containing the metal or the metal compound is formed before the probe. Even if a washing step is performed later, water can be used as a main solvent for the solution containing the metal or the metal compound.
[0097]
Specifically, an aqueous solution of a specific organometallic complex or a metal colloid, or a solution obtained by adding an appropriate humectant or surface tension retaining agent to these aqueous solutions can be used.
[0098]
As a forming method, the spot 603 is formed by discharging a spot 603 of a liquid containing a metal or a metal compound in advance and then drying the spot naturally or by heating the substrate and evaporating the solvent. Further, depending on the material of the substrate and the type of the organometallic complex, the substrate on which the spots 603 are formed is dried at a high temperature of 200 to 400 ° C. to remove the organic components in the complex and to be composed of fine particles of a metal or a metal compound. A spot 603 can be formed.
[0099]
Thereafter, in the same manner as the above-described simultaneous spot formation, a probe-containing spot 602 is formed by discharging a solution containing a probe and reacting the solution with the substrate.
[0100]
By using this formation method, more kinds of metals or metal compound substances can be used as the marker spots 603.
[0101]
When the spot 603 of the liquid containing the metal or the metal compound and the spot 602 of the probe are formed in the same step, the probe array is allowed to react with the target substance after the formation of both spots, and then washing may be performed. Considering the above, it is necessary to devise a solution containing the metal or the metal compound so that the spot 603 of the metal or the metal compound is not removed during the cleaning.
[0102]
As one method, the solvent of the solution containing the metal or the metal compound contains the metal or the metal compound during the binding time between the solution containing the probe and the substrate by reducing the humectant from the solution containing the probe. It is also effective to dry only the solvent of the solution so that the spot 603 is not removed during washing. The amount of the humectant is appropriately selected depending on the type of the main solvent other than the humectant.
[0103]
Next, an embodiment of a probe-bound substrate with a marker will be described.
[0104]
The position of the probe can be easily detected by forming a conductive film by the inkjet method and also forming a marker indicating the position of the probe as shown in FIG. 8, for example. In FIG. 8, reference numeral 801 denotes an insulating substrate; 802, a probe array spot; 803, a conductive film formed by overlapping the ends of dots made of a conductive material by an inkjet method; and 804, a marker.
[0105]
The substrate, spot, and conductive film are manufactured in the same manner as described above. Although the ink jet method is used in FIG. 8 for the conductive film, the invention is not limited thereto, and the above-described sputtering method, printing method, or the like may be used.
[0106]
Further, while electrically connected to the conductive film 803, ejection for the marker 804 is performed. The marker is set so that the X and Y axes are the same as the position where the probe is to be bound. Any marker can be used as long as it can be visualized with an optical microscope, a stereoscopic microscope, a CCD camera, etc., which are often attached to the analyzer. Since it can be detected with a microscope or a stereo microscope, it is suitable as a marker indicating the position of the probe array spot 802.
[0107]
In addition, the conductive film 803 and the marker 804 may be formed using the same liquid or different liquids. There is an advantage that only one type of material is required if the same liquid is used.
[0108]
On the other hand, for example, when the measurement is performed by an X-ray photoelectron spectroscopy (XPS) apparatus, the material of the conductive film is made of aluminum and the marker is made of silver. On the other hand, the marker made of silver emits a large amount of photoelectrons, so that the position of the marker can be more easily recognized, which is preferable.
[0109]
When the conductive film 803 and the probe array spot 802 are formed in the same step, alignment between the row or column of the probe spots and the conductive film 803 can be omitted, and measurement can be performed more quickly.
[0110]
<Analysis method> Furthermore, since these conductive films can be easily formed into a metal film or a metal compound film in an arbitrary shape by using the above-described inkjet method, in-line analysis at the time of manufacturing or shipping. Effective for pre-test analysis.
[0111]
The method for analyzing a probe-bound substrate according to the present invention will be described below.
[0112]
One example of the analysis method of the present invention is a method of analyzing the probe-bonded substrate of the present invention with an X-ray photoelectron spectrometer. First, the probe-bonded substrate of the present invention is introduced into a chamber.
[0113]
Since the probe array spot has a very small diameter since many types of spots need to be arranged in the substrate, it is preferable to use a monochromatic X-ray source for X-rays. However, since the X-ray beam from the monochromatic X-ray source is thinner than the X-ray beam of the non-monochromatic X-ray source, the X-ray beam is irradiated on the probe-bonded substrate formed on the insulating substrate. Then, charge-up sometimes occurred. This is because the substrate surface is tilted positively by irradiating the substrate with an X-ray beam and emitting photoelectrons from the surface of the probe-coupled substrate.
[0114]
At this time, charge-up can be avoided by attaching a metal cover for preventing charge-up on the substrate. A metal cover is a thin metal plate with a hole made in a part, placed on an insulative substrate, and a part of the metal cover is brought into contact with the ground to accumulate in a part of the insulative substrate. It works to release the charge. However, if the metal cover is used, only the hole portion can be measured, but the portion other than the hole region cannot be measured. For this reason, in order to measure all the probes, it is necessary to take out the substrate from the chamber and shift the metal cover every time the measurement is performed, and the analysis efficiency is extremely deteriorated.
[0115]
However, as shown in FIG. 6, in the probe connection substrate of the present invention in which the dots of the metal or the metal compound 603 are arranged so that the end of each dot overlaps the adjacent dot, the dots must be grounded in advance. Accordingly, the dots of the metal or metal compound 603 have the same function as the metal cover, so that all the probes can be measured at once without moving the metal cover, and the analysis efficiency is improved.
[0116]
Next, another embodiment of the probe binding substrate of the present invention will be described with reference to FIG. 701 is an insulating substrate, 702 is a probe array, and 703 is a conductive film.
[0117]
One example of the analysis method of the present invention is a method of analyzing the probe-bonded substrate of the present invention with a scanning electron microscope. First, the probe-bonded substrate of the present invention is introduced into a measurement chamber.
[0118]
When a probe-bonded substrate fabricated on an insulating substrate such as a glass substrate was observed with a scanning electron microscope, the substrate surface was charged up at the time of observation and could not be observed. That is, when observing the sample form with a scanning electron microscope, if a part or all of the sample is non-conductive, charges are accumulated on the sample because the electron beam irradiation point and the ground are not connected, so-called Charge-up occurs, and the trajectory and image of the electron beam are disturbed, making normal observation difficult.
[0119]
However, in the probe coupling substrate of the present invention in which the conductive film 703 is arranged around the probe array spot 702 as shown in FIG. 7, by connecting a part of the conductive film 703 to the ground in advance, Electric charges generated on a part of the substrate surface can be released, and charge-up can be prevented. That is, a probe-bonded substrate capable of observing the form without charging up is obtained.
[0120]
Further, the case where the probe-attached substrate with a marker of the present invention is analyzed by the above scanning electron microscope will be described below. The probe-bonded substrate with a marker has the structure shown in FIG. 8, and details are as described above.
[0121]
When the substrate is introduced into the scanning electron microscope, the whole image is confirmed in a low magnification mode in order to determine a measurement position on the substrate first introduced into the chamber. At this time, the probe array spot is an organic substance having a very small film thickness and a low secondary electron emission efficiency and does not include a metal or the like having a high secondary electron emission efficiency. In the case of the probe-bonded substrate, it was almost impossible to confirm the position of each probe array in the low magnification mode. However, in the probe-bonded substrate of the present invention, since the metal or metal compound 803 exists at the same position on the X and Y axes as the probe array, the measurement position is determined by following the position of the metal or metal compound. Can be.
[0122]
The analysis method using the probe-bonded substrate of the present invention has been described by using a photoelectron spectroscopy apparatus and a scanning electron microscope as examples, but is not limited thereto. For example, an electron beam microanalyzer, Auger electron spectroscopy, SIMS, etc. It is effective for the method of analyzing a probe-bound substrate using various analysis methods.
[0123]
【Example】
Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited to the examples.
[0124]
<Example 1>
FIG. 4 is a view showing an example of the present invention, and is a plan view of a probe array in which a plurality of probes having different base sequences from each other are bonded on a spot on a quartz substrate 401.
[0125]
A method of dissolving in a solvent containing the moisturizing component and the surface tension adjusting component to produce the same is preferable. Reference numeral 402 denotes a probe spot. Reference numeral 403 denotes a spot of palladium oxide, which is arranged at a position where the position of the spot 402 of the probe can be specified. Specifically, it is arranged at a position corresponding to each row and each column of the spots 402 of the probes 402 arranged in a matrix, so that the position of the spot 402 can be specified.
[0126]
The method for producing the probe-bonded substrate is described below.
[0127]
(1) Substrate cleaning
A 1-inch square quartz substrate was placed in a rack, and the glass plate was immersed in a 1 mol / L (= 1N) sodium hydroxide solution that had been heated to 60 ° C. in advance for 10 minutes. Then, after rinsing with pure water, ultrasonic treatment was further performed for 10 minutes in a container containing pure water.
[0128]
(2) Surface treatment
1 wt% aqueous solution of a silane coupling agent (trade name: KBM603; manufactured by Shin-Etsu Chemical Co., Ltd.) containing a silane compound having an amino group bonded thereto (N-β- (aminoethyl) -γ-aminopropyltrimethoxysilane) Was left at room temperature for 20 minutes to complete the silane coupling treatment, and an amino group was introduced to the substrate surface. Thereafter, the substrate was washed with pure water and subjected to a drying process at 160 ° C. for 1 hour.
[0129]
Then, 2.7 mg of N-maleimidocaproyloxysuccinimide (N- (6-maleimidocaproyloxy) succinimide; manufactured by Dojin Co.) (hereinafter abbreviated as EMCS) was weighed, and 2.7 mg of dimethyl sulfoxide (DMSO) / ethanol was added. An EMCS solution dissolved to a final concentration of 0.3 mg / ml was prepared. The quartz substrate subjected to the silane coupling treatment was immersed in this EMCS solution at room temperature for 30 minutes, and the amino group carried on the quartz substrate surface and the carboxyl group of the EMCS solution were reacted by the silane coupling treatment. The quartz substrate pulled up from the EMCS solution was sequentially washed with a mixed solvent of DMSO and ethanol and ethanol, and then dried under a nitrogen gas atmosphere.
[0130]
(3) Preparation of metal-containing solution
Next, a metal-containing solution was prepared. 0.84 g of palladium acetate-monoethanolamine (hereinafter abbreviated as PA-ME) was dissolved in 12 g of water, and polyvinyl alcohol (hereinafter abbreviated as PVA) was added thereto. An aqueous solution for application was obtained. In addition, PA-ME was synthesized as follows.
[0131]
200 g of 10 g of palladium acetate ³ Of isopropyl alcohol (hereinafter abbreviated as IPA), 16.6 g of monoethanolamine was further added, and the mixture was stirred at room temperature for 4 hours. After completion of the reaction, IPA was removed from the evaporate, ethanol was added to the solid, dissolved and filtered, and PA-ME was recrystallized from the filtrate.
[0132]
(4) Discharge of metal compound by BJ printer and bonding to substrate
The bubble jet applying solution thus prepared was filled in an ink tank for a bubble jet printer (trade name: BJC620; manufactured by Canon Inc.) and set in a bubble jet head. The bubble jet printer (trade name: BJC620; manufactured by Canon Inc.) used here was modified so that printing on a flat plate was possible. Thereafter, a metal-containing solution was applied on the quartz substrate subjected to the surface treatment as shown in FIG. 1 and dried.
[0133]
This was heated to 300 ° C. in an oven in an air atmosphere to cause the PA-ME and PVA to decompose and deposit on the substrate to form a metal compound spot composed of fine palladium oxide particles.
[0134]
(5) Preparation of probe DNA
DNAs (SEQ ID NOS: 1 to 25) shown below (manufactured by Vex Inc.) are prepared, and a thiol (Thol-Modifier) (manufactured by GlenResearch) is used at the 5 ′ end of the DNA. (SH) group was introduced. Subsequently, the DNA was recovered by ordinary deprotection, purified by high performance liquid chromatography, and used in the following experiments.
[0135]
[Table 1]

[0136]
(6) DNA ejection by BJ printer and binding to substrate
The 25 kinds of DNAs were dissolved in a TE solution (10 mmol / L Tris-HCl (pH 8) / 1 mmol / L EDTA aqueous solution) so that the final concentration was about 400 mg / ml, and single-stranded DNA solutions were prepared respectively ( The exact concentration is calculated from the absorption intensity).
[0137]
An aqueous solution containing 7.5 wt% of glycerin, 7.5 wt% of urea, 7.5 wt% of thiodiglycol, and 1 wt% of acetylene alcohol (trade name: acetylenol EH; manufactured by Kawaken Fine Chemical Co., Ltd.) is prepared. In addition to the solution, the final concentration of the single-stranded DNA was adjusted to 8 μmol / L. The surface tension of this liquid is in the range of 30 to 50 mN / m (30 to 50 dyne / cm), and the viscosity is 1.8 mPa · s (= 1.8 cps) (E-type viscometer: Tokyo Keiki Co., Ltd.) Made). This liquid was filled in an ink tank for a bubble jet printer (trade name: BJC620; manufactured by Canon Inc.) and attached to a bubble jet head. The bubble jet printer (trade name: BJC620; manufactured by Canon Inc.) used here can be printed on a flat plate, and has been modified so that each of 25 types of DNA can be introduced. This printer can print at a resolution of 360 × 720 dpi. Next, the quartz substrate treated in the above (2) was mounted on the printer, and the liquid containing the probe nucleic acid was spotted on the quartz substrate. The number of ejected droplets is 15 × 15 = 225, 5 × 5 = 25 as one unit, each unit has a different type of DNA probe, and each unit has the same DNA sequence. Structured. At this time, the palladium oxide dots were aligned with each other so that the positions of the DNA probes and the palladium oxide dots formed in the above (4) did not shift. After the completion of the spotting, the quartz substrate was allowed to stand in a humidified chamber for 30 minutes to react a maleimide group on the surface of the quartz substrate with a thiol group at the end of the nucleic acid probe. The ejection amount of the DNA probe solution per ejection operation of the printer was about 24 pL.
[0138]
(7) Spot position inspection
The discharge position of the DNA probe spot prepared in the above (6) was confirmed with an optical microscope with reference to the position of the palladium oxide dot. It was confirmed that each DNA probe spot was ejected to a desired position without displacement. Further, in each spot, the spot diameter was about 70 μm and a uniform shape was obtained without unnecessarily widening the spot.
[0139]
(8) Blocking reaction
After the reaction between the maleimide group and the thiol group is completed, the quartz substrate is washed with a 1 mol / L NaCl / 50 mmol / L phosphate buffer (pH 7.0) solution, and a liquid containing excess DNA not bound to the quartz substrate is removed. Washed off. Next, the quartz substrate was immersed in a 2% bovine serum albumin aqueous solution and left for 2 hours to perform a blocking reaction.
[0140]
(9) Spot film formation inspection
After the blocking reaction, the probe-bound substrate was washed with pure water and observed with a low vacuum scanning electron microscope. The low vacuum scanning electron microscope used was XL30 ESEM manufactured by FEI, and the observation was performed with an acceleration voltage of 15 kV and a degree of vacuum of 80 Pa (= 0.6 Torr).
[0141]
As a result of the observation, it was confirmed that, even after the washing and blocking reactions were performed on each DNA probe spot, the DNA spot was bonded to a desired position as it was at the time of ejection. In addition, it was confirmed that the washing and the blocking reaction did not cause any trouble such as deformation due to chipping of a part of the spot, and recombination of DNA to a region other than a desired place.
[0142]
(10) Result
In a conventional probe-bonded substrate consisting only of DNA probe spots, it is very difficult to find the spot position because the DNA probe is made of an organic material having a very thin and low secondary electron emission efficiency. Similarly, it was very difficult to find a marker spot on a probe-bound substrate using a fluorescent dye as a marker.
[0143]
However, in the probe-bonded substrate of the present invention, as described above, a position at which a DNA probe spot can be specified, specifically, a palladium oxide compound dot having the same X and Y axes is provided, thereby enabling inspection during production. It was possible to quickly and surely confirm whether there was no damage to the spot, whether there was a satellite spot, and whether dust or the like was covering the spot, for all of the manufactured DNA probe substrates.
[0144]
In addition, an inspection at the time of production could be performed without performing the hybridization treatment. As a result, all products that passed the inspection could be shipped without wasting the product by the sampling inspection. This is effective for quality assurance.
[0145]
<Comparative Example 1>
Except that the metal compound dots made of palladium oxide were not provided, the same probe-bonded substrate as in Example 1 was produced. In this case, since the colorless and transparent DNA probe was placed on the colorless and transparent quartz substrate, it was difficult to find the probe position, and it was also difficult to identify the position even if spot damage was found. Therefore, it is necessary to clean all the heads of the bubble jet printer, resulting in a large production loss.
[0146]
<Example 2>
A probe-bonded substrate was manufactured in the same manner as in Example 1, except that the metal compound was changed from palladium oxide to silver. The same effect as in Example 1 was obtained with this probe-bonded substrate.
[0147]
<Example 3>
In the present example, the same probe-bonded substrate as in Example 1 was prepared except that the X and Y addresses 504 were provided as shown in FIG. 5 and the ejection amount of the DNA probe spot was set to about 100 pL.
[0148]
The X and Y addresses 504 are obtained by forming the column numbers in the X direction and the row numbers in the Y direction outside the metal compound dots in advance. This address 504 is formed by filling the solution used for printing the metal compound marker into another nozzle.
With this probe-bonded substrate, not only the same effect as in Example 1 was obtained, but also the spot address can be confirmed in advance by the CCD image and the SEM image of the low vacuum scanning electron microscope. Then, it was possible to quickly find out which probe nozzle had an abnormality.
[0149]
<Example 4>
In this example, using the method of Example 1, metal compound dots and DNA probes were formed on a silicon substrate, and analyzed using an X-ray photoelectron spectrometer.
[0150]
FIG. 4 is a view showing one example of the present invention, and is a plan view of a probe-bonded substrate in which a plurality of probes having different base sequences from each other are bonded on a spot on a silicon substrate 401. Reference numeral 402 denotes a probe spot. A silver spot 403 is located at a position where the position of the spot 402 of the probe can be specified. Specifically, it is arranged at a position corresponding to each row and each column of the spots 402 of the probes 402 arranged in a matrix, so that the position of the spot 402 can be specified. The set values of the probe and the silver spot diameter were each 1 mm.
[0151]
This probe-bonded substrate was prepared in the same manner as in Example 1 except that the material of the metal spot was changed to silver, and then the substrate was placed in a chamber for X-ray photoelectron spectroscopy, and the spot of each probe was analyzed. . X-ray photoelectron spectroscopy used was μ-ESCA 250i-XL manufactured by VG Scientific, and monochromatic Al X-rays were used as X-rays. The X-ray filament current was 18 mA and the voltage was 17 kV.
[0152]
Usually, in a probe-bonded substrate consisting of only DNA probe spots, the position of the DNA probe could not be confirmed in the lens barrel because the DNA probe was colorless and transparent and had a very thin film.
[0153]
However, in the probe-bonded substrate of the present invention in which the silver spot 403 is arranged at a position where the position of the spot 402 of the probe can be specified as shown in FIG. 4, the analysis position can be specified by the position of the silver spot. By performing analysis in accordance with the specific position, analysis of each spot can be performed in a short time.
[0154]
As a result, it was confirmed that each probe was manufactured at a desired position on the substrate without peeling or ejection failure. That is, it was confirmed that a sufficiently reliable probe array for inspection was formed.
[0155]
<Example 5>
In this example, a palladium oxide spot and a protein antibody were prepared on a quartz substrate by an inkjet method, and analyzed by ToF-SIMS.
[0156]
FIG. 1 is a view showing an example of the present invention, in which antibodies against different proteins are arranged on a quartz substrate. After producing this substrate by the same manufacturing method as in Example 1, the substrate was placed in a chamber of ToF-SIMS, and the spot of each probe was analyzed. The ToF-SIMS used was ToF-SIMS IV manufactured by ION-TOF, and the emitter used was Ga. The acceleration voltage was 25 kV and the sample current was 2.4 pA.
[0157]
In a normal antibody-bound substrate, since the probe is transparent and thin, the position cannot be confirmed by a CCD camera or the like. Therefore, it is difficult to find the probe position and to specify the spot position. However, in the substrate of the present invention, since the analysis position can be specified by the position of the palladium oxide spot, each spot could be analyzed in a short time. As a result, it was confirmed that each probe was formed as designed without falling off.
[0158]
<Example 6>
In this example, palladium oxide dots and DNA probes were formed on a silicon substrate using the method of Example 1, and observation was performed with a normal high vacuum type scanning electron microscope.
[0159]
FIG. 4 is a view showing an example of the present invention. FIG. 4 is a plan view of a probe-bonded substrate in which a plurality of probes having different base sequences are bonded on a spot on a silicon base 401. 402 is a spot of the probe. Is a spot of palladium oxide, which is arranged at a position where the position of the spot 402 of the probe can be specified. Specifically, it is arranged at a position corresponding to each row and each column of the spots 402 of the probes 402 arranged in a matrix, so that the position of the spot 402 can be specified. The set values of the probe and the silver spot diameter were each 70 μm. The scanning electron microscope used Hitachi S-5000H and observed at an acceleration voltage of 1 kV.
[0160]
After producing this probe-bonded substrate by the same manufacturing method as in Example 1, the substrate was placed in a lens barrel of a scanning electron microscope, and the spots of each probe were observed in detail. Usually, since the DNA probe spot is colorless and transparent and has a very thin film, it is very difficult to confirm the position in the lens barrel.
[0161]
However, in the probe-bonded substrate of the present invention in which the palladium oxide spot 403 is arranged at a position where the position of the probe spot 402 can be specified as shown in FIG. 4, the approximate observation position is determined by the position of the palladium oxide spot. Since the specific position can be specified, it is possible to obtain a good image in a short time by observing the specific position in detail.
[0162]
As a result, it was confirmed that each probe was uniformly manufactured with a diameter of about 70 μm as set. Furthermore, it was possible to confirm a finer structure than with an optical microscope, and it was confirmed that a sufficiently reliable probe array for inspection was formed.
[0163]
<Example 7>
FIG. 7 is a view showing an example of the present invention, and is a plan view of a probe array in which probes are bonded on spots on a quartz substrate 701. FIG. Reference numeral 702 denotes a probe spot. Reference numeral 703 denotes a conductive film made of palladium oxide, which is electrically connected to an external ground electrode.
[0164]
The method for producing the probe-bonded substrate is described below.
[0165]
First, a nucleic acid probe array was prepared according to the method described in JP-A-11-187900 (Patent Document 4).
[0166]
(1) Substrate cleaning
A synthetic quartz substrate of 25.4 mm × 25.4 mm × 1 mm was put in a rack and immersed overnight in an ultrasonic cleaning detergent (Branson: GPIII) diluted to 10% with pure water. Thereafter, ultrasonic cleaning was performed in a detergent for 20 minutes, and then the detergent was removed by washing with water. After rinsing with pure water, ultrasonic treatment was further performed for 20 minutes in a container containing pure water. Next, the substrate was immersed in a 1 mol / L (1N) aqueous solution of sodium hydroxide preheated to 80 ° C. for 10 minutes. Subsequently, washing with water and washing with pure water were performed, and the resultant was directly used in the next step.
[0167]
(2) Surface treatment
A 1 wt% aqueous solution of an amino group-bonded silane coupling agent, N-β- (aminoethyl) -γ-aminopropyltrimethoxysilane KBM603 (Shin-Etsu Chemical Co., Ltd.) is stirred at room temperature for 2 hours, and the molecular weight of the silane compound is reduced. The methoxy group in was hydrolyzed. Next, the substrate obtained in the above (1) was immersed in this solution at room temperature for 1 hour, washed with pure water, and dried by spraying nitrogen gas on both sides of the substrate. Next, the substrate was baked for 1 hour in an oven heated to 120 ° C. to finally introduce amino groups on the substrate surface.
[0168]
Next, 2.7 mg of N-maleimidocaproyloxysuccinimide (Dojindo Laboratories: EMCS) was dissolved in a 1: 1 solution of dimethylsulfoxide (DMSO) / ethanol to a concentration of 0.3 mg / ml. The quartz substrate subjected to the silane coupling treatment was immersed in this EMCS solution at room temperature for 2 hours, so that the amino group carried on the substrate surface and the succinimide group of the EMCS solution were reacted by the silane coupling treatment. At this stage, a maleimide group derived from EMCS exists on the substrate surface. The substrate pulled up from the EMCS solution was sequentially washed with a mixed solvent of DMSO and ethanol and ethanol, and then dried by blowing nitrogen gas.
[0169]
(3) Preparation of metal-containing solution
Next, a metal-containing solution was prepared. 0.84 g of palladium acetate-monoethanolamine (hereinafter abbreviated as PA-ME) is dissolved in 12 g of water, and polyvinyl alcohol (hereinafter abbreviated as PVA) is further added to adjust the solution viscosity to 20 mPa · s (= 20 centipoise). The resulting solution was used as an aqueous solution for applying a bubble jet. In addition, PA-ME was synthesized as follows.
[0170]
200 g of 10 g of palladium acetate ³ Of isopropyl alcohol (hereinafter abbreviated as IPA), 16.6 g of monoethanolamine was further added, and the mixture was stirred at room temperature for 4 hours. After completion of the reaction, IPA was removed from the evaporate, ethanol was added to the solid, dissolved and filtered, and PA-ME was recrystallized from the filtrate.
[0171]
(4) Discharge of metal compound by BJ printer and bonding to substrate
The bubble jet applying solution thus prepared was filled in an ink tank for a bubble jet printer (trade name: BJC620; manufactured by Canon Inc.) and set in a bubble jet head. The bubble jet printer (trade name: BJC620; manufactured by Canon Inc.) used here was modified so that printing on a flat plate was possible. Thereafter, a metal-containing solution was applied to the surface-treated quartz substrate as shown in FIG. 6 and dried.
[0172]
This was heated to 300 ° C. in an oven in an air atmosphere to cause the PA-ME and PVA to decompose and deposit on the substrate to form a metal compound spot composed of fine palladium oxide particles.
[0173]
(5) Synthesis of probe DNA
A single-stranded nucleic acid of SEQ ID NO: 26 (40-mer of T) was synthesized at the request of a DNA synthesizer (Vex). A thiol (SH) group was introduced into the 5 ′ end of the single-stranded DNA of SEQ ID NO: 26 by using a thiol modifier (Glen Research) during synthesis. In addition, deprotection and recovery of DNA were performed by a conventional method, and HPLC was used for purification. A series of steps from synthesis to purification were all performed by a synthesis company.
SEQ ID NO: 26
5 'HS- (CH ₂ ) ₆ -O-PO ₂ -O-TTTTTTTTTTT TTTTTTTTTTT TTTTTTTTTTT TTTTTTTTTTT 3 '
(6) DNA ejection by thermal jet printer and bonding to substrate
The single-stranded DNA of SEQ ID NO: 26 was used at a concentration of 8 μmol / L at 7.5% by weight of glycerin, 7.5% by weight of urea, 7.5% by weight of thiodiglycol, and acetylene alcohol represented by the general formula (I) (commercially available). Name: acetylenol EH; 1% by weight of Kawaken Fine Chemical Co., Ltd.) was dissolved. A printer head BC-50 (Canon) for a bubble jet printer BJF-850 (Canon) using a bubble jet method, which is a kind of the thermal jet method, was modified so that several hundred μL of a solution can be discharged. It was mounted on a discharge drawing machine that was modified to discharge onto a quartz substrate. Several hundred μL of the above DNA solution was injected into the modified tank portion of the head, and an EMCS substrate was mounted on a discharge drawing machine, and spotting was performed here. In addition, the discharge amount at the time of spotting was 4 pL / droplet, and the spotting range was 200 dpi, that is, the pitch was 127 μm, in a range of 10 mm × 10 mm in the center of the substrate. Under these conditions, the spotted dots had a diameter of about 50 μm.
[0174]
After the completion of the spotting, the substrate was allowed to stand in a humidified chamber for 30 minutes to react the maleimide group on the surface of the glass plate with the thiol group at the terminal of the nucleic acid probe. Next, the substrate was washed with pure water and stored in pure water.
[0175]
(7) Spot film formation inspection
The stored DNA-bonded substrate (DNA chip) was dried by blowing nitrogen gas immediately before observation with a scanning electron microscope, and further dried in a vacuum desiccator. After drying, the sample is adhered to an aluminum sample base with a carbon paste, and the end of the conductive film 603 on the sample surface and the sample base are similarly connected with the carbon paste, so that the conductive film is grounded. Electrically connected. The scanning electron microscope was S-5000H manufactured by Hitachi, Ltd., and the observation was performed with an acceleration voltage of 5 kV.
[0176]
(8) Result
As a result of the observation, even at an acceleration voltage of 5 kV, observation could be performed without causing charge-up. In addition, it was confirmed that the DNA spots were bonded to desired positions as in the case of ejection even after the washing and blocking reactions were performed on each DNA probe spot. In addition, it was confirmed that the washing and the blocking reaction did not cause any trouble such as deformation due to chipping of a part of the spot, and recombination of DNA to a region other than a desired place.
[0177]
In the probe-bonded substrate of the present invention, as described above, by adopting a form in which the conductive film is disposed around, whether or not spots are damaged in the inspection at the time of manufacture, whether or not there are satellite spots, dust, etc. It was possible to quickly and surely confirm whether or not the sample covered the spot for all of the manufactured DNA probe substrates.
[0178]
In addition, an inspection at the time of production could be performed without performing the hybridization treatment. As a result, all products that passed the inspection could be shipped without wasting the product by the sampling inspection. This is effective for quality assurance.
[0179]
<Comparative Example 2>
Except that the conductive film made of palladium oxide was not provided, a probe-bonded substrate was manufactured in exactly the same manner as in Example 7, and observation was performed with a scanning electron microscope in the same manner as in Example 7. First, observation was performed at an accelerating voltage of 5 kV in the same manner as in Example 7. Since the DNA probe was arranged on an insulating quartz substrate, charge-up occurred due to electron beam irradiation, and a secondary electron image was not obtained. . Therefore, the probe-coupled substrate was once taken out of the lens barrel, introduced again into the lens barrel, and observed with the acceleration voltage lowered to an acceleration voltage of 1 kV. However, a detailed inspection of the probe could not be performed.
[0180]
Example 8
A probe-bonded substrate was produced in the same manner as in Example 7, except that the metal compound was changed from palladium oxide to silver. The same effect as in Example 7 was obtained with this probe-bonded substrate.
[0181]
<Example 9>
After forming the conductive film using a sputtering method as shown in FIG. 9, a probe was manufactured. In FIG. 9, reference numeral 901 denotes a blue plate substrate; 902, a probe array spot; and 903, a conductive film. Except for the conductive film, the manufacturing method was the same as in Example 7. Using this probe-bonded substrate, scanning electron microscopy was performed in the same manner as in Example 7, and the same effects as in Example 7 were obtained.
[0182]
<Example 10>
As shown in FIG. 8, a probe was manufactured by providing a marker electrically connected to the conductive film. 8, reference numeral 801 denotes a blue plate substrate; 802, a probe array spot; 803, a conductive film; and 804, a marker. As the conductive film 803, a continuous film was formed by an ink-jet method with a circular palladium oxide film having an overlapped end. Furthermore, the tip was connected to the ground, and electrically dropped to the ground. The marker 804 is a spot of palladium oxide, and is arranged at a position where the position of the spot 802 of the probe can be specified. Specifically, the probe spots 802 are arranged at positions corresponding to each row and each column of the probe spots 802 arranged in a matrix, so that the position of the spot 802 can be specified.
[0183]
Palladium oxide was formed by an ink-jet method, and the production method was the same as in Example 7, except for the marker.
[0184]
Using this probe-bonded substrate, scanning electron microscopy was performed in the same manner as in Example 7, and the same effects as in Example 7 were obtained.
[0185]
Further, the DNA probe spot is usually colorless and transparent, is a very thin film, and has a low secondary electron emission efficiency. Therefore, it is very difficult to confirm the position in the lens barrel. However, in the probe-bonded substrate of the present invention in which the palladium oxide spot 803 is located at a position where the position of the probe spot 802 can be specified as shown in FIG. 8, the position of the probe array spot 802 is referred to with reference to the position of the marker. Can reduce the time required to find the position of the probe array spot with low secondary electron emission efficiency, and by observing this specific position in detail, a good image can be obtained in a short time. And the analysis efficiency was improved.
[0186]
As a result, it was confirmed that each probe was formed uniformly with a diameter of about 70 μm. Furthermore, it was possible to confirm a finer structure than with an optical microscope, and it was confirmed that a sufficiently reliable probe array for inspection was formed.
[0187]
<Example 11>
In this example, using the method of Example 7, metal compound dots and DNA probes were formed on a quartz substrate, and analyzed using an X-ray photoelectron spectrometer.
[0188]
FIG. 12 shows an example of the present invention, and is a plan view of a probe-bonded substrate in which a plurality of probes having different base sequences from each other are bonded on a spot on a quartz substrate 1201. Reference numeral 1202 denotes a probe spot. Reference numeral 1203 denotes a silver spot, which is arranged at a position where the position of the spot 1202 of the probe can be specified. Specifically, they are arranged at positions corresponding to each row and each column of the spots 1202 of the probes arranged in a matrix, so that the positions of the spots 1202 can be specified. The set values of the probe and the silver spot diameter were each 1 mm.
[0189]
This probe-bonded substrate was prepared in the same manner as in Example 7 except that the material of the metal spot was changed to silver, and then the substrate was placed in an X-ray photoelectron spectroscopy chamber, and the spot of each probe was analyzed. . X-ray photoelectron spectroscopy used was μ-ESCA 250i-XL manufactured by VG Scientific, and monochromatic Al X-rays were used as X-rays. The X-ray filament current was 18 mA and the voltage was 17 kV.
[0190]
Usually, in X-ray photoelectron spectroscopy measurement of a probe-bound substrate consisting of only DNA probe spots, a metal mask is placed on the sample to prevent charge-up during measurement, and a neutralizing electron gun is used at the same time.
[0191]
At that time, in order to analyze a place other than the place where the hole of the metal mask is opened, the metal mask had to be gradually shifted according to the position of the probe array.
[0192]
However, in the probe-bonded array of the present invention shown in FIG. 11, since the conductive film plays the same role as the metal mask, the measurement could be performed without charge-up.
[0193]
As a result, it was confirmed that each probe was manufactured at a desired position on the substrate without peeling or ejection failure. That is, it was confirmed that a sufficiently reliable probe array for inspection was formed.
[0194]
【The invention's effect】
As described above, according to the present invention, it is possible to mark a probe-bonded substrate effective for various surface analyses. Further, by using the present invention, analysis for product inspection can be performed without performing a binding reaction with a target substance.
[0195]
[Sequence list]

[Brief description of the drawings]
FIG. 1 is a diagram showing an example of an embodiment of the present invention, Example 5.
FIG. 2 is a diagram showing another embodiment of the present invention.
FIG. 3 is a diagram showing another embodiment of the present invention.
FIG. 4 is a diagram showing an embodiment of the present invention.
FIG. 5 is a diagram showing a third embodiment of the present invention.
FIG. 6 is a diagram showing another embodiment of the present invention.
FIG. 7 is a diagram illustrating an example of an embodiment of the present invention and examples 7 and 8.
FIG. 8 is a diagram for explaining a tenth embodiment of the present invention.
FIG. 9 is a diagram illustrating an example of an embodiment of the present invention and a ninth embodiment.
FIG. 10 is a diagram illustrating another example of the embodiment of the present invention.
FIG. 11 is a diagram illustrating another embodiment of the present invention.
FIG. 12 is a diagram illustrating an eleventh embodiment of the present invention.
[Explanation of symbols]
101, 201, 301, 401, 501, 601, 701, 801, 901, 1001, 1101, 1201: substrate
102, 202, 302, 402, 502, 602, 702, 802, 902, 1002, 1102, 1202: DNA probe
103, 203, 303, 403, 503, 603, 703, 803, 903, 1003, 1103, 1203: Probe made of metal or metal compound
504: X, Y address
804: Marker

Claims (12)

A probe-binding substrate in which a probe capable of specifically binding to a target substance is bound to a surface of the substrate,
The probe is bonded to a first position on the surface, and a metal or a metal compound is arranged at a second position that can specify the first position by having a specific positional relationship with the first position. A probe-bonded substrate, characterized in that: The probe-bonded substrate according to claim 1, wherein the metal or metal compound is colored. The probe-bonded substrate according to claim 1, wherein the metal or the metal compound is a fine particle film. A method for manufacturing a probe-bonded substrate, comprising:
A first step of supplying a liquid having a probe capable of specifically binding to a target substance to a first position on a substrate surface;
A second step of disposing a metal or metal compound at a second position capable of specifying the first position by having a specific positional relationship with the first position,
A method for manufacturing a probe-bonded substrate, wherein the first step is performed after the second step. A method for manufacturing a probe-bonded substrate, comprising:
A first step of supplying a liquid having a probe capable of specifically binding to a target substance to a first position on a substrate surface;
A second step of supplying a metal or metal compound to a second position capable of specifying the first position by having a specific positional relationship with the first position,
A method for manufacturing a probe-bonded substrate, wherein the first step and the second step are performed simultaneously. The method according to claim 4, wherein the supply of the metal or the metal compound to the substrate surface is performed by discharging a liquid containing the metal or the metal compound by an inkjet method. A method for analyzing a probe array having, as a plurality of spots independent of each other, a probe that specifically binds to a target substance that may be contained in a measurement sample, comprising: A method for analyzing a probe array, comprising the step of specifying the position of a spot based on the position of a marker made of a metal or a metal compound arranged so as to specify the position of the spot. The method for analyzing a probe array according to claim 10, wherein an analyzer used for the analysis of the probe array is a scanning electron microscope. A method for detecting a target substance,
A step of preparing a probe array having probes that specifically bind to the target substance as a plurality of independent spots on a substrate,
Contacting a sample with each spot of the probe array,
Detecting the presence or absence of a reaction product of the probe and the target substance in any spot,
Specifying the position of the spot where the reactant is present based on the position of the metal or metal compound on the substrate surface when the reactant is detected. . A probe capable of specifically binding to a target substance, at least a surface of which is bonded to a surface of an insulating substrate, in a probe-bonded substrate,
A probe-bonded substrate, wherein a conductive film is disposed at least on the same surface as the probe. A method for producing a probe binding substrate having a probe capable of specifically binding to a target substance,
A first step of supplying a liquid having the probe to the substrate surface,
And a second step of disposing the conductive film on the substrate surface,
A method for manufacturing a probe-bonded substrate, wherein the first step and the second step are performed simultaneously. A method for analyzing a probe array having a probe specifically binding to a target substance as a plurality of independent spots on an insulating substrate, wherein a conductive film is disposed on the substrate surface, and the conductive film Analyzing the probe after electrically connecting the probe to ground.

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