JP4502167B2 - Microarray chip - Google Patents

Description

【００５８】
ただし、ｎは２以上の整数であり、Ｒ^１，Ｒ^２はそれぞれ炭素数１〜１０の置換もしくは非置換のアルキル、アルケニル、アリールあるいはシアノアルキル基であり、モル比で全体の４０％以下がビニル、フェニル、ハロゲン化フェニルである。また、Ｒ^１、Ｒ^２がメチル基のものが表面エネルギーが最も小さくなるので好ましく、モル比でメチル基が６０％以上であることが好ましい。また、鎖末端もしくは側鎖には、分子鎖中に少なくとも１個以上の水酸基等の反応性基を有する。
【００５９】
また、上記のオルガノポリシロキサンとともに、ジメチルポリシロキサンのような架橋反応をしない安定なオルガノシリコン化合物をバインダに混合してもよい。
【００６０】
本発明において光触媒含有層には上記の光触媒、バインダの他に、界面活性剤を含有させることができる。具体的には、日光ケミカルズ（株）製ＮＩＫＫＯＬＢＬ、ＢＣ、ＢＯ、ＢＢの各シリーズ等の炭化水素系、デュポン社製ＺＯＮＹＬ ＦＳＮ、ＦＳＯ、旭硝子（株）製サーフロンＳ−１４１、１４５、大日本インキ化学工業（株）製メガファックＦ−１４１、１４４、ネオス（株）製フタージェントＦ−２００、Ｆ２５１、ダイキン工業（株）製ユニダインＤＳ−４０１、４０２、スリーエム（株）製フロラードＦＣ−１７０、１７６等のフッ素系あるいはシリコーン系の非イオン界面活性剤を挙げることかでき、また、カチオン系界面活性剤、アニオン系界面活性剤、両性界面活性剤を用いることもできる。
【００６１】
また、光触媒含有層には上記の界面活性剤の他にも、ポリビニルアルコール、不飽和ポリエステル、アクリル樹脂、ポリエチレン、ジアリルフタレート、エチレンプロピレンジエンモノマー、エポキシ樹脂、フェノール樹脂、ポリウレタン、メラミン樹脂、ポリカーボネート、ポリ塩化ビニル、ポリアミド、ポリイミド、スチレンブタジエンゴム、クロロプレンゴム、ポリプロピレン、ポリブチレン、ポリスチレン、ポリ酢酸ビニル、ポリエステル、ポリブタジエン、ポリベンズイミダゾール、ポリアクリルニトリル、エピクロルヒドリン、ポリサルファイド、ポリイソプレン等のオリゴマー、ポリマー等を含有させることができる。
【００６２】
光触媒含有層中の光触媒の含有量は、５〜６０重量％、好ましくは２０〜４０重量％の範囲で設定することができる。また、光触媒含有層の厚みは、０．０５〜１０μｍの範囲内が好ましい。
【００６３】
上記光触媒含有層は、光触媒とバインダを必要に応じて他の添加剤とともに溶剤中に分散して塗布液を調製し、この塗布液を塗布することにより形成することができる。使用する溶剤としては、エタノール、イソプロパノール等のアルコール系の有機溶剤が好ましい。塗布はスピンコート、スプレーコート、ディッブコート、ロールコート、ビードコート等の公知の塗布方法により行うことができる。バインダとして紫外線硬化型の成分を含有している場合、紫外線を照射して硬化処理を行うことにより光触媒含有層を形成することかできる。
【００６４】
（濡れ性可変層上の親水性領域のパターン）
上記濡れ性可変層上における親水性領域のパターンの配置は、標識に由来する信号を検出する際に、親水性領域の位置（すなわち、その上に形成される固定化層および特異性生体高分子の位置）が確認可能である配置である限り、特に限定されるものではなく、例えば、分析目的に応じて、あるいは、標識に由来する信号を検出するのに用いる分析装置に応じて、適宜決定することができる。
【００６５】
このような親水性領域のパターンは、検出が容易である点に鑑みれば整列して配置されていることが好ましい。また、親水性領域の複数部分を、所定の間隔をあけて線状に一列に配置し、さらに、そのような列を相互に平行に複数列配置したパターンであることがより好ましい。このように親水性領域を配置したパターンであれば、特異性生体高分子と結合する高分子が有する標識に由来する信号の検出工程を自動化することが容易である。
【００６６】
上記濡れ性可変層上の親水性領域の間隔は、特異性生体高分子と結合する高分子が有する標識標識に由来する信号を検出する際に、隣接する親水性領域の影響を受けない程度に分離している限り、特に限定されるものではなく、例えば、隣接する親水性領域の縁部間の最短距離が、０．０１μｍ〜１ｃｍ、好ましくは０．０５μｍ〜５ｍｍの範囲内であることが好ましい。また、濡れ性可変層上の親水性領域の数も、特に限定されるものではなく、基材の大きさにも依存するが、例えば、数個〜数万個（一般には数十〜１乃至２万個）としてもよい。
【００６７】
濡れ性可変層が光触媒含有層である場合の、上記親水性領域のパターンの形成方法は、フォトマスクを用いたエネルギーのパターン照射により行うことが可能である。また、後述するように基材に遮光部が形成されているような場合は、光触媒含有層が形成されていない面側からの全面露光（エネルギー照射）を行なうようにしてもよい。
【００６８】
３．固定化層
本発明においては、上述した濡れ性可変層上にパターン状に形成された親水性領域上に、後述する特異性生体高分子を固定化するための固定化層が形成される。
【００６９】
この固定化層に用いられる固定化剤としては、特異性生体高分子の固定化手段に応じて種々のものを用いることができる。
【００７０】
このような固定化手段としては、当業者において周知の結合方法を使用することができ、例えば、静電結合による固定化方法、共有結合による固定化方法等を使用することができる。
【００７１】
上記静電結合による固定化手段の場合は、特異性生体高分子が有する電荷と反対の電荷を有する物質を固定化剤として溶解した溶液を固定化層形成用塗工液として調製し、これを上記親水性領域がパターン状に形成された濡れ性可変層上に塗布することにより固定化層を形成する。
【００７２】
例えば、特異性生体高分子がオリゴヌクレオチドの場合は、オリゴヌクレオチドが陰イオン（アニオン）性を有するものであるので、陽イオン（カチオン）性を有する物質を固定化剤とした塗工液が用いられ、これを塗布して乾燥することにより固定化層が形成される。
【００７３】
この際、用いられる陽イオン性を有する固定化剤としては、ポリＬ‐リシン、ポリ（アリルアミンヒドロクロリド）、ポリ（エチレンイミン）、ポリ（ジアリルジメチルアンモニウムクロリド）等を挙げることができ、中でもポリＬ‐リシンが好ましい。
【００７４】
一方、共有結合（化学結合）による固定化法としては、官能基（例えば、カルボキシル基、アミノ基、又は水酸基）を有する化合物を固定化剤として固定化剤形成用塗料に用いて固定化層を形成し、これに特異性生体高分子を結合させる方法を挙げることができる。このような固定化剤としては、官能基としてカルボキシル基又は第一級アミノ基をもつ化合物を挙げることができる。
【００７５】
具体的には、アミノ基を含むシランカップリング剤を、濡れ性可変層上にパターン状に形成された親水性領域に付着させる。これは、上記シランカップリング剤を含む溶液を用い、ディップコーティング法、転写法、スピンコーティング法等により全面に塗布し、親水性領域のみにシランカップリング剤を付着させる方法、インクジェット、ディスペンサ等を用いて親水性領域のみを狙って塗布する方法を挙げることができる。コーティングされたアミノ基を含むシランカップリング剤は、親水性領域に濡れ広がり、撥水性領域に残留することはない。コーティング後、必要に応じて加熱することにより、親水性領域と固定化剤とを結合させることも可能である。そして、このようにして形成した固定化層上に特異性生体高分子を結合させることにより、マイクロアレイチップを形成することができる。
【００７６】
上記固定化剤であるシランカップリング剤のアミノ基と、末端をアミノ基で修飾した生体高分子、具体的にはオリゴヌクレオチド等の末端アミノ基とを共有結合させることにより強固に膜に固定化することができる。
【００７７】
この際、用いられるアミノ基を有するシランカップリング剤としては、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、Ｎ−β（アミノエチル）γ−アミノプロピルメチルジメトキシシラン、γ−アミノプロピルトリエトキシシラン、Ｎ−フェニル−γ−アミノプロピルトリメトキシシラン等を挙げることができる。
【００７８】
また、固定化層と特異性生体高分子との共有結合による別の固定化法としては、例えば、生体高分子がオリゴヌクレオチドの場合、その５'末端を上記官能基に共有結合で結合させる方法が知られている。具体的には、まず、オリゴヌクレオチドの５'末端とマレイミド化合物とを反応させ、オリゴヌクレオチドの５'末端にマレイミド基を導入する。好適なマレイミド化合物としては、スルホスクシンイミジル−４−（Ｎ−マレイミドメチル）シクロヘキサン−１−カルボキシレート等を挙げることができる。上記反応とは別に、アミノ基を官能基として有する固定化剤とスクシンイミジル−Ｓ−アセチルチオアセテートとを反応させた後に、ヒドロキシラミンを用いて脱アセチル化を行なうことにより、上記固定化剤にＳＨ基を付与する。こうして付与した固定化剤上のＳＨ基と、５'末端にマレイミド基を導入して調製した上記オリゴヌクレオチドの５'末端に導入したマレイミド基とを反応させることにより、オリゴヌクレオチドと固定化剤とを結合させることができ、特異性生体高分子を固定化層に固定することができるのである。
【００７９】
その他、上述した方法の他にも、多くの固定化方法、例えば、ビオチン−アビジン系を用いる方法も使用することができる。
【００８０】
本発明に用いられる固定化層は、後述するように、その上に特異性生体高分子を含む液体を付着させた際に、この液体が固定化層より広がらないことが好ましい。したがって、固定化層の表面の濡れ性が、周囲の濡れ性可変層の領域（撥水性領域）より、水との接触角で１度以上、好ましくは５度以上小さくなるような固定化剤が選択されて用いられることが好ましい。
【００８１】
４．特異性生体高分子
本発明のマイクロアレイチップは、上記固定化層上に特異性生体高分子が固定化されて配置される。このような特異性生体高分子としては、所定の生体高分子と特異的に結合することができる生体高分子であれば特に限定されるものではない。
【００８２】
このような特異的に結合する場合としては、例えば、核酸（例えば、オリゴヌクレオチド又はポリヌクレオチド）とそれに相補的な核酸との組み合わせ、抗原と抗体（又は抗体フラグメント）との組み合わせ、受容体とそのリガンド（例えば、ホルモン、サイトカイン、神経伝達物質、又はレクチン）との組み合わせ、酵素とそのリガンド（例えば、酵素の基質アナログ、補酵素、調節因子、又は阻害剤）との組み合わせ、酵素アナログとその酵素アナログの元となる酵素の基質との組み合わせ、又はレクチンと糖との組み合わせ等を挙げることができる。なお、「酵素アナログ」とは、元の酵素に対する基質との特異的な親和性は高いものの、触媒活性は示さないものを意味する。また、上記の各組み合わせにおける各化合物は、それぞれ、いずれか一方が「特異性生体高分子」となり、他方が、検出される物質となることができる。例えば、「抗原と抗体との組み合わせ」では、抗原が「検出される物質」となる場合には、抗体が「特異性生体高分子」となることができ、逆に、抗体が「検出される物質」となる場合には、抗原が「特異性生体高分子」となることができる。
【００８３】
例えば、本発明のマイクロアレイチップを、核酸ハイブリダイゼーションアッセイに適用する場合には、上記特異性生体高分子として、検出される物質である核酸（例えば、オリゴヌクレオチド又はポリヌクレオチド）と相補的に結合することのできるオリゴヌクレオチド又はポリヌクレオチドを用いることができる。本明細書においては、「オリゴヌクレオチド」又は「ポリヌクレオチド」には、２'−デオキシリボ核酸（ＤＮＡ）、リボ核酸（ＲＮＡ）、及びペプチド核酸（ＰＮＡ）が含まれる。なお、ＰＮＡとは、ＤＮＡのホスホジエステル結合をペプチド結合に変換した人工核酸である。上記不溶性粒子に結合されるオリゴヌクレオチド又はポリヌクレオチドの鎖長は、分析目的に応じて適宜選択することができ、例えば、捕捉しようとするＤＮＡ、ＲＮＡ、又はＰＮＡの相補的配列の鎖長に基づいて決定することができる。
【００８４】
上記特異性生体高分子として用いるオリゴヌクレオチドの合成は、自動合成装置を用いて一般的に行なうことができる。また、必要に応じて、自動合成装置を用いて、末端アミノ基又は他の基が修飾されたオリゴヌクレオチドを製造することもできる。あるいは、自動合成装置を用いて未修飾オリゴヌクレオチドを予め合成しておき、必要に応じて、上記の未修飾オリゴヌクレオチドを修飾することもできる。オリゴヌクレオチドを修飾する方法は周知であり、例えば、アミン類やフルオレセイン等を付加することができる。このように修飾されたオリゴヌクレオチドは、未修飾のオリゴヌクレオチドと比べて、固定化層に対する親和性が大きい。また、特異性生体高分子として用いるポリヌクレオチド（例えば、ｃＤＮＡ）の合成は、通常の遺伝子工学的手法、例えば、ＰＣＲ（ポリメラーゼ連鎖反応）法を用いて行なうことができる。
【００８５】
また、本発明のマイクロアレイチップを免疫学的アッセイに適用する場合には、上記特異性生体高分子として、検出される物質と特異的に結合する抗原（ハプテンを含む）又は抗体を用いることができる。この場合に、上記検出される物質としては、被検試料中に一般的に含まれている成分で、しかも、免疫学的に検出することのできる物質あれば、特に制限されない。一例を挙げれば、各種タンパク質、多糖類、脂質、菌体、又は各種環境物質等を挙げることができる。より詳細には、免疫グロブリン（例えば、ＩｇＧ、ＩｇＭ、又はＩｇＡ）、感染症関連マーカー（例えば、ＨＢｓ抗原、ＨＢｓ抗体、ＨＩＶ−１抗体、ＨＩＶ−２抗体、ＨＴＬＶ−１抗体、又はトレポネーマ抗体）、腫瘍関連抗原（例えば、ＡＦＰ、ＣＲＰ、又はＣＥＡ）、凝固線溶マーカー（例えば、プラスミノーゲン、アンチトロンビン−III、Ｄ−ダイマー、ＴＡＴ、又はＰＰＩ）、抗てんかん薬（例えば、ホルモン）、各種薬剤（例えば、ジゴキシン）、菌体（例えば、Ｏ−１５７又はサルモネラ）若しくはそれらの菌体内毒素若しくは菌体外毒素、微生物類、酵素類、残留農薬、又は環境ホルモン等を挙げることができる。上記特異性生体高分子として用いる抗体としては、周知の方法で得られるポリクローナル抗体又はモノクローナル抗体のいずれをも使用することができる。さらに、上記抗体は、タンパク質［例えば、酵素（例えば、ペプシン又はパパイン）］処理したもの［例えば、抗体フラグメント（例えば、Ｆａｂ、Ｆａｂ'、Ｆ（ａｂ'）₂、又はＦｖ）］を用いることもできる。
【００８６】
５．遮光部
本発明のマイクロアレイチップにおいては、上記固定化層が形成される部分を区切るように遮光部が形成されていてもよい。このように遮光部が形成されていることにより、後述するようにマイクロアレイチップを再生する際に効率的に再生することが可能となる。
【００８７】
すなわち、本発明のマイクロアレイチップのうち、濡れ性可変層として光触媒含有層を有するものにおいては、マイクロアレイチップを用いて検出等を行なった後、このマイクロアレイチップの固定化層が形成されている領域、すなわち光触媒含有層が親水性領域とされている部分のみにエネルギーを照射することにより、光触媒含有層中の光触媒の作用により固定化層および固定化層上に固定化された特異性生体高分子が分解され除去される。この際、マイクロアレイチップの固定化層が形成される部分を区切るように遮光部を形成することにより、マスク等を用いることなく全面にエネルギーを照射することにより、容易にマイクロアレイチップの再生を行なうことが可能となるからである。
【００８８】
本発明における遮光部は、スパッタリング法、真空蒸着法等により厚み１０００〜２０００Å程度のクロム等の金属薄膜を形成し、この薄膜をパターニングすることにより形成されたものを用いることができる。
【００８９】
また、樹脂バインダ中にカーボン微粒子、金属酸化物、無機顔料、有機顔料等の遮光性粒子を含有させた層を遮光部として用いてもよい。用いられる樹脂バインダとしては、ポリイミド樹脂、アクリル樹脂、エポキシ樹脂、ポリアクリルアミド、ポリビニルアルコール、ゼラチン、カゼイン、セルロース等の樹脂を１種または２種以上混合したものや、感光性樹脂、さらにはＯ／Ｗエマルジョン型の樹脂組成物、例えば、反応性シリコーンをエマルジョン化したもの等を用いることができる。このような樹脂製遮光部の厚みとしては、０．５〜１０μｍの範囲内で設定することができる。このよう樹脂製遮光部のパターニングの方法は、フォトリソ法、印刷法等一般的に用いられている方法を用いることができる。
【００９０】
本発明における遮光部は、基材上であればいかなる位置に形成されていてもよい。具体的には、基材上に遮光部が形成され、その上から光触媒含有層が形成されたものであってもよいし、基材上に光触媒含有層が形成され、その上に遮光部が形成されたものであってもよい。しかしながら、製造が容易である等の観点から、基材と光触媒含有層との間に遮光部が形成される構成を採ることが好ましい。
【００９１】
なお、マイクロアレイチップの再生に際してのエネルギーの照射は、遮光部が光触媒含有層と基材との間に形成されている場合は、裏面、すなわち基材の光触媒含有層が形成されていない面からのエネルギーの照射となる。したがって、この場合の基材は照射されるエネルギーを透過する材質で形成されていることが好ましい。
【００９２】
Ｂ．マイクロアレイチップの製造方法
次に、本発明のマイクロアレイチップの製造方法について説明する。本発明のマイクロアレイチップの製造方法は、
（１）基材上に露光により露光部分の濡れ性を水との接触角が低下する方向に変化する光触媒含有層を設ける工程と、
（２）上記基材上に設けられた光触媒含有層上の固定化層を形成する部位に、エネルギーをパターン照射して固定化層用露光部を形成する工程と、
（３）上記固定化層用露光部に固定化層を付着させる工程と、
（４）上記固定化層上に特異性生体高分子を固定させる工程と
を含むことを特徴とするものである。
【００９３】
図２は、このようなマイクロアレイチップの製造方法の一例を説明するためのものである。この例においては、まず、基材１上に光触媒含有層２が形成される（図２（ａ））。この光触媒含有層２の形成は、上述したような光触媒とバインダとを必要に応じて他の添加剤とともに溶剤中に分散して塗布液を調製し、この塗布液を塗布した後、加熱等を行うことにより、加水分解、重縮合反応を進行させてバインダ中に光触媒を強固に固定することにより形成される。使用する溶剤としては、エタノール、イソプロルパノール等のアルコール系の有機溶剤が好ましく、塗布はスピンコート、スプレーコート、ディップコート、ロールコート、ビードコート等の公知の塗布方法により行うことかできる（上記工程（１））。
【００９４】
次に、図２（ｂ）に示すように光触媒含有層２が形成された基材１に対して、紫外光等のエネルギー６をフォトマスク７を介してパターン照射する。これにより、図２（ｃ）に示すように、光触媒含有層２上の固定化層が形成される部位を、光触媒含有層５内の光触媒の作用により親水性領域とした固定化層用露光部３が形成される（上記工程（２））。
【００９５】
本発明において、光触媒含有層に照射するエネルギーとしては、紫外光を含む光を用いることができる。このような紫外光を含む光源としては、例えば、水銀ランプ、メタルハライドランプ、キセノンランプ等を挙げることができる。この露光に用いる光の波長は４００ｎｍ以下の範囲、好ましくは３８０ｎｍ以下の範囲から設定することができ、また、露光に際しての光の照射量は、露光された部位が光触媒の作用により親水性を発現するのに必要な照射量とすることができる。
【００９６】
エネルギーの照射に際してのパターン照射は、上述したような光源を用い、フォトマスクを介したパターン照射により行うことができるが、他の方法として、エキシマ、ＹＡＧ等のレーザーを用いてパターン状に描画照射する方法を用いることも可能である。
【００９７】
このようにして親水性領域に形成された固定化層用露光部３内に、固定化層形成用塗工液を塗布して固化させることにより、図２（ｄ）に示すように固定化層４を形成する。この際の固定化層形成用塗工液の塗布方法は、例えばディッピング法等により基材全面に塗布し、親水性領域のみに付着させる方法であってもよく、またインクジェット法等の吐出法を用いて固定化層用露光部のみに固定化層形成用塗工液を塗布するようにしてもよい。
【００９８】
そして、図２（ｅ）に示すように、この固定化層４上に特異性生体高分子５を付着させることにより、マイクロアレイチップとすることができる。この際の特異性生体高分子５は、通常は特異性生体高分子を含む液体を、インクジェット法により塗布したり、スタンプピンでスポットすることにより塗布され、これを乾燥させることにより形成される。この際、必要であれば、特異性生体高分子を固定化層に固定化する処理を行なってもよい。
【００９９】
なお、上記マイクロアレイチップの製造方法における、基材、光触媒含有層、固定化層、および特異性生体高分子は、上述したマイクロチップで説明したものと同様であるので、ここでの説明は省略する。
【０１００】
Ｃ．マイクロアレイチップの再生方法
本発明においては、上述したマイクロアレイチップを用いて検出等の使用した後、上記固定化層および特異性生体高分子が付着した部分のみパターン状にエネルギー照射を行なうことにより再生することができる。
【０１０１】
本発明のマイクロアレイチップは、このように再生可能であることから、複数回にわたって使用することが可能であり、最終的なコストを低減させることができるという利点を有するものである。
【０１０２】
図３は、本発明のマイクロアレイチップの再生方法の一例を説明するためのものである。図３（ａ）は、生体高分子の検出等の使用が終わった後のマイクロアレイチップを示すものである。本発明のマイクロアレイチップの再生方法においては、図３（ｂ）に示すように、このような使用済みのマイクロアレイチップに対して、その固定化層４および特異性生体高分子５が配置されている部分のみ、すなわち固定化層用露光部３のみが露光されるように、紫外光等のエネルギー６をフォトマスク７を介して照射する。エネルギーが照射された部分の光触媒含有層２は、その光触媒の作用により有機成分を分解する。したがって固定化層用露光部３上の固定化層４および特異性生体高分子５は、光触媒の作用により分解される。一方、エネルギーが照射されない部分はそのまま撥水性領域として残存する。そして、必要であれば洗浄等を行うことにより、図３（ｃ）に示すように、光触媒含有層２上にパターン状に固定化層用露光部３が形成された基材１を得る。
【０１０３】
このような基材に対して、上述した図２（ｄ）および（ｅ）に示す工程を行なうことにより、マイクロチップアレイとして再度利用することが可能となる。
【０１０４】
図４は、本発明のマイクロチップアレイの再生方法の他の例を示すものである。このマイクロチップアレイには、基材１と光触媒含有層２との間に遮光部８が形成されており、この遮光部８は固定化層が形成される部分を区切る位置に形成されている（図４（ａ））。このようなマイクロアレイチップを再生する場合は、図４（ｂ）に示すように、基材１の光触媒含有層２が形成されていない面側からエネルギー６を全面照射することにより、遮光部８が形成されていない部分、すなわち固定化層４および特異性生体高分子５が付着している部分のみエネルギーが照射されることになり、これらは光触媒の作用により除去され、図４（ｃ）に示すように光触媒含有層２にパターン状に固定化層用露光部３が形成された基材１を得る。この場合も、上述した例と同様に、この基材に対して、上述した図２（ｄ）および（ｅ）に示す工程を行なうことにより、マイクロチップアレイとして再度利用することが可能となる。
【０１０５】
なお、上記マイクロアレイチップの再生方法における、基材、光触媒含有層、固定化層、特異性生体高分子、及び遮光部は、上述したマイクロチップで説明したものと同様であるので、ここでの説明は省略する。
【０１０６】
D．マイクロアレイチップ用基材
最後に、本発明のマイクロアレイチップ用基材について説明する。本発明のマイクロアレイチップ用基材は、基材と、上記基材上に形成され、少なくとも光触媒とバインダとからなり、かつエネルギーの照射により水との接触角が低下するように濡れ性が変化する層である光触媒含有層とを有し、上記光触媒含有層は生体高分子を固定化するための固定化層が形成される部位のみ他の部分より水との接触角が低い親水性領域とされていることを特徴とする。
【０１０７】
このような基材を用いることにより、パターン状に形成された親水性領域に固定化層形成用塗工液を塗布すれば、容易に固定化層をパターン状に形成することが可能となる。そして、この上に特異性生体高分子を付着させれば、固定化層周囲は撥水性領域であるので、形状の整った特異性生体高分子パターンを基材上に有するマイクロアレイチップとすることが可能となり、検出精度の高いマイクロアレイチップとすることができる。
【０１０８】
本発明のマイクロアレイチップ用基材の例としては、上記図３（ｃ）および図４（ｃ）に示すものを挙げることができる。
【０１０９】
本発明においては、上記マイクロアレイチップの再生方法においても説明したように、マイクロアレイチップを効率的に再生できることから、上記図４（ｃ）に示すように、基材１上に固定化層が形成される位置を区切るように形成された遮光部８を有し、さらにその上に光触媒含有層２が形成されたマイクロアレイチップ用基材であってもよい。この場合光触媒含有層には、固定化層が形成される領域に予め親水性領域とされた固定化層用露光部３が形成されている。
【０１１０】
なお、上記マイクロアレイチップ用基材における、基材、光触媒含有層、固定化層、特異性生体高分子、及び遮光部は、上述したマイクロチップで説明したものと同様であるので、ここでの説明は省略する。
【０１１１】
以上、本発明のマイクロアレイチップ、その製造方法、その再生方法、およびマイクロチップアレイ用基材を説明したが、本発明は、上記実施形態に限定されるものではない。上記実施形態は、例示であり、本発明の特許請求の範囲に記載された技術的思想と実質的に同一な構成を有し、同様な作用効果を奏するものは、いかなるものであっても本発明の技術的範囲に包含される。
【０１１２】
【実施例】
以下、本発明について、実施例を通じてさらに詳述する。
【０１１３】
［実施例１］
イソプロピルアルコール30gにフルオロアルキルシランが主成分であるMF-160E（商品名、トーケムプロダクツ（株）製）0.4gとトリメトキシメチルシラン（東芝シリコーン（株）製、商品名TSL8113）3gと光触媒である二酸化チタンの水分散体であるSTS-01（商品名、石原産業（株）製）20gとを混合し、100℃で20分間攪拌し、光触媒含有層用組成物とした。
【０１１４】
クロムにより遮光部が形成されたスライドガラス（開口部50μm径100μmピッチ）上に上記組成物をスピンコーターにより塗布し、120℃で10分間の乾燥処理を行うことにより、厚さ0.2μmの光触媒含有層を形成した。光触媒含有層表面の水の接触角を測定したところ90°であった。
【０１１５】
上記基板のスライドガラス面側から露光（365nm 500mJ/cm²）し、開口部上の光触媒含有層表面の濡れ性を水の接触角で10°以下に変化させ、固定化層用露光部を形成した。
【０１１６】
上記基板をポリL−リシンの3％水溶液に浸漬し引き上げることにより、露光部のみに固定化層を形成した。
【０１１７】
上記固定化層上に各種DNA溶液をインクジェット法にて吐出し、固定させることによりマイクロアレイチップを作製した。
【０１１８】
［実施例２］
上記使用済みマイクロアレイチップをガラス基板側から露光（365nm 1000mJ/cm²）して、ポリL−リシン、ＤＮＡを分解除去した。
【０１１９】
これを上記と同様にしてポリL−リシン、ＤＮＡを形成し、マイクロアレイチップとした。
【０１２０】
【発明の効果】
本発明のマイクロアレイチップによれば、パターン状に形成された固定化層上にのみ特異性生体高分子を有し、その他の領域は撥水性領域となっているので、固定化層上にスポットされた特異性生体高分子が濡れ広がり、隣同士のスポットが接触してしまうといった不具合が生じることがない。したがって、特異性生体高分子の形状が乱れ、マイクロアレイチップの検出精度が低下するといった問題が無く、検出精度を向上させることができるといった効果を奏するものである。
【図面の簡単な説明】
【図１】本発明のマイクロアレイチップの一例を示す概略断面図である。
【図２】本発明のマイクロアレイチップの製造方法の一例を説明するための工程図である。
【図３】本発明のマイクロアレイチップの再生方法の一例を説明するための工程図である。
【図４】本発明のマイクロアレイチップの再生方法の他の例を説明するための工程図である。
【符号の説明】
１…基材
２…光触媒含有層
３…固定化層用露光部
４…固定化層
５…特異性生体高分子
８…遮光部[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a microarray chip in which a large number of specific biopolymers that specifically bind to specific DNA or protein are arranged.
[0002]
[Prior art]
In the field of molecular biology, microarray chips in which biopolymers such as DNA and proteins are immobilized at a high density have attracted rapid attention in recent years. In an experiment using a microarray chip, a large amount of a support (chip) on which a large number of samples (samples) are immobilized is replicated in large quantities, and the sample on each chip is reacted with a sample (target) for investigation control. . It is possible to detect a difference in reaction between samples on one chip by one experiment and a difference in reaction between the same sample set among a plurality of chips by repeating an experiment with different conditions.
[0003]
As a method for producing such a microarray chip, first, a method developed by Affimetrix can be mentioned. This method synthesizes oligonucleotides at high density for every several tens of μm rectangles on a substrate using protective groups that are selectively removed by light irradiation and photolithography technology used in semiconductor manufacturing. . The microarray chip obtained by this method is currently the only standardized standard in the world, but the price is very high and there is a problem that only some researchers can use it sufficiently. there were.
[0004]
On the other hand, as another method for manufacturing a microarray chip, a method developed by P. Brown et al. This method is very simple compared to the Affimetrix method described above, in which pre-prepared DNA is dispensed into 96 or 384 well microplates that have been chemically treated on the surface, resulting in high density spots. This is a method of spotting with high density on a slide glass with a possible robot. According to this method, the manufacturing cost is low, and custom chips for many researchers can be created. Therefore, this method is used in many research institutions.
[0005]
Such a Stanford method is usually produced by applying a fixing agent for immobilizing DNA on a glass substrate and spotting the target DNA at a high density thereon. However, in the case where the surface of the immobilizing agent is hydrophilic, there is a possibility that the spotted DNA is diffused and the adjacent spots come into contact with each other.
[0006]
[Problems to be solved by the invention]
The present invention has been made in view of the above problems, can be easily formed, and even when DNA is spotted at a high density regardless of the type of immobilizing agent, the spotted DNA is wet. The main purpose is to provide a microarray chip that does not have a problem of spreading.
[0007]
[Means for Solving the Problems]
In order to achieve the above object, the present invention provides a substrate according to claim 1, a wettability variable layer formed on the substrate and capable of changing wettability by an external stimulus, and the wettability. An immobilization layer for immobilizing a biopolymer formed on a variable layer, and a specific biopolymer that is immobilized on the immobilization layer and specifically binds to a target biopolymer. The wettability variable layer is a layer in which a hydrophilic region is formed in a pattern by an external stimulus, and the immobilization layer is formed on the hydrophilic region on the wettability variable layer. A microarray chip is provided.
[0008]
The microarray chip of the present invention has a specific biopolymer only on the immobilization layer formed in a pattern, and the other region is a water-repellent region. The problem that the conductive biopolymer wets and spreads and the adjacent spots come into contact with each other does not occur.
[0009]
In the invention described in claim 1, as described in claim 2, the wettability variable layer is a photocatalyst-containing layer composed of at least a photocatalyst and a binder, and contacts with water by energy irradiation. A layer whose wettability changes so that the corners are lowered is preferable.
[0010]
Thus, if a photocatalyst-containing layer whose wettability changes so that the contact angle with water is reduced by energy irradiation is formed, the wettability of this layer can be easily made into a pattern by performing energy pattern irradiation. It is possible to change and form a hydrophilic region having a small contact angle with water in a pattern. Therefore, a microarray chip can be easily formed by applying a fixing agent to the hydrophilic region formed in this pattern to form an immobilization layer and immobilizing a specific biopolymer thereon. It is.
[0011]
In the invention described in claim 2, as described in claim 3, the photocatalyst is made of titanium oxide (TiO 2).₂), Zinc oxide (ZnO), tin oxide (SnO)₂), Strontium titanate (SrTiO)₃), Tungsten oxide (WO₃), Bismuth oxide (Bi₂O₃), And iron oxide (Fe₂O₃It is preferable that it is 1 type, or 2 or more types of substances selected from these. Among them, as described in claim 4, titanium oxide (TiO₂) Is preferable. This is because titanium oxide is effective as a photocatalyst because of its high band gap energy, is chemically stable, has no toxicity, and is readily available.
[0012]
On the other hand, the binder, which is another component constituting the photocatalyst-containing layer, is Y as described in claim 5._nSiX_(4-n)Wherein Y represents an alkyl group, a fluoroalkyl group, a vinyl group, an amino group, a phenyl group or an epoxy group, X represents an alkoxyl group or a halogen, and n is an integer from 0 to 3. It is preferable that it is the organopolysiloxane which is a 1 type, or 2 or more types of hydrolyzed condensate or cohydrolyzed condensate of the silicon compound. This is because a photocatalyst-containing layer having good physical properties in which a photocatalyst is firmly bonded to the inside can be obtained.
[0013]
In the invention according to any one of claims 1 to 5, as described in claim 6, the immobilization layer is preferably formed of a substance having a cationic property. . This is because a substance having a normal cationic property can electrostatically immobilize an anionic biopolymer such as an oligonucleotide.
[0014]
In the invention described in any one of claims 1 to 6, as described in claim 7, the specific biopolymer specifically binds to a target nucleic acid. It is preferable that the probe DNA. This is because a detection method using probe DNA and utilizing hybridization with a fluorescently labeled target DNA has been established.
[0015]
In the invention described in any one of claims 2 to 7, as described in claim 8, the portion where the fixing layer is formed is separated on the base material. It is preferable that a light-shielding portion is formed. This is because the microarray chip regeneration method described later can be used without a mask.
[0016]
In the present invention, as described in claim 9,
(1) A step of providing a photocatalyst-containing layer that changes the wettability of an exposed portion by exposure on a substrate in a direction in which the contact angle with water decreases;
(2) A step of irradiating the pattern with energy to form a fixed layer exposed portion on a portion where the fixed layer on the photocatalyst-containing layer provided on the substrate is formed;
(3) attaching the fixing layer to the exposure part for the fixing layer;
(4) a step of immobilizing a specific biopolymer on the immobilization layer;
A method for manufacturing a microarray chip is provided.
[0017]
According to the present invention, since the exposed portion for the fixed layer, which is a hydrophilic region formed in a pattern on the photocatalyst-containing layer, is formed, the fixed layer can be easily formed in a pattern by, for example, dipping can do. A microarray chip can be easily formed by attaching a specific biopolymer on the immobilization layer formed in this manner. In addition, since the region where the immobilization layer is not formed is a water-repellent region, even when a liquid containing DNA was dropped on the immobilization layer, the immobilization layer was dripped without spreading. It is possible to prevent problems such as the shape of DNA being deformed and the detection accuracy being lowered.
[0018]
The present invention also provides, as described in claim 10, a part to which the specific biopolymer is attached after using the microarray chip described in any one of claims 2 to 8. There is provided a method for reproducing a microarray chip, wherein only a pattern is exposed.
[0019]
In the present invention, the immobilizing agent and the specific biopolymer are formed on the photocatalyst containing layer. Therefore, it is possible to remove the fixing agent and the specific biopolymer by the action of the photocatalyst by exposing only the hydrophilic region formed in a pattern, that is, the exposure part for the immobilization layer in a pattern. Thereby, the microarray chip can be easily regenerated and reused.
[0020]
Furthermore, in the present invention, as described in claim 11, the substrate is formed on the substrate and is composed of at least a photocatalyst and a binder, and the contact angle with water is lowered by energy irradiation. A photocatalyst-containing layer that is a layer whose wettability changes, and the photocatalyst-containing layer has a contact angle with water from other portions only at a site where an immobilization layer for immobilizing a biopolymer is formed. Provided is a substrate for a microarray chip, characterized by being a low hydrophilic region. By using such a microarray chip substrate, for example, it is possible to easily form an immobilization layer by dipping or the like, and the specific biopolymer immobilized on the immobilization layer is surrounded by Since it is surrounded by the water-repellent region, it does not spread out and the shape of the microarray chip is not disturbed. As a result, the detection accuracy can be improved.
[0021]
DETAILED DESCRIPTION OF THE INVENTION
A. Microarray chip
First, the microarray chip of the present invention will be described in detail. FIG. 1 shows an example of such a microarray chip of the present invention. In the microarray chip of this example, a photocatalyst containing layer 2 is formed as a wettability variable layer on a glass substrate 1 which is a base material, and the surface of the photocatalyst containing layer 2 is irradiated with energy in a pattern to form a hydrophilic region. An immobilization layer exposure portion 3 is formed, and an immobilization layer 4 is formed on the immobilization layer exposure portion 3, and a specific living body height is formed on the immobilization layer 4. Molecule 5 is fixed.
[0022]
In the microarray chip of this example, the immobilization layer 4 is formed in a pattern, the specific biopolymer 5 is arranged thereon, and the region where the immobilization layer 4 is not formed is water-repellent. It is an area. Therefore, for example, when the specific biopolymer 5 is disposed on the immobilization layer 4 by an ink jet method or the like, the specific biopolymer 5 does not spread out beyond the region of the immobilization layer 4, and the specificity The shape of the biopolymer 5 can be made uniform, and the final detection sensitivity can be improved.
[0023]
Hereinafter, each configuration of the microarray chip of the present invention as shown in this example will be described in detail.
[0024]
1. Base material
The substrate used in the microarray chip of the present invention includes an organic or inorganic material that is substantially insoluble in water or an organic solvent generally used in nucleic acid hybridization assays or immunological assays, such as silicon. Glass, magnetic metal or non-magnetic metal, or plastic. In particular, silicon, various plastics (for example, polycarbonate, polystyrene, polypropylene, or the like), quartz, glass, or the like is preferably used. The shape of such a substrate is not particularly limited as long as it has at least one flat surface, but it can be said that it is preferably a plate shape or a film shape.
[0025]
2. Variable wettability layer
The wettability variable layer used in the present invention is not particularly limited as long as the wettability of the surface can be changed by an external stimulus such as a physical stimulus or a chemical stimulus. For example, the surface roughness may be changed by acid or alkali, and the wettability may be changed. The wettability variable layer may be formed by irradiation with energy such as ultraviolet rays, visible light, or heat. It may be a layer or the like in which the wettability is changed by changing the substance inside.
[0026]
As for the change in wettability, the contact angle with water is large before the stimulus is applied (poor wettability), and the contact angle with water becomes small after the stimulus is applied (improves wettability). It may be a wettability variable layer that changes to, or conversely, the contact angle with water is small before the stimulus is applied (good wettability), and after the stimulus is applied, It may be a wettability variable layer that greatly changes the contact angle (deteriorates wettability).
[0027]
(Photocatalyst containing layer)
In the present invention, the wettability variable layer is preferably a photocatalyst-containing layer whose wettability changes so that the contact angle with water is reduced by energy irradiation. Thus, the wettability is reduced so that the contact angle with water is reduced by exposure (in the present invention, not only light is irradiated but also energy is irradiated). By providing a photocatalyst-containing layer that changes, the wettability can be easily changed by performing pattern irradiation of energy, etc., and a hydrophilic region with a small contact angle with water can be obtained, and the photocatalyst-containing layer is removed. Only a hydrophilic region can be easily formed. Therefore, the microarray chip can be manufactured efficiently, which is advantageous in terms of cost.
[0028]
The energy referred to in the present invention is not particularly limited as long as it is an energy capable of making the photocatalyst-containing layer into a hydrophilic region, but generally light including ultraviolet light is preferably used.
[0029]
Further, the hydrophilic region is a region having a small contact angle with water, and refers to a region having good wettability with respect to an immobilization layer forming coating solution for forming an immobilization layer described later. . Further, the water-repellent region is a region having a large contact angle with water, and refers to a region having poor wettability with respect to an immobilization layer forming coating solution or a solution containing a biopolymer, which will be described later. . In the present invention, the region is referred to as a hydrophilic region if the contact angle with water is 1 degree or more smaller than the adjacent region, and conversely, the contact angle with water is larger than the region adjacent to the region. If it is larger than once, it will be a water-repellent region.
[0030]
The photocatalyst-containing layer in the present invention is a photocatalyst-containing layer in which a contact angle with water in a portion not irradiated with energy is a contact angle that is 1 degree or more larger than a contact angle with water in a portion irradiated with energy. The photocatalyst-containing layer is particularly preferably used at 5 ° or more, most preferably 10 ° or more.
[0031]
If the difference between the contact angle with water in the part not irradiated with energy and the contact angle with water in the part irradiated with energy is less than the specified range, immobilization is performed using the difference in wettability. This is because it becomes difficult to apply the layer-forming coating solution in a pattern, and it becomes difficult to fix the specific biopolymer in a pattern.
[0032]
As a specific contact angle with water in such a photocatalyst-containing layer, it is preferable that a contact angle with water in an unexposed portion is 30 degrees or more, particularly 60 degrees or more, and particularly preferably 90 degrees or more. A photocatalyst-containing layer having such a contact angle with water is preferably used. This is the portion that is not exposed in the present invention where water repellency is required. Therefore, when the contact angle with water is small, the water repellency is not sufficient, and there is a possibility that a fixing layer forming coating liquid, which will be described later, may remain even in an unnecessary part. This is because the shape of the specific biopolymer to be converted may be disturbed and the detection accuracy of the microarray chip may be lowered.
[0033]
Further, the contact angle with water when the photocatalyst-containing layer is exposed is specifically preferably a photocatalyst-containing layer that is less than 30 degrees, particularly 20 degrees or less, especially 10 degrees or less. If the contact angle of the exposed part with water is high, there is a possibility that the spreading of the immobilization layer forming coating solution for forming the immobilization layer in this part may be inferior, and in the region where the immobilization layer should be formed. This is because there is a possibility that the detection accuracy of the resulting microarray chip is lowered without being all wet and spread.
[0034]
In addition, the contact angle with water here is measured by using a contact angle measuring device (CA-Z type manufactured by Kyowa Interface Science Co., Ltd.) with a contact angle with water (dropping a water drop from a microsyringe for 30 seconds. And later).
[0035]
The photocatalyst-containing layer is preferably composed of at least a photocatalyst and a binder. This is because, by using such a layer, it becomes possible to increase the critical surface tension by the action of the photocatalyst by energy irradiation, and to reduce the contact angle with water.
[0036]
In such a photocatalyst-containing layer, the mechanism of action of a photocatalyst represented by titanium oxide as described later is not necessarily clear, but carriers generated by light irradiation react directly with nearby compounds, or It is considered that the active oxygen species generated in the presence of oxygen and water change the chemical structure of organic matter.
[0037]
When a photocatalyst-containing layer is used as the wettability variable layer in the present invention, the photocatalyst is used to change the wettability of the energy irradiation part using the action of oxidation, decomposition, etc. of organic groups and additives that are part of the binder. Thus, it can be made hydrophilic and a great difference in wettability with an unirradiated part can be produced. Therefore, by improving the acceptability (hydrophilicity) and the repellent property (water repellency) with the coating solution for forming the immobilization layer to which the specific biopolymer is attached, the accuracy is good and the cost is advantageous. A microarray chip can be obtained.
[0038]
Further, by utilizing the action of such a photocatalyst, the microarray chip of the present invention can be reused as will be described later.
[0039]
Further, when such a photocatalyst containing layer is used in the present invention, the photocatalyst containing layer contains at least a photocatalyst and fluorine, and the fluorine content on the surface of the photocatalyst containing layer irradiates the photocatalyst containing layer with energy. In this case, the photocatalyst-containing layer may be formed so as to be lower than that before energy irradiation due to the action of the photocatalyst.
[0040]
In the microarray chip having such a feature, a pattern composed of a portion having a small fluorine content can be easily formed by pattern irradiation with energy. Here, fluorine has an extremely low surface energy. Therefore, the surface of a substance containing a large amount of fluorine has a smaller critical surface tension. Therefore, the critical surface tension of the portion having a small fluorine content is larger than the critical surface tension of the surface of the portion having a large fluorine content. This means that the portion with a low fluorine content is a hydrophilic region compared to the portion with a high fluorine content. Therefore, forming a pattern composed of a portion having a lower fluorine content than the surrounding surface forms a hydrophilic region pattern in the water-repellent region.
[0041]
Therefore, when such a photocatalyst-containing layer is used, the pattern of the hydrophilic region can be easily formed in the water-repellent region by irradiating the pattern with energy. An immobilization layer forming coating solution or the like for immobilizing a biopolymer can be easily applied, and a microarray chip with good accuracy can be obtained.
[0042]
The fluorine content in the hydrophilic region having a low fluorine content formed by irradiation with energy is 10 or less, preferably 5 or less, particularly preferably 1 when the fluorine content of the portion not irradiated with energy is defined as 100. The following is preferable.
[0043]
By setting it within such a range, it is possible to make a large difference in hydrophilicity between the energy irradiated portion and the unirradiated portion. Therefore, by applying an alkaline solution or the like that dissolves the photocatalyst-containing layer to such a photocatalyst-containing layer, it is possible to accurately dissolve only the hydrophilic region having a reduced fluorine content, and to obtain a microarray chip with high accuracy. Because you can. This rate of decrease is based on weight.
[0044]
For the measurement of the fluorine content in the photocatalyst-containing layer, various commonly used methods can be used. For example, X-ray photoelectron spectroscopy (ES-ray photoelectron spectroscopy, ESCA) for Chemical Analysis)), and any method that can quantitatively measure the amount of fluorine on the surface, such as X-ray fluorescence analysis and mass spectrometry, is not particularly limited.
[0045]
Examples of the photocatalyst used in the present invention include titanium oxide (TiO 2), which is known as an optical semiconductor.₂), Zinc oxide (ZnO), tin oxide (SnO)₂), Strontium titanate (SrTiO)₃), Tungsten oxide (WO₃), Bismuth oxide (Bi₂O₃), And iron oxide (Fe₂O₃1) or a mixture of two or more selected from these.
[0046]
In the present invention, titanium oxide is particularly preferably used because it has a high band gap energy, is chemically stable, has no toxicity, and is easily available. Titanium oxide includes anatase type and rutile type, and both can be used in the present invention, but anatase type titanium oxide is preferable. Anatase type titanium oxide has an excitation wavelength of 380 nm or less.
[0047]
Examples of such anatase-type titanium oxide include hydrochloric acid peptizer-type anatase-type titania sol (STS-02 manufactured by Ishihara Sangyo Co., Ltd. (average particle size 7 nm), ST-K01 manufactured by Ishihara Sangyo Co., Ltd.), nitric acid solution Examples include glue-type anatase-type titania sol (TA-15 (average particle size: 12 nm) manufactured by Nissan Chemical Co., Ltd.).
[0048]
The smaller the particle size of the photocatalyst, the more effective the photocatalytic reaction occurs. The average particle size is preferably 50 nm or less, and it is particularly preferable to use a photocatalyst of 20 nm or less. Further, the smaller the particle size of the photocatalyst, the smaller the surface roughness of the formed photocatalyst-containing layer, which is preferable. When the particle size of the photocatalyst exceeds 100 nm, the centerline average surface roughness of the photocatalyst-containing layer becomes coarse, and the photocatalyst This is not preferable because the water repellency of the non-exposed portion of the containing layer is lowered and the hydrophilicity of the exposed portion becomes insufficient.
[0049]
The microarray chip of the present invention contains fluorine on the surface of the photocatalyst containing layer as described above, and reduces the fluorine content on the surface of the photocatalyst containing layer by pattern irradiating energy on the surface of the photocatalyst containing layer. A hydrophilic region pattern is formed in the region, and an immobilization layer forming coating solution for immobilizing the specific biopolymer is applied to the region, and the specificity is formed on the immobilization layer thus formed. It may be a microarray chip obtained by immobilizing a biopolymer. Even in this case, it is preferable to use titanium dioxide as described above as a photocatalyst. However, when titanium dioxide is used in this way, the fluorine content contained in the surface of the photocatalyst containing layer is X-ray photoelectron. When analyzed and quantified by spectroscopy, when the titanium (Ti) element is defined as 100, the fluorine (F) element is in a ratio such that the fluorine (F) element is 500 or more, preferably 800 or more, particularly preferably 1200 or more. Is preferably contained on the surface of the photocatalyst-containing layer.
[0050]
Fluorine (F) is included in the surface of the photocatalyst containing layer to such an extent that the critical surface tension on the surface of the photocatalyst containing layer can be made sufficiently low, so that water repellency on the surface can be secured, thereby pattern irradiation with energy. As a result, the difference in wettability with the hydrophilic region on the surface of the pattern portion where the fluorine content is reduced can be increased, and the quality of the microarray chip of the photocatalyst-containing layer finally obtained can be improved. It is.
[0051]
Further, in such a microarray chip, the fluorine content in the hydrophilic region formed by pattern irradiation of energy is 50 or less when the titanium (Ti) element is 100, preferably It is preferably contained in a ratio of 20 or less, particularly preferably 10 or less.
[0052]
If the fluorine content in the photocatalyst-containing layer can be reduced to this extent, an alkaline solution for dissolving the photocatalyst-containing layer can be attached, and sufficient hydrophilicity can be obtained to sufficiently spread the region. The pattern of the photocatalyst-containing layer can be formed with high accuracy due to the difference in wettability with the water repellency of the portion where the energy is not irradiated, and a microarray chip with good quality can be obtained.
[0053]
In the present invention, the binder used in the photocatalyst-containing layer preferably has a high binding energy such that the main skeleton is not decomposed by the photoexcitation of the photocatalyst. For example, (1) chloro or alkoxysilane or the like by sol-gel reaction or the like. Examples include organopolysiloxanes that exhibit high strength by hydrolysis and polycondensation, and (2) organopolysiloxanes crosslinked with reactive silicones that are excellent in water repellency and oil repellency.
[0054]
In the case of (1) above, the general formula:
Y_nSiX_(4-n)
(Here, Y represents an alkyl group, a fluoroalkyl group, a vinyl group, an amino group, a phenyl group or an epoxy group, X represents an alkoxyl group, an acetyl group or a halogen. N is an integer from 0 to 3. )
It is preferable that it is the organopolysiloxane which is a 1 type, or 2 or more types of hydrolysis condensate or cohydrolysis condensate of the silicon compound shown by these. Here, the number of carbon atoms of the group represented by Y is preferably in the range of 1 to 20, and the alkoxy group represented by X is a methoxy group, an ethoxy group, a propoxy group, or a butoxy group. preferable.
[0055]
As the binder, polysiloxane containing a fluoroalkyl group can be preferably used. Specifically, one or more hydrolyzed condensates and cohydrolyzed condensates of fluoroalkylsilanes can be mentioned. In general, those known as fluorine-based silane coupling agents can be used. By using such a polysiloxane containing a fluoroalkyl group as a binder, the water repellency of the non-irradiated part of the photocatalyst-containing layer is greatly improved, and the function of preventing the fixing layer forming coating liquid from adhering is exhibited. .
[0056]
Examples of the reactive silicone (2) include compounds having a skeleton represented by the following general formula.
[0057]
[Chemical 1]

[0058]
However, n is an integer greater than or equal to 2, R¹, R²Each represents a substituted or unsubstituted alkyl, alkenyl, aryl or cyanoalkyl group having 1 to 10 carbon atoms, and 40% or less of the total is vinyl, phenyl or phenyl halide in a molar ratio. R¹, R²Is preferably a methyl group because the surface energy becomes the smallest, and the methyl group is preferably 60% or more by molar ratio. In addition, the chain end or side chain has at least one reactive group such as a hydroxyl group in the molecular chain.
[0059]
In addition to the above organopolysiloxane, a stable organosilicon compound that does not undergo a crosslinking reaction, such as dimethylpolysiloxane, may be mixed in the binder.
[0060]
In the present invention, the photocatalyst-containing layer can contain a surfactant in addition to the above-mentioned photocatalyst and binder. Specifically, hydrocarbons such as NIKKOLBL, BC, BO, BB series manufactured by Nikko Chemicals Co., Ltd., ZONYL FSN, FSO manufactured by DuPont, Surflon S-141, 145 manufactured by Asahi Glass Co., Ltd., Dainippon Ink Chemical industry Co., Ltd. Megafax F-141, 144, Neos Co., Ltd., Fategent F-200, F251, Daikin Industries Co., Ltd. Unidyne DS-401, 402, 3M Co., Ltd. Fluorado FC-170, Fluorine-based or silicone-based nonionic surfactants such as 176 can be mentioned, and cationic surfactants, anionic surfactants, and amphoteric surfactants can also be used.
[0061]
In addition to the above surfactants, the photocatalyst-containing layer includes polyvinyl alcohol, unsaturated polyester, acrylic resin, polyethylene, diallyl phthalate, ethylene propylene diene monomer, epoxy resin, phenol resin, polyurethane, melamine resin, polycarbonate, Polyvinyl chloride, polyamide, polyimide, styrene butadiene rubber, chloroprene rubber, polypropylene, polybutylene, polystyrene, polyvinyl acetate, polyester, polybutadiene, polybenzimidazole, polyacrylonitrile, epichlorohydrin, polysulfide, polyisoprene, oligomers, polymers, etc. It can be included.
[0062]
The content of the photocatalyst in the photocatalyst containing layer can be set in the range of 5 to 60% by weight, preferably 20 to 40% by weight. The thickness of the photocatalyst containing layer is preferably in the range of 0.05 to 10 μm.
[0063]
The photocatalyst-containing layer can be formed by dispersing a photocatalyst and a binder in a solvent together with other additives as necessary to prepare a coating solution, and applying the coating solution. As the solvent to be used, alcohol-based organic solvents such as ethanol and isopropanol are preferable. The coating can be performed by a known coating method such as spin coating, spray coating, dip coating, roll coating or bead coating. When an ultraviolet curable component is contained as the binder, the photocatalyst-containing layer can be formed by irradiating ultraviolet rays and performing a curing treatment.
[0064]
(Pattern of hydrophilic region on wettability variable layer)
The arrangement of the hydrophilic region pattern on the wettability variable layer is determined based on the position of the hydrophilic region (that is, the immobilization layer and the specific biopolymer formed thereon) when detecting a signal derived from the label. The position is not particularly limited as long as the position can be confirmed. For example, the position is appropriately determined according to the purpose of analysis or according to the analyzer used to detect the signal derived from the label. can do.
[0065]
Such a hydrophilic region pattern is preferably arranged in alignment in view of easy detection. In addition, it is more preferable that a plurality of portions of the hydrophilic region are arranged in a line in a line at a predetermined interval, and a plurality of such lines are arranged in parallel to each other. With such a pattern in which hydrophilic regions are arranged, it is easy to automate the process of detecting a signal derived from a label possessed by a polymer that binds to a specific biopolymer.
[0066]
The interval between the hydrophilic regions on the wettability variable layer is such that it is not affected by the adjacent hydrophilic region when detecting a signal derived from the labeling label possessed by the polymer that binds to the specific biopolymer. For example, the shortest distance between the edges of adjacent hydrophilic regions is in the range of 0.01 μm to 1 cm, preferably 0.05 μm to 5 mm. preferable. Further, the number of hydrophilic regions on the wettability variable layer is not particularly limited, and depends on the size of the base material, for example, several to tens of thousands (generally several tens to one to one to 20,000).
[0067]
When the wettability variable layer is a photocatalyst-containing layer, the method for forming the hydrophilic region pattern can be performed by energy pattern irradiation using a photomask. Further, as described later, when a light shielding part is formed on the base material, the entire surface exposure (energy irradiation) from the surface side where the photocatalyst containing layer is not formed may be performed.
[0068]
3. Immobilization layer
In the present invention, an immobilization layer for immobilizing a specific biopolymer described later is formed on the hydrophilic region formed in a pattern on the wettability variable layer described above.
[0069]
As the immobilizing agent used in the immobilizing layer, various agents can be used according to the immobilizing means for the specific biopolymer.
[0070]
As such an immobilization means, a binding method known to those skilled in the art can be used. For example, an immobilization method by electrostatic coupling, an immobilization method by covalent bonding, or the like can be used.
[0071]
In the case of the above-mentioned immobilization means by electrostatic binding, a solution in which a substance having a charge opposite to that of the specific biopolymer is dissolved as a fixing agent is prepared as an immobilization layer-forming coating solution. The immobilization layer is formed by applying the hydrophilic region on the wettability variable layer in which the pattern is formed.
[0072]
For example, when the specific biopolymer is an oligonucleotide, since the oligonucleotide has an anionic property, a coating solution using a substance having a cationic property as a fixing agent is used. The immobilization layer is formed by applying this and drying.
[0073]
In this case, examples of the cationic immobilizing agent to be used include poly L-lysine, poly (allylamine hydrochloride), poly (ethyleneimine), poly (diallyldimethylammonium chloride) and the like. L-lysine is preferred.
[0074]
On the other hand, as an immobilization method using a covalent bond (chemical bond), a compound having a functional group (for example, a carboxyl group, an amino group, or a hydroxyl group) is used as an immobilizing agent for an immobilizing agent-forming coating material. A method of forming and binding a specific biopolymer to this can be mentioned. Examples of such a fixing agent include compounds having a carboxyl group or a primary amino group as a functional group.
[0075]
Specifically, a silane coupling agent containing an amino group is attached to the hydrophilic region formed in a pattern on the wettability variable layer. This uses a solution containing the above silane coupling agent, and is applied to the entire surface by a dip coating method, a transfer method, a spin coating method, etc., and a silane coupling agent is attached only to the hydrophilic region, an inkjet, a dispenser, etc. It is possible to use a method of applying only the hydrophilic region. The silane coupling agent containing a coated amino group spreads in the hydrophilic region and does not remain in the water-repellent region. After coating, the hydrophilic region and the immobilizing agent can be bonded by heating as necessary. A microarray chip can be formed by binding a specific biopolymer on the immobilization layer thus formed.
[0076]
Strongly immobilized on the membrane by covalently bonding the amino group of the silane coupling agent, which is the immobilizing agent, to a biopolymer whose end is modified with an amino group, specifically, the terminal amino group of an oligonucleotide or the like can do.
[0077]
In this case, as the silane coupling agent having an amino group to be used, N-β (aminoethyl) γ-aminopropyltrimethoxysilane, N-β (aminoethyl) γ-aminopropylmethyldimethoxysilane, γ-aminopropyl is used. Examples include triethoxysilane, N-phenyl-γ-aminopropyltrimethoxysilane, and the like.
[0078]
In addition, as another immobilization method by covalent bond between the immobilization layer and the specific biopolymer, for example, when the biopolymer is an oligonucleotide, a method of covalently bonding the 5 ′ end to the functional group It has been known. Specifically, first, the 5 ′ end of the oligonucleotide is reacted with a maleimide compound to introduce a maleimide group at the 5 ′ end of the oligonucleotide. Suitable maleimide compounds include sulfosuccinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate and the like. Separately from the above reaction, the immobilization agent having an amino group as a functional group and succinimidyl-S-acetylthioacetate are reacted, and then deacetylation is performed using hydroxyramine, whereby the immobilization agent is converted into SH. Giving a group. By reacting the SH group on the immobilizing agent thus imparted with the maleimide group introduced at the 5 ′ end of the oligonucleotide prepared by introducing a maleimide group at the 5 ′ end, the oligonucleotide and the immobilizing agent are reacted. The specific biopolymer can be immobilized on the immobilization layer.
[0079]
In addition to the methods described above, many immobilization methods such as a method using a biotin-avidin system can also be used.
[0080]
As will be described later, the immobilization layer used in the present invention preferably does not spread from the immobilization layer when a liquid containing a specific biopolymer is attached thereon. Therefore, there is provided an immobilizing agent in which the wettability of the surface of the immobilization layer is smaller than the surrounding wettability variable layer region (water repellent region) by 1 degree or more, preferably 5 degrees or more in contact angle with water. It is preferably selected and used.
[0081]
4). Specific biopolymer
In the microarray chip of the present invention, a specific biopolymer is immobilized on the immobilization layer. Such a specific biopolymer is not particularly limited as long as it is a biopolymer capable of specifically binding to a predetermined biopolymer.
[0082]
Such specific binding includes, for example, a combination of a nucleic acid (eg, oligonucleotide or polynucleotide) and a complementary nucleic acid, a combination of an antigen and an antibody (or antibody fragment), a receptor and its Combinations with ligands (eg hormones, cytokines, neurotransmitters or lectins), combinations of enzymes with their ligands (eg enzyme substrate analogs, coenzymes, modulators or inhibitors), enzyme analogs and their enzymes The combination with the substrate of the enzyme used as the base of the analog, or the combination of lectin and sugar can be mentioned. The “enzyme analog” means a substance having a high specific affinity with a substrate for the original enzyme but showing no catalytic activity. In addition, each of the compounds in each of the above combinations can be a “specific biopolymer” and the other can be a substance to be detected. For example, in the “combination of antigen and antibody”, when the antigen is “a substance to be detected”, the antibody can be “specific biopolymer”, and conversely, the antibody is “detected” In the case of “substance”, the antigen can be “specific biopolymer”.
[0083]
For example, when the microarray chip of the present invention is applied to a nucleic acid hybridization assay, it binds complementarily to a nucleic acid (for example, an oligonucleotide or a polynucleotide) that is a substance to be detected as the specific biopolymer. Oligonucleotides or polynucleotides that can be used. As used herein, “oligonucleotide” or “polynucleotide” includes 2′-deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and peptide nucleic acid (PNA). PNA is an artificial nucleic acid obtained by converting a DNA phosphodiester bond into a peptide bond. The chain length of the oligonucleotide or polynucleotide bound to the insoluble particle can be appropriately selected depending on the purpose of analysis, and is based on, for example, the chain length of the complementary sequence of DNA, RNA, or PNA to be captured. Can be determined.
[0084]
Synthesis of the oligonucleotide used as the specific biopolymer can be generally performed using an automatic synthesizer. In addition, if necessary, an oligonucleotide having a modified terminal amino group or other group can be produced using an automatic synthesizer. Alternatively, an unmodified oligonucleotide can be synthesized in advance using an automatic synthesizer, and the above-mentioned unmodified oligonucleotide can be modified as necessary. Methods for modifying oligonucleotides are well known, and for example, amines and fluorescein can be added. Oligonucleotides modified in this way have a greater affinity for the immobilization layer than unmodified oligonucleotides. In addition, synthesis of a polynucleotide (for example, cDNA) used as a specific biopolymer can be performed using a normal genetic engineering technique, for example, a PCR (polymerase chain reaction) method.
[0085]
When the microarray chip of the present invention is applied to an immunological assay, an antigen (including a hapten) or an antibody that specifically binds to a substance to be detected can be used as the specific biopolymer. . In this case, the substance to be detected is not particularly limited as long as it is a component generally contained in a test sample and can be detected immunologically. For example, various proteins, polysaccharides, lipids, fungal bodies, various environmental substances, and the like can be given. More specifically, an immunoglobulin (eg, IgG, IgM, or IgA), an infection-related marker (eg, HBs antigen, HBs antibody, HIV-1 antibody, HIV-2 antibody, HTLV-1 antibody, or treponema antibody) Tumor associated antigens (eg, AFP, CRP, or CEA), coagulation fibrinolytic markers (eg, plasminogen, antithrombin-III, D-dimer, TAT, or PPI), antiepileptic drugs (eg, hormones), Examples include various drugs (for example, digoxin), bacterial cells (for example, O-157 or Salmonella) or their endotoxins or exotoxins, microorganisms, enzymes, residual agricultural chemicals, environmental hormones, and the like. As the antibody used as the specific biopolymer, either a polyclonal antibody or a monoclonal antibody obtained by a well-known method can be used. In addition, the antibody may be a protein [eg, enzyme (eg, pepsin or papain)] [eg, antibody fragment (eg, Fab, Fab ′, F (ab ′)₂Or Fv)] can also be used.
[0086]
5. Shading part
In the microarray chip of the present invention, a light shielding portion may be formed so as to divide the portion where the fixing layer is formed. Since the light shielding portion is formed in this way, it is possible to efficiently reproduce the microarray chip as will be described later.
[0087]
That is, in the microarray chip of the present invention, in the one having a photocatalyst-containing layer as the wettability variable layer, after performing detection or the like using the microarray chip, the region where the immobilized layer of the microarray chip is formed, That is, by irradiating energy only to the portion where the photocatalyst-containing layer is a hydrophilic region, the specific biopolymer immobilized on the immobilization layer and the immobilization layer by the action of the photocatalyst in the photocatalyst-containing layer Disassembled and removed. At this time, the microarray chip can be easily reproduced by irradiating the entire surface without using a mask or the like by forming a light-shielding portion so as to divide the portion of the microarray chip where the fixing layer is formed. This is because it becomes possible.
[0088]
The light-shielding portion in the present invention can be formed by forming a metal thin film of chromium or the like having a thickness of about 1000 to 2000 mm by sputtering, vacuum vapor deposition or the like, and patterning this thin film.
[0089]
Further, a layer containing light shielding particles such as carbon fine particles, metal oxides, inorganic pigments, and organic pigments in a resin binder may be used as the light shielding portion. As the resin binder to be used, polyimide resin, acrylic resin, epoxy resin, polyacrylamide, polyvinyl alcohol, gelatin, casein, cellulose, or a mixture of one or more resins, photosensitive resin, or O / A W emulsion type resin composition, for example, an emulsion of a reactive silicone can be used. The thickness of such a resin light-shielding portion can be set within a range of 0.5 to 10 μm. As a method for patterning the resin light-shielding portion, a generally used method such as a photolithography method or a printing method can be used.
[0090]
The light shielding part in the present invention may be formed at any position on the base material. Specifically, the light shielding part may be formed on the substrate, and the photocatalyst containing layer may be formed thereon, or the photocatalyst containing layer may be formed on the substrate, and the light shielding part may be formed thereon. It may be formed. However, it is preferable to adopt a configuration in which a light-shielding portion is formed between the base material and the photocatalyst-containing layer from the viewpoint of easy production.
[0091]
When the light-shielding part is formed between the photocatalyst-containing layer and the base material, the energy irradiation during the regeneration of the microarray chip is performed from the back surface, that is, the surface of the base material on which the photocatalyst-containing layer is not formed. It becomes irradiation of energy. Therefore, the base material in this case is preferably formed of a material that transmits the irradiated energy.
[0092]
B. Manufacturing method of microarray chip
Next, the manufacturing method of the microarray chip of this invention is demonstrated. The manufacturing method of the microarray chip of the present invention includes:
(1) A step of providing a photocatalyst-containing layer that changes the wettability of an exposed portion by exposure on a substrate in a direction in which the contact angle with water decreases;
(2) A step of irradiating the pattern with energy to form a fixed layer exposed portion on a portion where the fixed layer on the photocatalyst-containing layer provided on the substrate is formed;
(3) attaching the fixing layer to the exposure part for the fixing layer;
(4) a step of immobilizing a specific biopolymer on the immobilization layer;
It is characterized by including.
[0093]
FIG. 2 is a view for explaining an example of a manufacturing method of such a microarray chip. In this example, first, the photocatalyst containing layer 2 is formed on the base material 1 (FIG. 2A). The photocatalyst-containing layer 2 is formed by dispersing a photocatalyst and a binder as described above in a solvent together with other additives as necessary to prepare a coating solution, and after applying this coating solution, heating or the like is performed. By performing the hydrolysis and polycondensation reaction, the photocatalyst is firmly fixed in the binder. The solvent to be used is preferably an alcohol-based organic solvent such as ethanol or isopropylol, and the coating can be performed by a known coating method such as spin coating, spray coating, dip coating, roll coating, bead coating, etc. Step (1)).
[0094]
Next, the substrate 1 on which the photocatalyst containing layer 2 is formed as shown in FIG. 2B is irradiated with a pattern of energy 6 such as ultraviolet light through a photomask 7. As a result, as shown in FIG. 2 (c), the portion for forming the immobilization layer on the photocatalyst-containing layer 2 is used as a hydrophilic region by the action of the photocatalyst in the photocatalyst-containing layer 5. 3 is formed (step (2) above).
[0095]
In the present invention, light including ultraviolet light can be used as the energy applied to the photocatalyst-containing layer. Examples of such a light source including ultraviolet light include a mercury lamp, a metal halide lamp, and a xenon lamp. The wavelength of light used for this exposure can be set in the range of 400 nm or less, preferably in the range of 380 nm or less, and the amount of light irradiation during the exposure exhibits hydrophilicity by the action of the photocatalyst. It is possible to make the irradiation dose necessary to do.
[0096]
Pattern irradiation upon energy irradiation can be performed by pattern irradiation through a photomask using a light source as described above. As another method, pattern irradiation is performed using a laser such as excimer or YAG. It is also possible to use a method to do this.
[0097]
In this way, the immobilization layer forming coating solution is applied and solidified in the immobilization layer exposure part 3 formed in the hydrophilic region, thereby fixing the immobilization layer as shown in FIG. 4 is formed. The application method of the fixing layer forming coating liquid at this time may be, for example, a method in which the entire surface of the substrate is applied by a dipping method or the like and adhered only to the hydrophilic region, or a discharge method such as an ink jet method may be used. Alternatively, the immobilization layer forming coating solution may be applied only to the immobilization layer exposure part.
[0098]
And as shown in FIG.2 (e), it can be set as a microarray chip | tip by attaching the specific biopolymer 5 on this fixed layer 4. FIG. The specific biopolymer 5 at this time is usually formed by applying a liquid containing the specific biopolymer by an ink-jet method or by spotting with a stamp pin and drying it. At this time, if necessary, a treatment for immobilizing the specific biopolymer on the immobilization layer may be performed.
[0099]
In addition, since the base material, the photocatalyst containing layer, the immobilization layer, and the specific biopolymer in the microarray chip manufacturing method are the same as those described for the microchip described above, description thereof is omitted here. .
[0100]
C. Microarray chip regeneration method
In the present invention, after using the above-described microarray chip for detection or the like, only the portion where the immobilized layer and the specific biopolymer are attached can be regenerated by irradiating energy in a pattern.
[0101]
Since the microarray chip of the present invention is reproducible in this way, it can be used a plurality of times and has the advantage that the final cost can be reduced.
[0102]
FIG. 3 is a view for explaining an example of a method for reproducing a microarray chip of the present invention. FIG. 3A shows the microarray chip after use of detection of biopolymers and the like is finished. In the microarray chip regeneration method of the present invention, as shown in FIG. 3B, the immobilized layer 4 and the specific biopolymer 5 are arranged on such a used microarray chip. Irradiation of energy 6 such as ultraviolet light through a photomask 7 is performed so that only a portion, that is, only the fixed layer exposure portion 3 is exposed. The portion of the photocatalyst containing layer 2 irradiated with energy decomposes the organic component by the action of the photocatalyst. Therefore, the immobilization layer 4 and the specific biopolymer 5 on the immobilization layer exposure unit 3 are decomposed by the action of the photocatalyst. On the other hand, the portion not irradiated with energy remains as a water-repellent region as it is. Then, if necessary, washing or the like is performed to obtain a base material 1 in which the immobilization layer exposure part 3 is formed in a pattern on the photocatalyst containing layer 2 as shown in FIG.
[0103]
By performing the above-described steps shown in FIGS. 2D and 2E on such a substrate, it can be reused as a microchip array.
[0104]
FIG. 4 shows another example of the method for reproducing the microchip array of the present invention. In this microchip array, a light shielding portion 8 is formed between the substrate 1 and the photocatalyst containing layer 2, and this light shielding portion 8 is formed at a position that delimits a portion where the immobilization layer is formed ( FIG. 4 (a)). When reproducing such a microarray chip, as shown in FIG. 4 (b), the light shielding portion 8 is formed by irradiating the entire surface with energy 6 from the side of the substrate 1 where the photocatalyst containing layer 2 is not formed. Energy is irradiated only to the portion where the immobilization layer 4 and the specific biopolymer 5 are attached, and these are removed by the action of the photocatalyst, as shown in FIG. 4 (c). Thus, the base material 1 in which the exposure part 3 for fixed layers was formed in the pattern form on the photocatalyst containing layer 2 is obtained. Also in this case, similar to the above-described example, this substrate can be reused as a microchip array by performing the above-described steps shown in FIGS. 2D and 2E.
[0105]
Note that the base material, photocatalyst-containing layer, immobilization layer, specific biopolymer, and light-shielding portion in the microarray chip regeneration method are the same as those described for the microchip described above, and are therefore described here. Is omitted.
[0106]
D. Microarray chip substrate
Finally, the microarray chip substrate of the present invention will be described. The substrate for a microarray chip of the present invention is formed on the substrate and the substrate, and comprises at least a photocatalyst and a binder, and the wettability changes so that the contact angle with water is reduced by energy irradiation. A photocatalyst-containing layer that is a layer, and the photocatalyst-containing layer is a hydrophilic region having a lower contact angle with water than other portions only in a portion where an immobilization layer for immobilizing a biopolymer is formed. It is characterized by.
[0107]
By using such a substrate, the immobilization layer can be easily formed in a pattern by applying the immobilization layer forming coating solution to the hydrophilic region formed in a pattern. If a specific biopolymer is attached on this, the periphery of the immobilization layer is a water-repellent region, so that a microarray chip having a specific biopolymer pattern with a uniform shape on the substrate can be obtained. Therefore, a microarray chip with high detection accuracy can be obtained.
[0108]
As an example of the base material for microarray chips of this invention, what is shown to the said FIG.3 (c) and FIG.4 (c) can be mentioned.
[0109]
In the present invention, as described in the microarray chip regeneration method, since the microarray chip can be efficiently regenerated, an immobilization layer is formed on the substrate 1 as shown in FIG. The base material for microarray chips which has the light-shielding part 8 formed so that it may divide the position where the photocatalyst containing layer 2 is further formed may be used. In this case, the photocatalyst-containing layer is formed with an immobilization layer exposure portion 3 that is previously made a hydrophilic region in a region where the immobilization layer is formed.
[0110]
In addition, since the base material, the photocatalyst-containing layer, the immobilization layer, the specific biopolymer, and the light-shielding part in the microarray chip base material are the same as those described in the microchip described above, the description here. Is omitted.
[0111]
As described above, the microarray chip, the manufacturing method thereof, the reproduction method thereof, and the microchip array substrate of the present invention have been described, but the present invention is not limited to the above embodiment. The above-described embodiment is an exemplification, and the present invention has substantially the same configuration as the technical idea described in the claims of the present invention, and any device that exhibits the same function and effect is the present invention. It is included in the technical scope of the invention.
[0112]
【Example】
Hereinafter, the present invention will be described in more detail through examples.
[0113]
[Example 1]
Photocatalyst with 0.4 g of MF-160E (trade name, manufactured by Tochem Products Co., Ltd.) and 30 g of trimethoxymethylsilane (trade name TSL8113, manufactured by Toshiba Silicone Co., Ltd.), whose main component is fluoroalkylsilane, in 30 g of isopropyl alcohol 20 g of STS-01 (trade name, manufactured by Ishihara Sangyo Co., Ltd.), which is an aqueous dispersion of titanium dioxide, was mixed and stirred at 100 ° C. for 20 minutes to obtain a composition for a photocatalyst-containing layer.
[0114]
A photocatalyst with a thickness of 0.2μm is contained by applying the above composition with a spin coater on a slide glass (opening 50μm diameter 100μm pitch) with a light-shielding part formed of chrome, and performing a drying treatment at 120 ° C. for 10 minutes. A layer was formed. The contact angle of water on the photocatalyst-containing layer surface was measured and found to be 90 °.
[0115]
Exposure from the slide glass side of the above substrate (365nm 500mJ / cm²Then, the wettability of the photocatalyst-containing layer surface on the opening was changed to 10 ° or less by the contact angle of water to form an immobilization layer exposure part.
[0116]
The substrate was dipped in a 3% aqueous solution of poly L-lysine and pulled up to form an immobilization layer only in the exposed area.
[0117]
A microarray chip was prepared by discharging various DNA solutions onto the immobilization layer by an ink jet method and immobilizing them.
[0118]
[Example 2]
Exposure of the above used microarray chip from the glass substrate side (365nm 1000mJ / cm²Then, poly L-lysine and DNA were decomposed and removed.
[0119]
In the same manner as above, poly L-lysine and DNA were formed to obtain a microarray chip.
[0120]
【The invention's effect】
According to the microarray chip of the present invention, since the specific biopolymer is present only on the immobilization layer formed in a pattern and the other region is a water-repellent region, it is spotted on the immobilization layer. There is no problem that the specific biopolymer wets and spreads and the adjacent spots come into contact with each other. Therefore, there is no problem that the shape of the specific biopolymer is disturbed and the detection accuracy of the microarray chip is lowered, and the detection accuracy can be improved.
[Brief description of the drawings]
FIG. 1 is a schematic cross-sectional view showing an example of a microarray chip of the present invention.
FIG. 2 is a process diagram for explaining an example of a method for producing a microarray chip of the present invention.
FIG. 3 is a process diagram for explaining an example of a method of reproducing a microarray chip according to the present invention.
FIG. 4 is a process diagram for explaining another example of the microarray chip reproduction method of the present invention.
[Explanation of symbols]
1 ... Base material
2 ... Photocatalyst containing layer
3 ... Exposure part for fixed layer
4 ... Immobilization layer
5 ... Specific biopolymer
8 ... Shading part

Claims (11)

A substrate, a wettability variable layer formed on the substrate and capable of changing wettability by an external stimulus, and formed on the wettability variable layer for immobilizing a biopolymer. An immobilization layer and a specific biopolymer that is immobilized on the immobilization layer and specifically binds to a target biopolymer; and the wettability variable layer is formed into a pattern by an external stimulus. A microarray chip comprising a hydrophilic region and a fixed layer formed on the hydrophilic region on the wettability variable layer. The wettability variable layer is a photocatalyst-containing layer composed of at least a photocatalyst and a binder, and is a layer whose wettability changes so that a contact angle with water is reduced by energy irradiation. 1. The microarray chip according to 1. The photocatalyst is composed of titanium oxide (TiO ₂ ), zinc oxide (ZnO), tin oxide (SnO ₂ ), strontium titanate (SrTiO ₃ ), tungsten oxide (WO ₃ ), bismuth oxide (Bi ₂ O ₃ ), and oxide. _3. The microarray chip according to claim 2, wherein the microarray chip is one or more substances selected from iron (Fe ₂ O ₃ ). Microarray chip of claim 3, wherein the photocatalyst is titanium oxide (TiO _2). The binder is Y _n SiX _(4-n) (wherein Y represents an alkyl group, a fluoroalkyl group, a vinyl group, an amino group, a phenyl group or an epoxy group, X represents an alkoxyl group or a halogen, and n represents It is an integer from 0 to 3. This is an organopolysiloxane that is one or more of the hydrolytic condensates or cohydrolytic condensates of the silicon compound represented by (2) to (3). The microarray chip according to any one of claims 4 to 4. The microarray chip according to any one of claims 1 to 5, wherein the immobilization layer is formed of a cationic substance. The microarray chip according to any one of claims 1 to 6, wherein the specific biopolymer is a probe DNA that specifically binds to a target nucleic acid. The microarray chip according to any one of claims 2 to 7, wherein a light-shielding portion is formed on the base material so as to divide a portion where the immobilization layer is formed. . (1) A step of providing a photocatalyst-containing layer that changes the wettability of an exposed portion by exposure on a substrate in a direction in which the contact angle with water decreases;
(2) A step of forming an exposure part for the fixed layer by pattern irradiation with energy on a site for forming the fixed layer on the photocatalyst-containing layer provided on the substrate;
(3) attaching a fixing layer to the exposure part for the fixing layer;
(4) A method for producing a microarray chip, comprising a step of immobilizing a specific biopolymer on the immobilization layer. After the microarray chip according to any one of claims 2 to 8 is used, only a portion to which the specific biopolymer is attached is exposed in a pattern. Playback method. A base material, and a photocatalyst-containing layer that is formed on the base material and includes at least a photocatalyst and a binder, and is a layer whose wettability changes so that a contact angle with water is reduced by energy irradiation. In the microarray chip, the photocatalyst-containing layer is a hydrophilic region having a contact angle with water lower than other portions only in a portion where an immobilization layer for immobilizing a biopolymer is formed. Base material.

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