JP2004502138A - Composite array using hybridization chamber and microspheres - Google Patents

Abstract

The present invention relates to a sensor composition comprising a composite array of individual arrays to allow simultaneous processing of multiple samples. The present invention further provides methods of making and using composite arrays. The invention further provides a hybridization chamber for use in a composite array.

Description

【０１１１】
蛍光色は、デコーディングスキームにおいて使用するために特に都合のよい属性である。蛍光色は、ＩＢＬを認識するいずれの薬剤にも接着できて、標識されたＤＢＬを形成する。議論は、ＤＢＬとしてのオリゴヌクレオチド（核酸類似物を包含する）に向けられる。蛍光標識したオリゴヌクレオチドは、特異的、かつ、可逆的にビーズの任意の所望のサブセットを特定の色で、ハイブリダイゼーションおよびデハイブリダイゼーション（すなわち、相補的配列を有するＤＢＬに対して）の過程によって単純に「ペイント」（標識）することができるので、特に有用なＤＢＬである。さらに、蛍光は容易に画像化され、標準的な光学的ハードウェアおよびソフトウェアを用いて定量される。所定のビーズタイプを特定の色で「ペイント」するために、ビーズタイプは固有のハイブリッド形成可能なＤＮＡ配列（ＩＢＬ）で標識されなければならず、デコーディング溶液は該配列の色で標識された補体を含有しなければならない。
【０１１２】
デコーディングスキームの実施において１の考慮すべき事柄は、集められる画像の数を最小限にすることである。色に基づくスキームにおいて、集められる画像の数は色の数および段階の数の産物である。画像の数は、各所定の段階につき複数の色でビーズを「ペイント」することによって減らすことができる。複数の色をビーズに与えることにより、効果的なコードの数が増加する。例えば、コンピューターによって使用される２４ビットの３色のスキーム（例えば、赤、緑、青）カラーリングプロセスにおいて、全部で２５６^＊２５６^＊２５６＝１６７０００００個の異なる「色相」がたった３色（赤、緑、青）から生じることができる。
したがって、好ましい具体例において、ＤＢＬは、有色の蛍光体の組み合わせを用いて標識される。該方法はそれ自体、ほんの少数の異なる色素（色）を用いてＤＢＬを標識するために利用可能なコードの数を増やすことにおいて有用である。各デコーディング工程で利用可能なコードの数を増やすことは、所定のデコーディング過程において必要とされるデコーディング工程の数を大幅に減らすであろう。
【０１１３】
一例において、単一のＤＢＬをエンコードするオリゴヌクレオチドの集団は、ＤＢＬが結合する各ビーズが有色の蛍光体の組み合わせから公式化される特徴的な「色相」に基づいて同定されるように、所定の比率の色で標識される。好ましい具体例において、２つの別個の色を用いる。好ましい具体例において、３またはそれ以上の別個の色素（色）が利用可能である。該例において、単一のＤＢＬをエンコードしているオリゴヌクレオチドの集団をいずれかの所定の色で標識することによって生じた区別可能なコードの数は３である。しかしながら、標識化における色および色のレベルの組み合わせを考慮することによって、より多くのコードが生じる。
ハイブリダイゼーションによるデコーディングについて、識別可能な色調の好ましい数は２〜２０００であり、識別可能な色調のより好ましい数は２〜２００であり、識別可能な色調の最も好ましい数は２〜２０である。３つの異なる色調（強度）および３色を用いると、異なる色相の数が３^４＝８１になる。色相と連続的デコーディングの組み合わせにより、事実上無限数のコードを生じさせることができる。
【０１１４】
以前の記載のように、ＤＢＬは、ＩＢＬに結合するいずれかの薬剤であることができる。好ましい具体例において、単一のＤＢＬは予め決定された比率の色で標識される。該比率は、各ＤＢＬについて変化され、したがって、標識される各ＤＢＬ自体につき固有の「色相」が可能である。ビーズのＤＢＬでの処理後、ビーズを分析して、各ビーズと結合した「色相」を決定し、それにより、その結合した生物活性剤を有するビーズを同定する。
例えば、４つの主要な色および２つの強度レベル（色の存在または不在）を用いて、１５の異なる色相／段階が可能である。４つの色素および３つの異なる強度レベルを用いる場合（不在、半分存在、完全に存在）、７３の異なる色相／段階が可能である。この場合、たった４色の画像の獲得が、７３の異なるコーディングの色相についての情報を得るのに十分である。
【０１１５】
好ましい具体例において、本発明は、分離した部位を含む表面を有する第一の基体を含むアレイ組成物を提供する。好ましい具体例は、該部位に分布した微小球の集団を利用し、該集団は少なくとも第一および第二のサブ集団を含む。各サブ集団は、生物活性剤およびさらに、所定のｐＫａを有する少なくとも１個の光学的色素を含む。異なる光学的色素のｐＫａは異なる。
好ましい具体例において、例えば、アレイがクローン化した核酸を含む場合、アレイをデコードするために用いることのできるいくつかの方法がある。好ましい具体例において、クローン化核酸についてのいくつかの配列情報が知られている場合、本明細書に広く概説するように、特定のデコーディングプローブを作製することができる。
【０１１６】
好ましい態様において、「ランダム」なデコーディングプローブを作製することができる。前に概説したように、一連のハイブリダイゼーションまたは複数の標識の使用により、固有のハイブリダイゼーションパターンが各センサーエレメントに関して生じ得る。これにより、所定のクローンを示すビーズの全ては同じグループに属するとみなすことができる。一般に、これは、配列依存的であるが高程度に配列特異的ではない様式で結合するランダムまたは部分変性デコーディングプローブを用いて行う。該プロセスは各回、異なる標識物質を用いて何回も繰り返すことができ、ある意味特異的な相互作用に基づく異なるシグナルパターンを生じることができる。この方法において、各センサーエレメントに関して固有の光学的サインがついに確立される。パターン認識またはクラスター形成アルゴリズムを光学的サインに適用することにより、ビーズを同じサインを共有する（すなわち、同じプローブを有する）セットにグループ分けすることができる。
【０１１７】
クローンそのものの実際の配列を同定するためには、追加的方法が必要とされ、例えば、直接配列決定を行うことができる。斑点ｃＤＮＡアレイ（ａ ｓｐｏｔｔｅｄ ｃＤＮＡ）のような、クローンを含むオーダーアレイ（ｏｒｄｅｒｅｄ ａｒｒａｙ）を用いることにより、該セット内でのその位置が分かっている特定クローンにハイブリダイゼーションパターンを関係付ける「鍵」を生じることができる。この方法で、クローンを回収し、さらに特徴づけることができる。
別法では、クローンアレイは、ベクタータグを有する二元デコーディングを用いてデコードすることができる。例えば、部分的にランダム化したオリゴを核酸ベクター（例えばプラスミド、ファージなど）中にクローン化する。各オリゴヌクレオチド配列は限られたセットの配列のサブセットから成る。例えば、限られたセットが１０配列を含む場合、各オリゴヌクレオチドは幾つかのサブセット（または１０の全て）配列を有してもよい。つまり、１０配列のそれぞれがオリゴヌクレオチド中に存在することもできるし、あるいは存在しないことも可能である。それゆえ、２^１０または１０２４の可能な組み合わせが存在する。配列は重複してもよく、可能な組み合わせの数を増すために、副次的な変種を示すこともできる（例えばＡ、Ｃ、ＴおよびＧ置換）。核酸ライブラリーをランダムなコード配列を含むベクター中にクローン化する。別法では、ＰＣＲのような他の方法を用いてタグを付加することができる。この方法において、クローンのアレイをデコードするために、少数のオリゴデコーディングプローブを用いることができる。
【０１１８】
好ましい具体例において、判別分析およびクラスターアルゴリズムおよびコンピューター装置を用いて、本発明のアレイ由来のデコーディングデータを分析する。多段デコーディングプロセスにおいて異なる強度および「色相」の蛍光体の使用と組み合わせた本発明に用いられる潜在的に多数のコードは、データの良好な分類を必要とする。データ、特に強度データは、各段階にてビーズが異なる色または色の混合（「色相」）で可逆的に標識される（例えば、色素標識された相補的なデコーディングオリゴヌクレオチドをビーズ上のＩＢＬプローブにハイブリッド形成することによるか、または非核酸ＩＢＬ−ＤＢＬ対のための結合リガンド対の形成による）多段プロセスにおいてもたらされる。難題は、各工程でどの色がペイントされたかにしたがってビーズを正確に分類することである。標識が互いにより密接に関係すればするほど（光学的画像システムによって決定されるように）、分類が困難になる。
【０１１９】
画像システムによって示されるような色素の接近は、デコーディング色素のスペクトル特性および画像システムのスペクトルチャンネル分離によって決定される。より良好な色の分離は、狭い発光スペクトルを有する蛍光色素を用いることによって、および色素を「ピークで」励起し、「ピークで」その発光を測定するために設計された狭い帯域通過励起および発光フィルターを有する光学系を用いることによって達成される。ビーズ上の色素を光学的に画像化する過程は、目の中の３つの異なる円錐タイプにおける励起の比率を測定することによって脳が色を見るヒトの視覚過程に類似する。しかしながら、光学的画像システムを用いると、実際の色のチャンネルの数は、ヒトの目に存在する３つよりも非常に多い。ＣＣＤに基づく画像システムは、３５０ｎｍから８５０ｎｍまでの色を「見る」ことができるが、目の円錐体は５００−６００ｎｍの可視スペクトルに調律されている。
【０１２０】
ビーズアレイをデコーディングする問題は、本質的に、判別分析分類の問題である。したがって、好ましい具体例において、ハイパースペクトルαスペースにおける変動の分析は、ビーズの色または色相の既知のセットにおいて行われる。αスペースにおけるビーズクラスターの中心は、クラスターの重心と呼ばれ、クラスター内の点の散乱がクラスターの拡散を決定する。しっかりとした分類スキームは、異なるビーズクラス（色相）の重心間の距離がいずれかのクラスタークラスの拡散より非常に大きいことを必要とする。さらに、重心の場所は、ファイバー毎におよび実験毎に変化しないままであるべきである。
したがって、好ましい具体例において、色相「ゾーン」は、色相の重心の周囲であって、クラスターの拡散半径の外まで広がっているαスペース中の領域として定義される。色相の重心および拡散半径の対照セットを用いると、経験的に決定されるように、データの新規なセットの分類は、所定のビーズ点が色相クラスターの「ゾーン」の近くまたは中に落ちるかどうかを求めることによって達成できる。これは、異なる色相クラスの重心からビーズ点のマハラノビス距離（この場合、それは単純にユークリッド距離計量である）を計算することによって達成される。図３に示されるデータについて、重心の場所およびそれらの互いからの距離を表２に示す。
【０１２１】
【表２】

【０１２２】
特定の色相クラスに異なるビーズを分類するために、０．３のユークリッド距離カットオフを選択した。最も近い２つの重心、Ｂｏｄ−Ｒ６ＧおよびＢｏｄ−５６４（距離＝０．５５）は、０．３のユークリッドまたはマハラノビス距離を用いる場合、デコーディングゾーンにおいてわずかに重複する。分類における改善は、この距離を減らし、異なる座標軸に適当に重みを与えることによって達成される。
したがって、本発明は、ビーズの色を分析および分類するコンピューター法を提供する。ビーズの色の分類は、ハイパースペクトル「α」スペース中においてビーズを調べることによって行われ（ａ_１＝Ｉ_１／ＳＩ_ｉ，ａ_２＝Ｉ_２／ＳＩ_ｉ，ａ_３＝Ｉ_３／ＳＩ_ｉ，など）、ここに、各座標軸は、所定の画像チャンネル内のビーズ強度のフラクションを示す。例えば、４つの画像チャンネルを用いてビーズを画像化する場合、ビーズの色または色相は、３−Ｄαスペース中の点によって示すことができる（Ｓａ_ｉ＝１なので、第４の次元は必要ない）。ビーズを標識する異なる主要な色素のセットを用いる場合、色素は比率の変化および組み合わせパターンの変化において組み合わせることができるので、これらの色素から生じることのできる色相の数は際限がない。実際の色相の数は、ハイパースペクトルαスペース中における異なる色相クラスターの分離によって、実験的に決定される。
【０１２３】
図３は、４つの分離した画像チャンネルにおいて画像化される４つの異なる色相で標識されたビーズのハイパースペクトルαプロットを示す。ビーズが４つの別個のクラスターを形成することに注目すること。これらの４つのクラスターがよく分離しているという事実は、しっかりとしたデコード分類スキームの実行を可能にする。
好ましい具体例において、デコーディング過程の品質管理分析を行う。これは、各デコーディング段階につきαスペースのクラスター分析を行うことによって達成される。決定されるクラスターの数は、色相の予想数によって固定されるであろう。クラスター重心の位置はモニターされ、予想される位置からのいずれかの偏りが注目されるであろう。
【０１２４】
したがって、本発明は、本発明のアレイをデコーディングするための装置を提供する。本明細書に概説される組成物に加えて、装置は、メモリーおよび入力／出力装置のセット（例えば、キーボード、マウス、モニター、プリンターなど）とバスを介して通信する中央処理装置を包含する。中央処理装置、メモリー、入力／出力装置およびバスの間の一般的な相互作用は当該分野で既知である。本発明の１の態様は、メモリー中に貯蓄されたハイパースペクトル「α」スペース分類システムに向けられている。
分類システムプログラムは、光学的リーダーまたは共焦顕微鏡（または他の画像システム）からデータを受けるデータ獲得モジュールを包含する。一般に、分類プログラムはまた、ハイパースペクトルαスペース中の変化を分析でき、クラスターの重心を計算でき、クラスターの散乱（拡散）を計算でき、色相ゾーンおよび距離カットオフを明らかにすることのできる分析モジュールを包含する。一般に、分析モジュールはさらに、マハラノビス距離を計算することによって、データ点が色相ゾーン内に落ちるかどうかを決定するであろう。
【０１２５】
最終的に、分析モジュールは、最終的に生物活性剤をビーズ場所に与えるために、異なる一連のデコーディング情報を分析するであろう。
このように、各工程が判別分析計算を用いる一連のデコーディング工程を行って、各工程にて、アレイ中の各ビーズを色相クラスターに与える。一連のデコーディング情報の蓄積は、ビーズの場所とそこに含まれる化学的性質の相互関係を与える。
一度作成されると、本発明の組成物は、多くの応用において使用できる。好ましい態様において、存在する標的アナライトの量の定量化を含め、標的アナライトの存在または不在に関してサンプル溶液をプローブするために該組成物を用いる。本明細書中の「標的アナライト」または「アナライト」または文法的に等しいものにより、結合パートナーについて検出あるいは評価されるいずれかの原子、分子、イオン、分子イオン、化合物または粒子を意味する。当業者には明らかであるように、多くのアナライトを本発明に用いることができ、基本的には、生物活性剤を結合するいずれのアナライトも、または、結合パートナー（すなわち薬物候補物質）が求められているいずれのアナライトも用いることができる。
【０１２６】
適当なアナライトには、生物分子を含む有機および無機分子が含まれる。標的アナライトの検出を行う場合、適当な標的アナライトには、制限されるものではないが、環境汚染物質（農薬、殺虫剤、毒素などを含む）、化学物質（溶媒、ポリマー、有機物質などを含む）、治療分子（治療薬および濫用薬物、抗生物質などを含む）、生物分子（ホルモン、サイトカイン、タンパク質、核酸、脂質、炭水化物、細胞膜抗原およびレセプター（神経、ホルモン、栄養素および細胞表面レセプター）またはそれらのリガンドなどを含む）、全細胞（原核細胞（病原性細菌など）および真核細胞、哺乳動物の腫瘍細胞を含む）、ウイルス（レトロウイルス、ヘルペスウイルス、アデノウイルス、レンチウイルスなどを含む）および胞子などが含まれる。特に好ましいアナライトは、核酸およびタンパク質である。
【０１２７】
好ましい具体例において、標的アナライトはタンパク質である。当業者には明らかであるように、本発明を用いて結合パートナーについて検出または評価することができる、多数の可能性のあるタンパク質性標的アナライトがある。適当なタンパク質標的アナライトには、制限されるものではないが、（１）免疫グロブリン；（２）酵素（および他のタンパク質）；（３）ホルモンおよびサイトカイン（その多くは細胞レセプターのリガンドとして役立つ）および（４）他のタンパク質が含まれる。
好ましい具体例において、標的アナライトは核酸である。米国特許番号６０／１６００２７；６０／１６１１４８号；０９／４２５６３３および６０／１６０９１７（全て、出典明示により明らかに本明細書の一部とされる）に広く概説されるように、これらのアッセイは、幅広い範囲の適用に用いられる。
【０１２８】
好ましい具体例において、プローブは遺伝子診断に用いられる。例えば、プローブを本明細書中に開示する方法を用いて作成し、非ポリープ性の結腸癌、ＢＲＣＡ１乳癌遺伝子、様々な癌に関連する遺伝子であるＰ５３、アルツハイマー病のより大きな危険性を示すＡｐｏＥ４遺伝子のような標的配列を検出することができ、患者、嚢胞性繊維症遺伝子、シトクロムｐ４５０ｓまたは当業者によく知られる他のいずれかの変異を容易に前駆症状スクリーニング（ｐｒｅｓｙｍｐｔｏｍａｔｉｃ ｓｃｒｅｅｎｉｎｇ）することが可能となる。
さらなる具体例において、ウイルスおよび細菌の検出を、本発明の複合体を用いて行う。この態様において、様々な細菌およびウイルス由来の標的配列を検出するためにプローブを設計する。例えば、最新の血液スクリーニング技術は抗ＨＩＶ抗体の検出による。本明細書中に開示する方法により、ＨＩＶ核酸配列、特に高度に保存されたＨＩＶ配列を検出するための臨床サンプルの直接スクリーニングが可能となる。加えて、抗ウイルス治療の効能を評価する改良法のように、これにより患者の体内に循環しているウイルスを直接測定することが可能となる。同様に、白血病、ＨＴＬＶ−ＩおよびＨＴＬＶ−ＩＩと関連するウイルスをこの方法で検出することができる。結核、クラミジアおよび他の性的伝染病のような細菌感染を検出することもできる。
【０１２９】
好ましい具体例において、本発明の核酸は、水および食物サンプルのスクリーニングにおいて毒性細菌のためのプローブとして用いる。例えば、サンプルを処理して細菌を溶解し、その核酸を放出させ、次いで、サルモネラ（Ｓａｌｍｏｎｅｌｌａ）、カンピロバクター（Ｃａｍｐｙｌｏｂａｃｔｅｒ）、ビブリオコレラ（Ｖｉｂｒｉｏ ｃｈｏｌｅｒａｅ）、リーシュマニア（Ｌｅｉｓｈｍａｎｉａ）、イー．コリのエンテロトキシン菌株および在郷軍人病細菌のような病原性菌株を含むがこれらには制限されない細菌株を認識するプローブを設計することができる。同様に、バイオレメディエーション法を本発明の組成物を用いて評価することができる。
さらなる具体例において、犠牲者および容疑者から採取したサンプルに対して犯罪事件のＤＮＡ（ｃｒｉｍｅ−ｓｃｅｎｅ ＤＮＡ）を対応させるための法医学「ＤＮＡフィンガープリント法」のために該プローブを用いる。
さらなる具体例において、アレイ中のプローブをハイブリダイゼーションによる配列決定のために用いる。
【０１３０】
本発明はまた、標的核酸配列における変異またはミスマッチの検出のための方法として用いる。例えば、多形ＤＮＡマーカーを使用することにより遺伝子変異と表現型の間の関係を分析することが近年注目されている。これまでの研究は、多形位置マーカーとして短いタンデムリピート（ＳＴＲ）を利用したが、近年は、単一ヌクレオチド多形（ｐｏｌｙｍｏｒｐｈｉｓｍｓ）（ＳＮＰ）の使用が注目されている。共通のＳＮＰｓは、ヒトのゲノムＤＮＡ１キロベースあたり１以上の平均頻度で生じる。いくらかのＳＮＰ、特にコーディング配列内およびその付近のものは、治療的適切な表現型変種の直接原因であるようである。臨床的に重要な表現型を引き起こす多くの周知の多形があり、例えば、ａｐｏＥ２／３／４変種はアルツハイマー病および他の疾患の異なる相対リスクと関連する（Ｃｏｒｄｏｒ ｅｔ ａｌ．， Ｓｃｉｅｎｃｅ ２６１（１９９３）を参照されたい）。オリゴヌクレオチドアレイに対するその後のハイブリダイゼーションを用いたＳＮＰ位の多重ＰＣＲ増幅は、少なくとも何百ものＳＮＰを同時にゲノタイピング（ｇｅｎｏｔｙｐｉｎｇ）する迅速かつ信頼できる方法であることが示されている：Ｗａｎｇ ｅｔ ａｌ．， Ｓｃｉｅｎｃｅ， ２８０：１０７７（１９９８）を参照されたい；さらに、Ｓｃｈａｆｅｒ ｅｔ ａｌ．， Ｎａｔｕｒｅ Ｂｉｏｔｅｃｈｎｏｌｏｇｙ １６：３３−３９（１９９８）も参照されたい。本発明の組成物は従来技術のアレイに容易に取って代わることができる。特に、単一塩基伸長（ｓｉｎｇｌｅ ｂａｓｅ ｅｘｔｅｎｓｉｏｎ（ＳＢＥ））およびパイロシークエンシング技術が特に本発明の組成物に有用である。
【０１３１】
好ましい態様において、本発明の組成物を用いて生物活性剤をスクリーニングし、標的分子に結合して好ましくはその機能を修飾する薬剤を見出す。前記のように、当業者には明らかなように、幅広い種類の異なるアッセイフォーマットを作動させてもよい。一般に結合パートナーが所望される標的アナライトを標識し、標的アナライトを生物活性剤に結合させて標識をビーズに補充し、続いて検出する。
好ましい態様において、生物活性剤と標的アナライトの結合は特異的である；つまり、生物活性剤は標的アナライトに特異的に結合する。本明細書中、「特異的結合」は、アナライトと、試験サンプルの他の成分または汚染物質を区別するのに十分特異的に物質がアナライトに結合することを意味する。しかし、当業者には明らかなように、それほど特異的でない結合を用いてアナライトを検出することができるであろう。例えば、該システムは異なる結合リガンド、例えば異なるリガンドのアレイを用いてもよく、特定アナライトのいずれの検出も結合リガンドのパネルに結合するその「サイン」によるものであり、「電子ノーズ（ｅｌｅｃｔｒｏｎｉｃ ｎｏｓｅ）」が働く様式と類似している。これは化学アナライトの検出において特に利用できる。結合は、非特異的結合を除去するための洗浄工程を含む（いくらかの態様においては、洗浄工程は必要とされないが）アッセイの条件下で結合を維持するのに、すなわち低いアフィニティーの結合パートナーを検出するのに十分であるべきである。いくらかの態様、例えばある種の生物分子の検出においては、アナライトの結合リガンドへの分離定数は約１０^−４−１０^{− ６}Ｍ^−１より小さく、約１０^{− ５}−１０^{− ９}Ｍ^{− １}より小さいことが好ましく、１０^−７−１０^−９Ｍ^−１より小さいことが特により好ましい。
【０１３２】
一般に、（標的アナライトの検出のための、または、標的アナライトの結合パートナーをスクリーニングするための）標的アナライトを含むサンプルを、少なくとも一つの生物活性剤に対する標的アナライトの結合に適した条件下、すなわち、一般に生理的条件下でアレイに添加する。標的アナライトの存在または不在を次いで検出する。当業者には明らかなように、これは様々な方法で、一般に光学シグナルの変化を用いて行うことが出来る。この変化は、多くの異なるメカニズムを介して起こることができる。少数の例としては、ビーズへの色素タグを付加したアナライトの結合、ビーズ上または付近での色素種の産生、存在する色素種の破壊、ビーズ上の色素とのアナライト相互作用における光学的サインの変化、またはその他の光学的に応答指令信号を送信可能な事象が含まれる。
【０１３３】
好ましい例においては、光学的シグナルの変化は、直接的または間接的に検出可能な標識（好適には蛍光色素のような光学的標識）で標識された標的アナライトの結合の結果として生じる。したがって、例えば、タンパク質性標的アナライトを用いる場合、例えば標識した抗体の使用によって蛍光（ｆｌｕｏｒ）で直接標識するか、または非直接的に標識できる。同様に、核酸は、当該分野で既知のように例えばＰＣＲ増幅を通じて、蛍光色素で容易に標識される。別法として、標的配列の結合に基づき、ハイブリダイゼーション指示薬を標識として用いることができる。ハイブリダイゼーション指示薬は通常可逆的に二本鎖核酸と優先的に結合する。ハイブリダイゼーション指示薬は、インターカレーター（ｉｎｔｅｒｃａｌａｔｏｒ）ならびに副溝および／または主溝結合部を包含する。好適な一具体例においては、インターカレーターを用いることができる；インターカレーションは通常二本鎖核酸の存在下においてのみ起こるので、標的ハイブリダイゼーションの存在下でのみ標識が明るくなる。したがって、標的アナライトの生物活性剤への結合に基づいてこの部位に新たな光学的シグナルが発生し、検出できる。
【０１３４】
別法として、いくつかの場合において、上記のように、酵素のような標的アナライトは直接的または間接的に光学的に検出できる種を生じる。
さらに、いくつかの具体例では、光学的サインの変化は光学的シグナルに基づきうる。例えば、ある化学的標的アナライトとビーズ上のある蛍光色素との相互作用は光学的サインを変化させることができ、したがって異なる光学的シグナルを生じる。
当業者に理解されるように、いくつかの具体例では、標的アナライトの存在または不在は、他の光学的または非光学的シグナル（表面増強ラマン分光法、表面プラズモン共鳴、放射活性等を含むが、これに限定されるものではない）における変化を用いてなされうる。
【０１３５】
当業者に理解されるように、アッセイは様々な実験条件下で実施できる。スクリーニングアッセイにおいて、種々の他の試薬が含まれうる。これらは最適なタンパク質−タンパク質結合を促進し、および／または非特異的もしくはバックグラウンドの相互作用を減少させるために用いることができる塩、中性タンパク質（例、アルブミン、界面活性剤等）のような試薬を包含する。また、プロテアーゼ阻害剤、ヌクレアーゼ阻害剤、抗微生物剤等のような、別の方法でアッセイの効率を改良する試薬も用いることができる。成分の混合物は、必要な結合を与える任意の順番で添加できる。当業者に知られているように、種々のブロッキング工程および洗浄工程を用いることができる。
【０１３６】
好ましい例においては、２色拮抗ハイブリダイゼーションアッセイを実行する。これらのアッセイは慣用のサンドイッチアッセイに基づくことができる。ビーズはＳＮＰの片側（上流または下流）に位置する捕獲配列を含み、標的配列を捕獲できる。各々異なる蛍光体で標識した２つのＳＮＰ対立遺伝子特異的プローブを標的配列にハイブリダイズさせる。通常、より良い結合を示す較正配列を用いて、２つのシグナルの比率から遺伝子型を得ることができる。これは、標識される必要のない標的配列自体において有利である。さらに、プローブは拮抗するので、これは結合条件を最適化する必要がないことを意味する。ミスマッチのプローブが安定して結合する条件下で、マッチしたプローブをさらに置き換えることができる。したがって、拮抗アッセイはそれらの条件下でより良い識別を与えることができる。多くのアッセイを平行して実施するので、全てのプローブについて条件を同時に最適化することはできない。したがって、拮抗アッセイシステムを用いて、ミスマッチ識別についての非最適条件を補うことを補助するために用いることができる。
【０１３７】
好適な一具体例においては、本発明の組成物を用いてジデオキシヌクレオチド チェーン ターミネーション配列決定を行う。この具体例においては、ＤＮＡポリメラーゼにより蛍光的に標識したｄｄＮＴＰを用いてプライマーを伸長する。該プライマーの３’末端はＳＮＰ部位に隣接する。この方法において、単一塩基伸長は該ＳＮＰ部位の配列に相補的である。各塩基について１つの、４つの異なる蛍光体を用い、４つの塩基特異的シグナルを比較することによりＳＮＰの配列を導き出すことができる。これはいくつかの方法で行うことができる。第一の具体例では、捕捉プローブを伸長できる；このアプローチでは、プローブはビーズ上で５’−３’合成されるか、または５’末端に結合されて、ポリメラーゼ伸長のための遊離３’末端を与えなければならない。別法として、サンドイッチ型アッセイを用いることができる；この具体例では、プローブによって標的をビーズ上に捕捉し、ついでプライマーをアニーリングし、伸長する。また、後者の場合、標的配列は標識されていなくてもよい。さらに、サンドイッチアッセイは２つの特異的相互作用を必要とするので、これは複合サンプルの分析に特に有益なストリンジェンシーを増加させる。
【０１３８】
さらに、標的アナライトおよびＤＢＬの双方が薬剤に結合する場合、デコーディングの拮抗を介して非標識化標的アナライトの検出を行うことも可能である。 好適な一具体例においては、本発明の方法はアレイ品質管理において有益である。本発明以前には、あらゆるアレイ上のあらゆるプローブの性能のポジティブ試験を提供する方法は開示されていない。アレイのデコーディングがこの試験を提供するだけではなく、そのデコーディング過程自体の間に生じたデータを利用することによってもそのようにする。したがって、さらなる実験作業は要求されない。本発明はソフトウエアにコードできる１組のデータ分析アルゴリズムのみを必要とする。
【０１３９】
品質管理処置はアレイ中の広範な体系的およびランダムな問題を同定できる。例えば、塵または他の汚染物質のランダムな斑点はいくつかのセンサーに正しくないシグナルを与えさせうる−これはデコーディングの間に検出できる。複数のアレイに由来する１以上の薬剤の遺漏もまた検出できる。この品質管理処置の利点は、アッセイ自体の直前に実行でき、個々のセンサーの真の機能試験であることである。したがって、アレイアセンブリ（ａｓｓｅｍｂｌｙ）と実際の使用との間で生じうるあらゆる問題を検出できる。非常に高レベルの信頼性が必要で、および／または実験手順の間にセンサーの故障の重大な可能性がある場合の応用において、デコーディングと品質管理は実際のサンプル分析の前後両方で実施できる。
【０１４０】
好適な一具体例においては、アレイを用いて試薬品質管理を行うことができる。多くの例では、生物学的な巨大分子が試薬として用いられ、品質管理されなければならない。例えば、オリゴヌクレオチドプローブの大きなセットが試薬として提供されうる。多数の異なる生物学的な巨大分子において品質管理を実行することは通常困難である。本明細書に記載したアプローチを用いて、アレイの代わりに変化可能である試薬（ＤＢＬとして定式化した）を処理することによってこれをなすことができる。
好適な実施例においては、本明細書に記載した方法をアレイ較正に用いる。ｍＲＮＡ定量のような多くの応用の場合、標的アナライトの濃度に対して線形応答であるシグナルを有するか、または別法として、非線形の場合、濃度とシグナルとの間の関係を決定し標的アナライトの濃度を評価できることが望ましい。したがって、本発明は、アレイにおける複数のビーズについて平行して較正曲線を作成する方法を提供する。分析するサンプルの複雑さをシミュレートする条件下で較正曲線を作成できる。各曲線は他の曲線（例えば、異なる濃度範囲について）と独立して、アレイについての他の全曲線と同時に構築できる。したがって、該具体例において、一連のデコーディングスキームは、異なる蛍光体よりもむしろ、異なる濃度をコード「標識」として用いて実行される。この方法において、濃度に対する応答としてのシグナルは各ビーズについて測定できる。該較正は、アレイの使用の直前に実施でき、その結果、あらゆるアレイ上のあらゆるプローブが個々に、必要に応じて較正される。
【０１４１】
好適な一具体例においては、本発明の方法を同様にアッセイ開発に用いることができる。したがって、例えば、該方法は良いプローブおよび悪いプローブの同定を可能にする；当業者に理解されるように、いくつかのプローブはうまくハイブリダイズしないかまたは１以上の配列とクロスハイブリッド形成するので、うまく機能しない。これらの問題はデコーディングの間に容易に検出される。プローブの性能を迅速に評価する能力はアッセイ開発の時間と費用を大きく減少させる可能性を有する。
同様に、好適な一具体例において、本発明の方法はアッセイ開発での定量において有益である。多くのアッセイの主要なチャレンジはサンプル間のアナライト濃度における相違を検出する能力、これらの相違を定量する能力、およびアナライト、関係するアナライトの複合混合物の存在下での全て、の絶対濃度の測定である。この課題の例は全細胞性ｍＲＮＡの存在下における特異的ｍＲＮＡの定量である。ｍＲＮＡ定量の基礎として開発された１つのアプローチは、複数のマッチしたプローブ対とミスマッチのプローブ対を利用する（Ｌｏｃｋｈａｒｔら、１９９６）（出典明示によってその内容を本発明の一部とする）。このアプローチは単純であるが、比較的多数のプローブを必要とする。このアプローチにおいて、濃度に対する定量的応答は、目的の遺伝子または配列に対する１組の異なるプローブ由来のシグナルを平均することによって得られる。いくつかのプローブのみが定量的に応答し、これらのプローブを確実に予測することはできないので、このことは必須である。事前の知識がない場合、適切に選択されたプローブのコレクションの平均応答のみが定量的である。しかしながら、本発明においては、他のアッセイと同様に核酸に基づくアレイへの一般的な適用が可能である。要するに、そのアプローチは特定のアッセイにおいて定量的に応答するプローブを同定することであり、他のプローブとの平均ではない。これは、濃度に基づいたコードを用いる上記のアレイ較正スキームによって行われる。各および全配列を有効な方法で試験できるので、このアプローチの利点には；より少いプローブしか必要でないこと；測定の正確さが用いられるプローブの数にあまり依存しないこと；およびセンサーの応答が高度な確実性で知られることがある。特に複雑な配列混合物中において、プローブ性能の予測の困難性と不確実性を避ける、うまく機能するプローブが経験的に選択されることに注目することは重要である。対照的に、オーダーアレイを用いる、これまでに記載された実験においては、混合物へ既知のｍＲＮＡを添加する定量的なスパイク（ｓｐｉｋｉｎｇ）実験の実施によって比較的少数の配列がチェックされる。
【０１４２】
好適な具体例においては、ＲＮＡ発現プロファイリングのためにｃＤＮＡアレイを作成する。この具体例においては、宿主ベクター系において増殖させたｃＤＮＡライブラリーから個々のｃＤＮＡクローンを増幅する（例えば、ＰＣＲを用いて）。各増幅ＤＮＡをビーズの集団に結合させる。異なる集団を一緒に混合し、ｃＤＮＡライブラリーを示すビーズのコレクションを作成する。該ビーズをアレイにし、上記のようにデコードし、アッセイに用いる（本明細書中に記載したが、同様にデコーディングがアッセイ後に起きてもよい）。抽出され、必要に応じて標識され、アレイにハイブリダイズしたＲＮＡサンプル（全細胞またはｍＲＮＡ）を用いてアッセイを行う。拮抗分析は、個々のＲＮＡの発現レベルにおける相違の検出を可能にする。較正標準の適当なセットとの比較は、ＲＮＡの絶対量の定量を可能にする。
【０１４３】
また、ｃＤＮＡアレイをマッピングに用いて、例えば欠失／挿入をマッピングし、または例えば、腫瘍や他の組織サンプル由来のゲノム中のコピー数の変化をマッピングできる。これは、ゲノムＤＮＡのハイブリダイズによって行うことができる。ｃＤＮＡ（またはＥＳＴ等）の代わりに、ランダムゲノム断片を含むその他のＳＴＳ（配列タグ部位）をこの目的でアレイにすることもできる。
本明細書に引例した全ての参考文献は、出典明示によりその全てにおいて本明細書の一部とされる。
【図面の簡単な説明】
【図１】図１Ａ、１Ｂ、１Ｃ、１Ｄおよび１Ｅは本発明のいくつかの異なる「二成分」システムの例を示す。
【図２】図２Ａおよび２Ｂは二つの異なる「一成分」システムを表す。
【図３】図３はハイパースペクトルアルファ空間における凝集を表す（α_１＝Ｉ_１／ΣＩ_Ｉ、α_２＝Ｉ_２／ΣＩ_ｉ、α_３＝Ｉ_３／ΣＩ_ｉなど）。
【図４】図４は、ＦＡＭ−標識されたかまたはＣｙ３−標識されたｏｌｉｇｏ補体のいずれかをアレイ上の異なるビーズタイプを「染色（ｐａｉｎｔ）」（標識）するために使用する２色デコーディングプロセスを示す。
【図５】図５は４色および４つのデコード段階で１２８の異なるビーズタイプのデコーディングを示す（挿入図は４つの異なる色素を用いて１６のビーズタイプをデコードする単一のデコード段階を示す）。
【図６】図６は、１６種の異なるビーズのグレースケールデコーディングを表す。
【図７】図７は蓋およびベースプレートを概略的に表す。
【図８】図８は蓋１０およびベースプレート６０を含むハイブリダイゼーションチャンバーを概略的に表す。
【図９】図９は孔１０５を有するベースプレートを表す。
【図１０】図１０は圧力および／または真空により引き起こされる膜上の変化する溶液の体積および局在化を表す。
【図１１】図１１はハイブリダイゼーションチャンバーに関して有用である代表的アッセイスキームのフローチャートを表す。[0001]
No. 60/113968 filed on Dec. 28, 1998, US patent application Ser. No. 09 / 256,943 filed on Feb. 24, 1998, filed on Dec. 28, 1999. No. 09 / 473,904, and PCT / US99 / 31022 filed on Dec. 28, 1999, all of which are hereby incorporated by reference.
[0002]
(Technical field)
The present invention relates to a sensor composition comprising a composite array of individual arrays that allows for the simultaneous processing of multiple samples. The invention further provides methods for making and using the composite array. The invention further relates to an apparatus comprising a hybridization chamber for holding the composite array.
[0003]
(Prior art)
There are many assays and sensors for detecting the presence and / or concentration of a particular substance in fluids and gases. Many of these rely on specific ligand / antiligand reactions as detection mechanisms. That is, it is known that a pair of substances (ie, a binding pair or a ligand / antiligand) bind to each other, but little or no other substance. This has been the focus of many technologies that utilize these binding pairs for complex detection. These generally label one component of the complex in some way, for example, to produce an entire complex that is detectable using radioisotopes, fluorescent and other optically active molecules, enzymes, and the like. This is done by:
[0004]
Particularly useful in these sensors is a detection mechanism that utilizes luminescence. In recent years, the use of optical fibers and optical fiber strands in combination with light absorbing dyes for chemical analysis measurements has developed rapidly, especially during the last decade. The use of optical fibers and technology such purposes, Milanovich et al., "Novel Optical Fiber Techniques For Medical Application", Proceedings of the SPIE 28th Annual International Technical Symposium On Optics and Electro-Optics, Volume 494, 1980; Seitz, W. R. , "Chemical Sensors Based On Immobilized Indicators and Fiber Optics" in C.I. R. C. Critical Reviews In Analytical Chemistry, Vol. 19, 1988, pp. 135-173; Wolfbeis, O .; S. , "Fiber Optical Fluorosensors In Analytical Chemistry" in Molecular Luminescence Spectroscopy, Methods and Applications, (S.G., N.S., N.I., N.S.G. M. , Spectroscopy 2 (4): 38 (1987); Walt et al., "Chemical Sensors and Microinstrumentation", ACS Symposium Series, Vol. 403, 1989, p. 252 and Wolfbeis, O.M. S. , Fiber Optic Chemical Sensors, Ed. CRC Press, Boca Raton, FL, 1991, 2nd Volume.
[0005]
More recently, fiber optic sensors have been made which allow the use of multiple dyes on a single, independent fiber optic bundle. U.S. Pat. Nos. 5,244,636 and 5,250,264 to Walt et al. Disclose systems for depositing a plurality of different dyes at the distal end of a bundle, the teachings of each of these patents being incorporated herein by reference. And The disclosed arrangement allows the separate optical fibers of the bundle to have optical access to the individual dyes. This occurs when the signal from two or more dyes, where each dye is sensitive to a different analyte, is combined and there is a significant overlap in the emission spectrum of the dye, the reflected light from each dye Avoid the problem of deconvolving separate signals during (returning light).
U.S. patent applications 08/818199 and 09/151877 disclose arrays utilizing microspheres or beads on the surface of a substrate, e.g., at the end of a fiber optic bundle where each individual fiber comprises a bead comprising an optical signature. A composition is described. Since the beads move randomly, they require a unique optical signature to "decode" the array. That is, after array preparation, the location of individual sites on the array can be correlated with beads or bioactive agents at specific sites. This means that the beads may be randomly distributed on the array and is a quick and inexpensive method compared to conventional in situ synthesis or spotting techniques. Once the array is loaded with beads, the array can be decoded or used, as described in more detail below, and complete or partial decoding occurs after testing.
In addition, a composition comprising a silicon wafer containing a plurality of probe arrays in a microtiter plate is described in US Pat. No. 5,545,531.
[0006]
(Disclosure of the Invention)
In accordance with the above objects, the present invention provides a composite array composition comprising a first substrate having a surface comprising a plurality of assay locations, wherein each assay location comprises a plurality of independent sites. The substrate further comprises a population of microspheres comprising at least a first and a second subpopulation, wherein each subpopulation comprises a bioactive agent. The microspheres are distributed on each assay site.
In another embodiment, the invention provides a composite array composition comprising a first substrate having a surface comprising a plurality of assay locations, and a second substrate comprising a plurality of array locations, wherein each array location comprises an independent site. provide. The composition further comprises a population of microspheres comprising at least a first and a second subpopulation, wherein each subpopulation comprises a bioactive agent. The microspheres are distributed on each array location.
In yet another embodiment, the invention provides an array composition as outlined above, wherein a plurality of decoding binding ligands are added to the composite array composition to at least a plurality of bioactive agent locations. A method for decoding an array composition comprising identifying
In yet another embodiment, the invention includes contacting a sample with a composition as described above to determine the presence of one or more target analytes in one or more samples comprising determining the presence or absence of the target analytes. Provide a way to make decisions.
[0007]
In yet another embodiment, the invention provides a hybridization chamber. The hybridization chamber includes a base plate and a lid. A sealant is placed between the lid and the base plate to provide a hermetic seal. When using a two-component array system, the chamber also includes a component port for immobilizing the array components in the lid. That is, the array components are inserted through the component inlet of the lid. The inlet may include a seal so that a hermetic seal is maintained. The chamber can also include clamps and alignment pins.
In yet another embodiment, the present invention provides a hybridization chamber, wherein the base plate includes a hole. The holes may be in a microplate array format. In one example, at least two holes are connected by a channel. In one example, a soft septum is placed on a base plate. When pressure, ie, vacuum, is applied to the membrane, wells are formed in the membrane at the locations of the holes in the base plate. The device also includes a pneumatic device for applying a vacuum or positive pressure to the membrane.
[0008]
In yet another embodiment, the invention provides a method of mixing samples in an array format. The method includes applying a vacuum to the film so that a well is formed. The solution is added to the membrane and at least one of the wells is filled with liquid. Thereafter, a vacuum is intermittently applied to the film, so that the liquid is mixed.
In yet another embodiment, the invention provides a hybridization chamber as described herein and any of the composite array compositions described herein.
In yet another aspect, the invention provides a method for performing decoding of an array composition described herein in a hybridization chamber described herein.
In yet another aspect, the invention provides a method for determining the presence of one or more target analytes in one or more samples in a hybridization chamber as described herein.
[0009]
(Brief description of drawings)
1A, 1B, 1C, 1D and 1E show examples of several different "two-component" systems of the present invention. FIG. 1A shows a bead array. First substrate 10 has an array location 20 having wells 25 and beads 30. The second substrate 40 has an assay location 45. An optical lens or filter 60 is also shown; as will be appreciated by those skilled in the art, this may also be internal to the substrate. FIG. 1B is similar except that no beads are used, but rather array location 20 has discrete portions 21, 22, 23, etc., that can be formed using spotting, printing, photolithographic techniques, and the like. 1C-F illustrate the use of multiple first substrates. FIG. 1C depicts a “bead of beads” having an additional use for the mixing function. FIG. 1D represents a plurality of bead arrays, and FIG. 1E represents a plurality of non-bead arrays. FIG. 1F illustrates the use of the binding function for the "target" first substrate 10 to a location on the second substrate 40; as will be appreciated by those skilled in the art, this is divided into flat second substrates or sections. Can be performed on the second substrate. FIG. 1F utilizes binding ligand pairs 70/70 ', 71/71', 72/72 ', and the like. These are either chemical or biological functions, such as those described for IBL / DBL pairs such as oligonucleotides.
[0010]
2A and 2B represent two different "one component" systems. FIG. 2A depicts a bead array having a substrate 50 having an assay location 45 having wells 25 containing beads 30. FIG. 2B shows a non-bead array, where each assay location 45 has independent sites 21, 22, 23, etc.
FIG. 3 shows aggregation in hyperspectral alpha space (α₁= I₁/ ΣI_I, Α₂= I₂/ ΣI_i, Α₃= I₃/ ΣI_iSuch). A complementary oligonucleotide labeled set of 128 different bead types present on the fiber bundle with four dyes: Bodipy-493, Bodipy-R6G, Bodipy-TXR, and Bod-564 (only one dye per oligonucleotide). With the hybridization set. Shown is the second stage of a four stage decode where 4013 beads were decoded. An ellipse is drawn around the region of the hue cluster.
[0011]
FIG. 4 shows a two-color decoding process that uses either FAM-labeled or Cy3-labeled oligo complement to “paint” (label) different bead types on the array. .
FIG. 5 illustrates the decoding of 128 different bead types in four colors and four decoding stages. (The inset shows a single decoding step using four different dyes to decode the 16 beads.)
FIG. 6 represents grayscale decoding of 16 different beads. (A) Combinatorial pooling scheme for complementary decoding oligo. (B) Two independent normalized images were obtained and the resulting bead strengths were compared. (C) The α values (ratio of the bead intensity at the indicated decoding stages to the intensity in the normalized image) are plotted for the three decoding stages described in (A).
[0012]
FIG. 7 schematically represents a lid and a base plate. A represents the lid 10 and the base plate 60 of the hybridization chamber. With the lid inlet 20, the fiber optic bundle 30 is inserted through the lid and makes contact with the sample in the wells of the microtiter plate 40 in the base cavity 50 of the base plate 60. B illustrates the base cavity 50 of the base plate 60.
FIG. 8 schematically illustrates a hybridization chamber including a lid 10 and a base plate 60. Also shown is the fiber optic bundle 30 inserted into the microtiter plate 40 through the annular seal 80, clamp 90 and clamp receiver 95, lid.
FIG. 9 shows a base plate having holes 105. A represents a hole 105 in the base plate. B represents the channel 100 connecting the holes 105.
[0013]
FIG. 10 illustrates the changing solution volume and localization on the membrane caused by pressure and / or vacuum. A. + P represents pressure; -P represents vacuum. The membrane deflects upward in response to pressure in all chambers and holes. B. When a vacuum is applied to the left chamber and pressure is applied to the middle and right chambers, the liquid moves to the left side of the membrane. C. The liquid fills the wells when a vacuum is first applied to the left section. Thereafter, vacuum is applied to the middle and right chambers to form empty wells. D. When a vacuum is applied to the center and pressure is applied to the left and right chambers, the liquid moves to the center of the membrane. E. FIG. Liquid fills wells formed by high vacuum at the center. When a low vacuum is applied, empty wells are formed on the left and right. F. A vacuum is applied to the right chamber and the liquid moves to the right when pressure is applied to the left and middle chambers.
FIG. 11 depicts a flowchart of an exemplary assay scheme that is useful for a hybridization chamber.
[0014]
(Best Mode for Carrying Out the Invention)
The present invention relates to the simultaneous analysis of large numbers of samples, that is, the formation of very dense arrays that can be processed in parallel rather than in serial. This is done by forming an "array of arrays", i.e., a composite array containing a number of individual arrays arranged to allow processing of multiple samples. For example, each individual array is present in each well of a microtiter plate. Thus, depending on the size of the microtiter plate and the size of the individual arrays, a very large number of assays can proceed simultaneously. For example, using 2000 individual formats of different formats (with high levels of overlapping integration) and a 96-well microtiter plate, 192,000 experiments can be performed at a time, with the same array in a 384 microtiter plate. 768,000 simultaneous experiments can be performed, and 3072,000 experiments can be performed on 1536 microtiter plates.
[0015]
Generally, the array compositions of the present invention can be arranged in several ways. In a preferred example, a "one-component" system is used, as described in detail below. That is, a first substrate, including a plurality of assay locations (sometimes referred to herein as "assay wells"), such as a microtiter plate, is positioned such that each assay location includes an individual array. That is, the assay location is the same as the array location. For example, the plastic material of a microtiter plate can be molded to include a plurality of "bead wells" at the bottom of each assay well. The beads containing the bioactive agent can then be placed into the bead wells at each assay location, as described in more detail below. Although the disclosure herein emphasizes the use of beads, it is not necessary to use beads in all examples of the invention, and the bioactive agent may be directly associated with the array location. For example, other types of arrays are well known and can be used in this format, and spot, print or photolithographic arrays are also well known. See, for example, WO 95/25116, WO 95/35505, PCT / US98 / 09163, U.S. Patent Nos. 5,700,637, 5,807,522, 5,445,934, and U.S. Patent Application Nos. 08/851203, 09/187289 and references therein. All of which are incorporated herein by reference. In one-component systems, where beads are not used, a preferred example utilizes a non-silicon wafer substrate.
[0016]
Alternatively, a "two-component" system can be used. In this example, the individual arrays are formed on a second substrate, which can then fit or “push” into the first microtiter plate substrate. As will be appreciated by those skilled in the art, various array formats and arrangements are available. Preferred examples utilize, as individual arrays, generally fiber optic bundles having "bead wells" etched on one surface of each fiber, so that beads containing bioactive agents are loaded onto the ends of the fiber optic bundle. ing. Thus, a composite array comprises a number of individual arrays arranged to fit within the wells of a microtiter plate. Alternatively, other types of array formats can be used in a two-component system. For example, an array of arrays, such as those made by spotting, printing or photolithographic techniques, can be placed on a second substrate as described above. In addition, as shown in FIGS. 1C-F, "pieces" of the array, either random or aligned, can be used as the first substrate.
[0017]
The present invention is generally based on beads that distribute beads carrying different chemical functions (also referred to as microspheres) on a substrate that includes a patterned surface of discrete sites capable of binding individual microspheres. Based on previous studies involving analytical chemistry systems. Generally, the beads are randomly placed on the substrate, so several different methods can be used to "decode" the array. In one example, a unique optical signature, generally a fluorescent dye, is incorporated into the bead and can be used to confirm the chemical function on any bead. This allows the synthesis of candidate agents (ie, compounds such as nucleic acids and antibodies) to be decoupled from their arrangement on the array. That is, the candidate agent can be synthesized on the beads, and the beads are then randomly distributed on the patterned surface. Since the beads are first coded by an optical signature, this means that the array can be later `` decoded '', i.e., after creating the array, correlating the individual positions on the array with the beads or candidate agents at each position. Means you can do it. This means that the beads can be randomly distributed on the array, a faster and cheaper method than conventional in situ synthesis or spotting techniques. These methods are generally described in PCT / US98 / 05025, PCT / US98 / 21193, PCT / US99 / 20914, PCT / US99 / 14387 and U.S. Patent Application Nos. 08/818199, 09/315584, and 09/151877. All of which are incorporated herein by reference. Also, the discussion herein generally relates to the use of beads, but the same configuration is applicable to cells and other particles. See, for example, PCT / US99 / 04473.
[0018]
In these systems, the placement of the bioactive agent is generally random, so the coding / decoding system needs to identify the bioactive agent at each location in the array. This can be accomplished in a variety of ways, as described in more detail below, and generally involves: a) generally directly labeled, which binds to an identifier binding ligand (IBL) attached to a bioactive agent or bead. B) using, for example, a bead arrangement (eg, a photoactivated or photocleavable moiety that allows for selective addition of beads to individual positions) Or the use of sub-bundles or selective loading of sites as described in more detail below; c) selective decoding in which only beads that bind to the target are decoded; Or d) any combination of these. Optionally, as described in more detail below, this decoding may occur on all beads, or only on beads bound to a particular target analyte. Similarly, this can occur before or after the addition of the target analyte.
Once the identity (ie, the actual drug) and location of each microsphere in the array has been fixed, the array is exposed to a sample containing the target analyte. As described below, this can be done before or during the analysis. The target analyte binds to the bioactive agent, as described in detail below, and thus changes the optical signature of a particular bead.
[0019]
In the present invention, "decoding" can use an optical signature, a decoding binding ligand added during the decoding step, or a combination of these methods. The decoding binding ligand binds to the unique identifier binding ligand partner located on the bead, or to the bioactive agent itself, for example, if the bead includes a single-stranded nucleic acid as the bioactive agent. The decoding binding ligand is directly or indirectly labeled, and decoding occurs by detecting the presence of the label. By using a pool of decoding binding ligands consecutively, the number of decoding steps required can be greatly reduced.
[0020]
Accordingly, the invention provides an array composition comprising at least a first substrate having a surface comprising a plurality of assay locations. As used herein, “array” refers to a plurality of candidate agents in an array format, the size of the array depending on the composition and end use of the array. Arrays containing from about 2 to millions of different bioactive agents (ie, different beads) can be made, and very large fiber optic arrays are possible. In general, arrays can contain from 2 to 1 billion or more, depending on the size of the beads and the substrate, and the end use of the array, and are therefore ultra-high, high, medium, low, ultra-low. It can be an array of density. A preferred range for ultra-dense arrays is from about 1,000,000 to about 2,000,000,000,000 (all numbers are in cm<sup>2</sup>From about 100,000,000 to about 100,000,000. High density arrays range from about 100,000 to about 100,000,000, with about 100000 to about 5,000,000 being particularly preferred. Medium density arrays preferably range from about 10,000 to about 100,000, with about 20,000 to about 50,000 being particularly preferred. Low density arrays are generally less than 10,000, with about 1000 to about 5000 being preferred. Very low density arrays are less than 1000, preferably about 10 to about 1000, and particularly preferably about 100 to about 500. In some instances, the compositions of the invention may not be in an array format, ie, in some instances, compositions comprising a single bioactive agent may be made as well. In addition, in some arrays, multiple substrates of different or identical compositions can be used. Thus, for example, a large array can include a plurality of smaller substrates.
[0021]
In addition, one advantage of the compositions of the present invention is that extremely high density arrays can be made, especially by fiber optic technology. For example, beads of 200 μm or less (200 nm beads are also possible) can be used, and very small fibers are known, so 1 mm<sup>2</sup>It is possible to have as many as 40,000 to 50,000 (possibly millions) different fibers and beads in a bundle of<sup>2</sup>In each case, a density of more than 15,000,000 individual beads or fibers (possibly 25000000 to 5000000) is obtained.
As used herein, "composite array" or "combination array" or grammatically equivalent terms mean a plurality of individual arrays as described above. Generally, the number of individual arrays is set by the size of the microtiter plate used, and 96-well, 384-well, and 1536-well microtiter plates utilize a composite array containing 96, 384, and 1536 individual arrays. As will be appreciated by the skilled artisan, it is not necessary that each microtiter well contains an individual array. It should be noted that composite arrays can include identical, similar or different individual arrays. That is, in some instances, it may be desirable to perform 2000 identical assays on 96 different samples, and it is desirable to perform 192000 experiments on the same sample (ie, the same sample in each of the 96 wells). In some cases. Alternatively, each row or column of the composite array may be the same for redundancy / quality control. As will be appreciated by those skilled in the art, there are various ways to make this system. Also, the randomness of the array means that the same population of beads can be applied to two different surfaces, resulting in an array that is substantially similar, but perhaps not identical.
[0022]
As used herein, "substrate" or "solid support" or other grammatical equivalents can be modified to include independent individual sites suitable for binding or associating beads, at least one Any substance that is sensitive to the detection method is meant. As will be apparent to those skilled in the art, the number of possible substrates is very large. Possible substrates include, but are not limited to, glass and modified or functionalized glass, plastics (acrylic, polystyrene and copolymers of styrene with other materials, polypropylene, polyethylene, polybutylene, polyurethane, Teflon) Etc.), polysaccharides, nylon or nitrocellulose, resins, silica-based materials including silica or silicon and modified silicon, carbon, metals, inorganic glasses, plastics, fiber optic bundles and various other polymers. Generally, the substrate allows for optical detection and does not itself fluoresce appreciably.
[0023]
Generally, the substrate is flat (planar), but as will be apparent to those skilled in the art, other forms of the substrate can be used as well, for example, beads in a plastic porous block that allows access to the beads of the sample. The three-dimensional morphology can also be used by embedding and using a confocal microscope for detection. Similarly, beads can be placed on the inner surface of a tube for flow-through sample analysis to minimize sample volume. Preferred substrates include fiber optic bundles, as described below, and planar substrates, such as glass, polystyrene and other plastics and acrylics. In some instances, a silicon wafer substrate is not preferred. In one example, the substrate is in the form of or is a microscope slide.
The first substrate includes a surface that includes a plurality of assay locations, i.e., locations where an analysis is performed to detect a target analyte. Assay locations are generally physically separated from each other, for example, as wells in a microtiter plate, but other forms (hydrophobic / hydrophilic, etc.) can be used to separate assay locations.
[0024]
In a preferred embodiment, the second substrate is a fiber optic bundle or array as generally described in U.S. patent application Ser. Nos. 08 / 944,850 and 08 / 519,062 and PCT US98 / 05025 and PCT US98 / 09163. It is herewith incorporated by reference. The preferred embodiment utilizes a preformed unitary fiber optic array. As used herein, a "preformed unitary fiber optic array" refers to an array of discrete fiber optic bundles arranged coaxially and united along their length. The fiber strands are generally individually coated. However, one thing that distinguishes other fiber optic formats from a pre-formed monolithic array is that the fibers cannot be physically manipulated individually, i.e., generally, one strand can have any length along its length. In that it cannot be physically separated from another fiber strand.
However, in some "two-component" examples, the second substrate is not an optical fiber array.
[0025]
In a preferred embodiment, an assay location ("one component") or an array location ("two component") comprises a plurality of independent sites. For example, in the former case, the assay location is the same as the array location as described herein. In the latter case, the array locations will fit separately into the assay locations. In these examples, at least one surface of the substrate is to include discrete individual sites for subsequent association with the microspheres (or for the binding of a bioactive agent if microspheres are not used). Qualify. These sites may be physically modified sites, i.e., physical forms such as wells or small depressions in a substrate that hold the beads and allow the microspheres to rest therein, or other forces (magnetic or magnetic). Compression) or chemically modified or activated sites such as chemically functionalized sites, electrostatically modified sites, hydrophobically / hydrophilically functionalized sites, adhesion It can include spots and the like.
[0026]
The sites may be in a pattern, ie, a regular design or form, or may be randomly distributed. In a preferred embodiment, a regular pattern of parts is used so that the parts can be addressed in the XY coordinate plane. A "pattern" in this sense includes a repeating unit cell, preferably one that allows for a high density of beads on the substrate. However, it should be noted that these sites need not be independent sites. Thus, for example, it is also possible to use an adhesive or a uniform surface of chemical functionality that allows the beads to be adhered at any location. That is, the surface of the substrate is modified so that the microspheres can be adhered to the individual sites, whether the individual sites are continuous or discontinuous with other sites. Thus, the surface of the substrate can be modified to form independent sites with only a single associated bead, or alternatively, the surface of the substrate can be modified so that the beads descend everywhere, Ends with the part you did.
[0027]
In a preferred embodiment, the surface of the substrate is modified to include wells, ie, depressions in the surface of the substrate. This may be accomplished by a variety of techniques including, but not limited to, photolithography, stamping techniques, molding techniques and microetching techniques in a manner generally known to those skilled in the art. Can be. As will be apparent to those skilled in the art, the technique used will depend on the composition and the shape of the substrate. If the first substrate contains both the assay site and the individual arrays, a preferred method utilizes a molding technique that forms bead wells at the bottom of the assay wells of a microtiter plate. Similarly, a preferred embodiment utilizes a molded second substrate comprising "fingers" or protrusions in an array format, each finger comprising a bead well.
In a preferred embodiment, a physical change is made to the surface of the substrate to create the site. In a preferred embodiment, for example, where the second substrate is a fiber optic bundle, the surface of the substrate is generally described in U.S. Patent Application Serial Nos. 08/818199 and 09/151877, both of which are hereby incorporated by reference. One end of the fiber bundle described. In this embodiment, the wells are formed at the distal or distal end of a fiber optic bundle containing individual fibers. In this embodiment, the cores of the individual fibers are etched for cladding to form small wells or depressions at one end of the fiber. The required depth of the well depends on the size of the beads added to the well.
Generally, in this embodiment, the microspheres are not covalently attached to the wells, but the wells are further chemically functionalized as outlined below, and a crosslinker can be used, or a physical barrier, That is, a film or membrane on beads can be used.
[0028]
In a preferred embodiment, modified sites, particularly chemically modified sites, that can be used to attach the microspheres of the invention, either covalently or non-covalently, to independent sites or locations on a substrate. The surface of the substrate is modified to contain the site. In this context, a “chemically modified moiety” is an amino, carboxy, oxo group that can be used to covalently attach microspheres that also typically contain a corresponding reactive functional group. And addition of one pattern of chemical functional groups, including thiol groups; addition of one pattern of adhesive that can be used to attach the microspheres (chemically functionalized first to add the adhesive). Or the direct addition of an adhesive); the addition of a pattern of charged groups (similar to chemical functionalization) to electrostatically attach the microspheres, ie, the microspheres The charged site opposite to that of the microsphere; under appropriate experimental conditions, the site is similarly characterized such that the addition of a hydrophobic or hydrophilic microsphere results in the association of the microsphere to the site based on hydroaffinity. To encompass the addition of chemical functional groups of a single pattern to hydrophobic or hydrophilic, but is not limited thereto. For example, in an aqueous system, if a hydrophobic site is used for the hydrophobic beads, the association of the beads will predominate on the site.
In addition, biologically modified sites can be used to attach the beads to the substrate. For example, binding ligand pairs as generally described herein can be used. One partner is on the beads and the other is on the substrate. Particularly preferred in this example are complementary nucleic acid strands and antigen / antibody pairs.
[0029]
In addition, the use of biological moieties in this method can also form composite arrays. This is similar to the system shown in FIG. In this example, the population of beads contains one binding partner, and sub-populations of this population have different bioactive agents. By using different populations with different binding partners and substrates containing different assays or array locations with spatially separated binding partners, a composite array can be generated. This example also reuses the code, as each separate array of the composite array can use the same code, as described generally below.
As mentioned above, "pattern" in this sense includes the use of a uniform treatment of the surface to adhere the beads at discrete sites, as well as treating the surface to obtain discrete sites. This can be done in various ways, as will be apparent to those skilled in the art.
[0030]
As will be appreciated by those skilled in the art, there are many possible forms of the system as shown generally in the drawings. In addition to the standard formats described herein, various other formats can be used. For example, as shown in FIGS. 1C-1F, "portions" of the substrate that are not interconnected can be used. Again, they may be the same or different arrays. These portions can be made individually or as large units on a single substrate, after which the substrate can be cut or separated into different individual substrates. Thus, for example, FIGS. 1C and 1D show multiple bead arrays added to the wells of the second substrate, and FIG. 1C is a “bead of beads” to maximize mixing. FIG. 1D utilizes a plurality of planar first substrates, which may or may not be coupled to a second substrate, as will be appreciated by those skilled in the art. In one example, no special bonding means is used and instead various bonding techniques are used. For example, covalent or non-covalent forces can be used, including the use of adhesives, chemistry, hydrophobic / hydrophilic interactions, etc., as described for bonding beads to a substrate. The substrate may be magnetic or magnetically held (mixed if desired). That is, for example, as shown in FIG. 1F, linking groups can be used, which are covalent or non-covalent. These can be used simply for binding, or to target the first substrate array to individual locations in or on the second substrate. That is, for example, different oligonucleotides can be used to direct and attach the first substrate to the second substrate.
[0031]
In a preferred embodiment, there are optical components incorporated into the substrate used for imaging. Thus, for example, in either a one-component or two-component system, a "lensing" capability can be incorporated into the substrate. For example, in a one-component system, the bottom of one or more assay locations may have unique or special optical elements such as lenses, filters, and the like.
Also, preferred embodiments utilize configurations to facilitate mixing of the assay reactions. For example, a preferred embodiment utilizes a two-component system that allows for mixing. That is, in some instances, the array protrudes from the block and can be used as a "stick", such as agitating the reaction to promote good mixing of assay components, increasing the rate of the reaction, and the like. This can be done in various ways, as will be appreciated by those skilled in the art. In a preferred embodiment, the first and second substrates move in either the XY coordinate plane, the XZ coordinate plane, the YZ coordinate plane, or three dimensions (XYZ) with respect to each other. It is arranged so that it can be. The preferred embodiment utilizes a block jig in which the block is free to move in the plane of the plate or orthogonal to the plate plane. This is particularly useful in such situations, as standard mixing conditions often do not work well in these situations.
[0032]
Additionally or alternatively, further mixing elements may be present as part of the system. For example, extraneous mixed particles may be added, one example utilizing magnetic particles with, for example, a moving magnet to mix. For example, a small electromagnetic stir bar and plate can be used.
Alternatively, mixing in a one- or two-component system can be accomplished by sealing the system and shaking using standard techniques, optionally with mixed particles.
In a preferred example, the system is arranged to reduce evaporation and facilitate small sample size and handling. That is, the system is hermetically sealed or sealed by enclosing a limited space to maintain regulation of the small sample volume. In this regard, the present invention provides a hybridization chamber that contains or encapsulates an array and / or sample. As outlined in more detail below, the preferred examples utilize a hybridization chamber that includes a base plate and an alignment moiety that are particularly useful in a two-component system, but they are also useful in a one-component system.
[0033]
One of the advantages of the sealing system is that it reduces or reduces vibration. That is, due to the small sample volume, the array is susceptible to vibrations, eg, disturbances caused by platform shaking, motor vibrations, or air circulation. By enclosing the array and placing the array on a baseplate, the sample and array are less susceptible to vibration-induced disturbances because the baseplate reduces vibration.
A further advantage of this aspect of the invention is that the enclosed array allows for the use of increasingly smaller volumes. In open array formats, small sample volumes evaporate, causing a variety of problems including sample diversity, variation, and inconsistencies in solute concentrations in solution. For example, if small sample volumes are present at different assay locations, the solute concentration will change dramatically as a result of differential evaporation of the solution. Such differences alter the rate of hybridization, for example, making it difficult to interpret and compare the results obtained from such open arrays. However, by enclosing the array in, for example, a hybridization chamber as outlined herein, such sample variability is minimized, and the data obtained from the encapsulated array is more reliable. Become something. Thus, any of the methods described herein are useful for hybridization chambers.
[0034]
In addition, the encapsulated array allows for an extended assay / incubation time as compared to the incubation time in an open array. Again, the sealed or encapsulated array prevents evaporation of the sample and allows for an extended incubation period.
In addition, the encapsulated array facilitates sample mixing if necessary. In general, when using small sample volumes, it is difficult to achieve proper mixing of the sample. However, as outlined in more detail below, in one example, when a soft septum is used for a pneumatic device providing vacuum and / or pressure, the hybridization chamber facilitates mixing.
[0035]
If a "two-component" system is used, a hybridization chamber can be used. That is, both components, a substrate including a plurality of assay locations and a substrate including a plurality of array locations, are encapsulated in a hybridization chamber. In a preferred example, these components include, but are not limited to, a fiber optic array and a multi-well microtiter plate enclosed in a hybridization chamber.
In a preferred example, the hybridization chamber includes a baseplate on or in which one of the components is located. By baseplate is meant any platform or holder on which one of the array components is placed. The base plate can be made of plastic, glass or metal or any of the materials outlined herein for the substrate, if the base plate is metal, it is preferably made of aluminum. Aluminum provides a lightweight but strong base plate. In addition, aluminum allows efficient and / or rapid heat transfer for the chamber. However, if the base plate is made of plastic or glass, the components come into direct contact with the base plate. Alternatively, a metal sheet or template can be inserted into the base plate or placed on the base plate. The metal sheet or template can be designed to hold any of a variety of forms to accommodate the size and / or shape of the various components. As already mentioned, metal offers the advantage of being a fast and efficient heat conductor.
[0036]
In one example, the base plate includes at least one depression or base cavity in which the array components are located. That is, when a microtiter plate is a component, the depressions or base cavities are preferably to avoid extra vibration and allow for efficient heat transfer so that the microtiter plate can be placed directly therein. It is formed so that it may adhere to. Recesses can be formed in the base plate. In addition, the baseplate may include multiple depressions or voids such that multiple discrete array components are located on a single baseplate.
Alternatively, the base plate may be flat and preferably includes hooks or other attachment portions to hold the array in place.
In addition, a preferred embodiment utilizes a lid for hybridization. The lid may be made of any material (again, as listed herein for the substrate), but is preferably glass, plastic or metal. The lid preferably mates with the base plate so that a closed chamber is formed when the lid is placed on the base plate.
In another example, the lid includes at least one component placement hole. The component arrangement hole means a site in the lid where the component is fixed. That is, one of the components is attached to the lid by the placement hole. In a preferred example, the hole is a hole in the lid through which the component is inserted. For example, if a fiber optic bundle is a component, the bundle is inserted through the hole. In this example, the aperture further includes a sealant around the attachment site, forming an airtight seal between the component, the distal end of the fiber optic bundle, and the lid. The sealant may be any material, such as any outlined, including silicone, rubber, plastic, and the like. Alternatively, the seal may be a gel-based material, such as petrolatum, or a film-based material, such as PARAFILM.
[0037]
In yet another example, the lid includes a plurality of holes in the lid. That is, when multiple components are used, it is necessary to have separate pores for each component. For example, if multiple fiber optic bundles are used, each fiber optic bundle is located in a separate hole. However, while it is possible to insert each optical fiber bundle into one hole at a time, it is also possible to continuously insert the same optical fiber bundle into different holes. That is, there is no limitation on the number of holes into which components are continuously inserted. For example, as shown in FIG. 7A, the lid 10 includes a plurality of holes 20 in which the optical fiber bundle 30 is installed. The lid is then placed on the microtiter plate 40 in the base cavity 50 of the base plate 60. The base plate 60 is shown in FIG. 7b, showing the base plate 60 and the base cavity 50.
[0038]
In a preferred example, the hole seal reduces or prevents solution cross-contamination. That is, the seals around the individual pores / components form a seal to the base plate or array components so that the solution from the sample corresponding to a particular pore / component is separated or sealed from other components.
In another example, not all pores are always filled with components. If it is appropriate or desirable to have less than the maximum filling of the hole, a plug can be inserted into the hole that does not contain the component. In this way, the lid still forms a gas-tight seal with the base plate, despite the presence of component-free holes. The plug can be in the form of a rubber stopper, gasket, film, gel or the like.
[0039]
In a preferred example, a sealant is present around the perimeter of the chamber between the lid and the base plate. The sealant is made of any material that provides an airtight seal formed between the lid and the base plate. In the preferred example, the sealant is made of rubber or rubber, such as a silicone gasket or O-ring 80 (see FIG. 8). The sealant can be secured to either the lid or the base plate. For this purpose, the sealant can be permanently attached to the lid. Alternatively, the sealant can be fitted into a groove in either the lid or the base plate. In this way, the sealant is fixed to the lid or base plate, but this fixation need not be permanent. Alternatively, the sealant may be formed from a liquid sealant, such as petrolatum or a flexible film material, such as PARAFILM or other wax.
[0040]
In a preferred example, if a two-component system is used, the hybridization chamber will further include an alignment moiety. By alignment part is meant a mechanism of the chamber that facilitates alignment of the lid with the base plate. The importance of the alignment part is not only in the alignment of one lid and base plate, but also in the reproducible alignment of multiple lids and base plates. That is, the alignment portion facilitates physical alignment between any array component and any multiwell microtiter plate configuration. When the fiber optic bundle in the lid is aligned with the microtiter plate on the base plate, the alignment portion aligns the vertical central axis of the fiber bundle with its corresponding well central axis. In a preferred example, all fiber bundles are aligned so that they are distinct, ie, do not contact the inner wall of the well. This alignment is important for sequential imaging.
[0041]
In one example, the alignment portion is a complementary male / female fit. The male fit is attached to the lid or base plate, but need not be permanently attached. If a male fit is used as the alignment portion of the base plate or lid of the chamber, the opposite chamber portion includes a slot or hole into which the male fit is inserted (female fit). One skilled in the art will appreciate this male / female mating variation that is useful with the present invention. In this regard, the features may be indexer pins or bumps on the chamber portion and holes or complementary grooves on other portions.
In a preferred example, criteria are used. See U.S. Patent Application Nos. 60/119323, and 09/500555 and PCT / US00 / 03375, which are hereby incorporated by reference in their entirety.
[0042]
In another example, the chamber may also include a clamping mechanism to maintain secure contact between the lid and the base plate. The advantage of clamping is that it distributes the load evenly throughout the chamber to achieve uniform seal compression. A "clamp mechanism" or "clamp" is any mechanism that applies and maintains an increased pressure or seal between the lid and the base plate. In one example, the claim mechanism includes a rotating stud / receiving mechanism. That is, the stud 90 is inserted into the receiver 95 and rotated to push down the lid and the base plate together (see FIG. 8). Alternatively, the mechanism may include a hook and latch mechanism. One skilled in the art will recognize many clamping mechanisms useful in the present invention. In addition, those skilled in the art understand that the method of clamping is not limited to manual clamping. Therefore, it can be automated.
[0043]
In another example, the chamber includes features around the perimeter for handling of the chamber. In a preferred example, the mechanism is a slot that is wide enough to allow the user's finger to manually handle the chamber / array. In another example, the mechanism is a slot, groove, handle, etc., that is particularly useful in automatic or robotic operation of the chamber. These additional features may be asymmetrically distributed to further facilitate robotic operation.
As mentioned above, an advantage of the hybridization chamber is that a small sample volume can be used without losing the sample solution. In yet another example, the chamber may include one or more storage containers for holding additional solution. Therefore, the hybridization chamber also functions as a humidity chamber. The inclusion of additional solution in the storage container further prevents evaporation of the sample.
[0044]
In another example, for example, when using a microtiter plate, the sample can be applied to a membrane on the surface of the base plate. The advantages of using a membrane include the ease with which the membrane can be cleaned or discarded after each use, and the soft septum can make the tip of the pipette or fiber optic tip contact with the bottom of the sample well. Does not damage.
In this example, the base plate includes a series of small openings 105, eg, in a microplate format (FIG. 9A). Thus, the membrane is pushed down into the opening, forming a discrete assay site. Various membranes are useful with the present invention. The important thing is that the membrane is flexible. It may be desirable to have a chemically inert membrane, and it may be desirable to have a membrane with which the assay components interact, such as a nylon, nitrocellulose membrane, and the like.
[0045]
In a preferred example, a channel connects each opening (FIG. 9B). Channel 100 may be connected to a pneumatic device that creates a vacuum and / or pressure. Thus, when a vacuum is applied, the film deforms into openings 105 to form small pockets or wells. The sample is then added to the pocket. By applying different amounts of vacuum to the membrane through the openings, the volume and liquid level of the well formed by the deformed membrane can be changed. In addition, the liquid in the wells can be agitated or mixed by applying a vacuum to the membrane intermittently through the channels. Such a mixing method is advantageous because the entire system does not need to vibrate and no stir bar or tumbler is required. Further, if a subset of the openings is connected to different channels, the different subsets can be independently mixed in the same baseplate.
[0046]
When a positive pressure is applied, the membrane will deform upward or remain flat, depending on the magnitude of the pressure, whether there is a load on top of the membrane, and the size and shape of the opening. This is particularly advantageous in cleaning or cleaning the chamber.
If pressure and vacuum are applied to different holes in a certain order, a small amount of solution can be transferred to different parts of the membrane. That is, as shown in FIGS. 10A-F, when pressure and vacuum are applied differentially, the film bulges in some parts and dents in other parts. Thus, the solution applied to the membrane will migrate to the lower part of the membrane. This has the advantage that the incubation of the sample on the membrane proceeds for a precise time. After a period of time, the vacuum is released and, if necessary, pressure is applied to remove the solution. This allows incubation in small sections and achieves a uniform incubation time between the first and last wells throughout the array.
[0047]
Advantages of adjusting the sample volume by applying vacuum or pressure include reducing the consumption volume of reagents such as hybridization solution; increasing the agitation of small sample volumes; and improving the ease of cleaning the membrane. .
In a preferred example, the channels are connected to a common liquid handling device to pump or aspirate a sample solution or wash, such as a hybridization mixture. Again, in one example, all openings are connected to one channel. Thus, all wells are treated with the same solution. Alternatively, the subpopulations of the openings are connected to different channels and the solution is differentially applied to the subpopulations.
[0048]
If the channel is connected to a liquid handling device, this must include a mechanism for applying and removing liquid from the sample. That is, in order for liquid to be added and removed through openings in the base plate, the membrane must be permeable for the liquid to move. In this regard, needles are useful for piercing the membrane to add and remove liquid. When using a needle, it is necessary to use a resealable membrane or apply a sealant to the puncture site to prevent unwanted leakage of the solution.
The chamber may include a heat transfer mechanism. That is, if high temperatures are needed or desired, the chamber is designed to maintain the high temperatures. In one example, this involves applying insulation to the chamber. Next, the preheated solution is introduced into the chamber and the high temperature is maintained. That is, the solution can be easily heated outside the chamber before being pumped into the chamber. A simple chamber configuration facilitates maintaining equal temperatures between liquids in different wells.
[0049]
In another example, the chamber includes a heating mechanism to maintain an elevated temperature in the chamber. In one example, the chamber is uniformly heated by the heating device. In another example, the heating device independently heats different portions of the chamber.
As mentioned above, the use of a metal such as aluminum on the base plate promotes heat transfer, as the metal is a fast and effective conductor of heat.
If a "one component" system is used, a lid and sealing mechanism can be used. That is, as described above, the lid forms an airtight seal with the base plate. Thus, like the lids described above, lids of the "one-component" type also include a sealant between the lid and the base plate. In one example, the lid and base plate also include an alignment portion as described above for the "two-component" system. Alternatively, in one example, the one-component chamber does not include an alignment portion. In this regard, the need for tight alignment of the lid and base plate in one-component systems is lower than for two-component systems. That is, in one-component systems, the alignment is less stringent because the array components of the lid do not need to be aligned with the array locations on the baseplate. However, alignment is still important for imaging.
[0050]
Further, as mentioned above, the lid of the chamber in a one-component system can be made of glass, plastic or metal. Again, the use of metal facilitates maintaining the temperature, as metal is a rapid and effective heat conductor.
In addition, the system may include additional elements. These include holders for probes or fiber optic bundles. Such holders are disclosed in U.S. Patent Application Nos. 60 / 135,089 (filed May 20, 1999), and 09/574962 (filed May 19, 2000), and PCT US00 / 13772 (filed May 19, 2000). It is described in detail. In addition, the system may include cells as described in US Patent Application Nos. 09/033462 and 09/260963 and PCT / US99 / 04473. In addition, the system may include criteria as described in US Patent Application Nos. 60/119323 and 09/500555 and PCT / US00 / 03375, all of which are hereby incorporated by reference.
[0051]
In a preferred example, the methods and compositions of the present invention include a robotic system. Many systems generally use 96 (or more) microtiter plates, but any number of different plates or shapes can be used, as will be appreciated by those skilled in the art. In addition, any or all of the steps outlined herein can be automated; thus, for example, the system can be fully or partially automated.
As will be appreciated by those skilled in the art, but not limited to, one or more robotic arms; a plate handler for positioning microplates; an automated lid for removing and replacing well lids on non-cross-contaminated plates. Handler; tip assembly for sample distribution with disposable tips; washable tip assembly for sample distribution; 96-well loading block; chilled reagent rack; microtiter plate pipette position (optionally cooled); plate And stacking towers for chips; and various available components including computer systems.
[0052]
Complete robotic systems include automated liquid and particle manipulation including high-throughput pipetting to perform all steps of screening applications. This includes liquid and particle manipulations such as aspiration, dispensing, mixing, dilution, washing, precise volume transfer; correction, and disposal of pipette tips; and repetition of the same volume to dispense multiple times from one sample aspiration Including pipetting.
In a preferred example, chemically derivatized particles, plates, tubes, electromagnetic particles, or other solid matrices having specificity for ligands or muteins are used. The binding surface of the microplate, tube, or any solid phase matrix may be a non-polar surface, a highly polar surface, a modified dextran coating to facilitate covalent binding, an antibody coating, an affinity media for binding to the fusion protein or peptide, Other affinity matrices, including surface immobilized proteins, such as recombinant proteins A or G, nucleotide resins or coatings, are useful in the present invention.
[0053]
In preferred examples, multiwell plate platforms, multitubes, minitubes, deepwell plates, microcentrifuge tubes, cold vials, square well plates, filters, chips, optical fibers, beads, and other solid phase matrices of varying volumes or Platform applies to modular platforms that can be upgraded for additional capacity. The modular platform includes a variable speed orbital shaker, and a multi-position work deck for source samples, sample and reagent dilutions, assay plates, sample and reagent storage vessels, pipette tips, and an active wash station.
In a preferred example, a thermocycler and a thermoregulation system are used to stabilize the temperature of a heat exchanger such as a control block or platform to precisely control the temperature of the sample incubation from 4 ° C to 100 ° C.
[0054]
In a preferred example, a replaceable pipette head (single or multi-channel) with single or multiple electromagnetic probes, an affinity probe, or a pipettor manipulates liquids and particles by a robot. Multiwell or multitube electromagnetic separators or platforms operate on liquids and particles in single or multisample formats.
In some preferred examples, the apparatus includes a CCD camera for acquiring and converting data and images into a quantifiable format; and a computer workstation. These allow for data analysis.
Flexible hardware and software make the device applicable for multiple uses. Software program modules allow methods to be created, modified, and implemented. The system diagnostics module allows for equipment alignment, accurate connection, and motor operation. Customized tools, labware, and liquid and particle transfer patterns perform different applications. The database allows for method and parameter storage. Robot and computer interfaces allow communication between the devices.
[0055]
In a preferred example, the robotic workstation includes one or more heating or cooling components. Depending on the reactions and reagents, either cooling or heating is required, which can be done using known heating and cooling systems, including any number of Peltier systems.
In a preferred example, the robotic device includes a central processing unit that connects to a combination of memory and input / output devices (eg, keyboard, mouse, monitor, printer, etc.) via a bus. The general interaction between central processing units, memories, input / output devices, and buses is known in the art. Therefore, various different procedures are stored in the CPU depending on the experiment to be performed.
[0056]
In a preferred example, the composition of the present invention further comprises a population of microspheres. By “population” herein is meant a plurality of beads, as described above for the array. Within a population are separate sub-populations that are single microspheres or multiple identical microspheres. That is, in some examples, as described in more detail below, the array may only contain a single bead for each bioactive agent; a preferred example is a plurality of beads of each type. Use
As used herein, “microsphere” or “bead” or “particle” or grammatical equivalents means small, discrete particles. The composition of the beads will vary depending on the type of bioactive agent and the method of synthesis. Suitable bead compositions include, for example, plastics, ceramics, glass, polystyrene, methylstyrene, acrylic polymers, paramagnetic materials, triazoles, carbon graphite, titanium dioxide, latex or cross-linked dextrans, such as sepharose, cellulose, nylon, cross-linked micelles, and the like. All those used for the synthesis of peptides, nucleic acids and organics, including but not limited to Teflon, can be used. Bangs Laboratories, Fishers IN, "Microsphere Detection Guide" is a useful guidebook.
[0057]
The beads need not be spherical; irregular particles can be used. In addition, the beads may be porous, increasing the surface area of the beads available for bioactive agent binding or IBL binding. Bead sizes range from a few nanometers, ie, 100 nm, to a few millimeters, ie, 1 mm, with beads of about 0.2 microns to about 200 microns being preferred, and beads of about 0.5 to about 5 microns being particularly preferred. Although preferred, smaller beads may be used in some cases.
An important element of the present invention is to use a substrate / bead pairing that allows the beads to bind or attach to independent sites on the surface of the substrate so that the beads do not move during the assay.
[0058]
Each microsphere contains a bioactive agent, but as will be apparent to those skilled in the art, depending on the method of synthesis, there may be microspheres that do not contain a bioactive agent. As used herein, a "candidate bioactive agent" or "bioactive agent" or "chemical functional group" or "binding ligand" is any molecule capable of binding to a microsphere of the invention. For example, proteins, oligopeptides, small organic molecules, coordination complexes, polysaccharides, polynucleotides and the like. It is understood that the compositions of the present invention have two main uses. In a preferred embodiment, as described in more detail below, the composition is used to detect the presence of a particular target analyte, e.g., the presence or absence of a particular nucleotide sequence or a particular protein, e.g., an enzyme, antibody or antigen. Object is used. In another preferred embodiment, the composition can be used to screen a bioactive agent, ie, a drug candidate, for binding to a particular target analyte.
[0059]
Bioactive agents are typically organic molecules, preferably small organic compounds having a molecular weight greater than 100 daltons and less than about 2500 daltons, but encompass many species. Bioactive agents comprise functional groups essential for structural interactions with proteins, especially hydrogen bonding, and typically comprise at least one amino, carbonyl, hydroxyl or carboxyl group, preferably at least two chemical functional groups. including. Bioactive agents often comprise cyclical carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Bioactive agents are also found in biological molecules, including peptides, nucleic acids, sugars, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations. Nucleic acids and proteins are particularly preferred.
Bioactive agents can be obtained from a variety of sources, including libraries of synthetic or natural compounds. Many means are available for random and directed synthesis of many organic compounds and biomolecules, including, for example, expression of randomized oligonucleotides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or are readily produced. In addition, libraries and compounds generated naturally or synthetically are readily modified via conventional chemical, physical and biochemical means. Known pharmacological substances may be subjected to directed or random chemical modifications such as acylation, alkylation, esterification and / or amidation to produce structural analogs.
[0060]
In a preferred embodiment, the bioactive agent is a protein. As used herein, the term “protein” refers to a protein in which at least two amino acids are covalently bonded, and includes proteins, polypeptides, oligopeptides, and peptides. Proteins may be made up of naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic structures. Thus, "amino acid" or "peptide residue" as used herein refers to both naturally occurring amino acids and synthetic amino acids. For example, homo-phenylalanine, citrulline and norleucine are considered to be amino acids for the purposes of the present invention. The side chains may be in either the (R) or (S) configuration. In preferred embodiments, the amino acids are in the (S) or L-configuration. If non-naturally occurring side chains are used, non-amino acid substituents can be used, for example, to prevent or prevent in vivo degradation.
[0061]
In one preferred embodiment, the bioactive agent is a naturally occurring protein or a fragment of a naturally occurring protein. Thus, for example, cell extracts containing proteins or random or directed digests of proteinaceous cell extracts can be used. In this way, libraries of prokaryotic and eukaryotic proteins can be prepared for screening in the systems described herein. Particularly preferred in this embodiment are libraries of bacterial, fungal, viral and mammalian proteins, the latter being preferred, and human proteins being particularly preferred.
In a preferred embodiment, the bioactive agent is a peptide of about 5 to about 30 amino acids, with about 5 to about 20 amino acids being preferred, and about 7 to about 15 being particularly preferred. The peptide may be a digest of the naturally occurring protein described above, a random peptide or a "biased" random peptide. As used herein, "randomized" or grammatical equivalents means that the nucleic acid and peptide are each composed essentially of random nucleotides and amino acids. Generally, because these random peptides (or nucleic acids described below) are chemically synthesized, they can incorporate any nucleotide or amino acid at any position. Synthetic methods can be designed to produce randomized proteins or nucleic acids and form all or most possible combinations over the length of the sequence, thus providing a library of randomized bioactive proteinaceous materials. It is formed.
[0062]
In a preferred embodiment, a library of bioactive agents is used. The library provides a structurally diverse population of bioactive agents, and will likely bind to the target analyte to a sufficient extent. Thus, the interaction library must be large enough so that at least one of its members has a structure that confers affinity to the target analyte. Although it is difficult to determine the absolute size required of the interaction library, one clue is obtained in the immune response properties: 10<sup>7</sup>-10<sup>8</sup>The diversity of different antibodies provides at least one combination that has sufficient affinity to interact with the most likely antigen the organism will encounter. 10 published in vitro selection techniques<sup>7</sup>-10<sup>8</sup>Shows that the size of the library is sufficient to find structures with affinity for the target. Thus, in a preferred embodiment, at least 10<sup>6</sup>, Preferably at least 10<sup>7</sup>, More preferably at least 10<sup>8</sup>, Most preferably at least 10<sup>9</sup>Of different bioactive agents are analyzed simultaneously in the method of the invention. Preferred methods take full advantage of the size and diversity of the library.
[0063]
In a preferred embodiment, the library is sufficiently randomized that there is no sequence preference or constantity at any position. In another preferred embodiment, the library is biased. That is, certain positions in the sequence are selected from among a limited number of those that retain stationery or are likely. For example, in a preferred embodiment, a nucleotide or amino acid residue is phosphorylated to a certain type, eg, a hydrophobic amino acid, a hydrophilic residue, to crosslink to create cysteine, to a proline for the SH-3 domain. Sites are randomized to serine, threonine, tyrosine or histidine, etc., or within sterically biased (small or large) residues to purines.
[0064]
In a preferred embodiment, the bioactive agent is a nucleic acid (generally referred to herein as "probe nucleic acid" or "candidate probe"). As used herein, "nucleic acid" or "oligonucleotide" or grammatical equivalents means that at least two nucleotides are covalently linked together. The nucleic acids of the invention generally contain a phosphodiester bond, but in some cases, as described below, for example, phosphoramides (Beaucage et al., Tetrahedron, 49 (10): 1925 (1993) and references therein. Letsinger, J. Org. Chem., 35: 3800 (1970); Sprinzl et al., Eur. J. Biochem., 81: 579 (1977); Letsinger et al., Nucl. Acids Res., 14: 3487 (1986); Sawai et al., Chem. Lett., 805 (1984); Letsinger et al., J. Am. Chem. Soc., 110: 4470 (1988); and Pauwels et al., Chemica Scipra, 26: 141 (1986)), e. Holothioate (Mag et al., Nucleic Acids Res., 19: 1437 (1991); and U.S. Patent No. 5,644,048), phosphorodithioate (Briu et al., J. Am. Chem. Soc., 111: 2321 (1989)), O-methylphosphoramide linkages (see Eckstein, oligonucleotides and analogs: A Practical Approach, Oxford University Press) and peptide nucleic acid backbones and linkages (Eholm, J. Am. Chem. Soc., 114: 1895 (1992); Meier et al., Chem. Int. Ed. Engl., 31: 1008 (1992); Nielsen, Nature, 365: 566 (1993); Carlsson et al., Nature. 380: 207 (1996), all of which are hereby incorporated by reference). Other similar nucleic acids include positive scaffolds (Denpcy et al., Proc. Natl. Acad. Sci. USA, 92: 6097 (1995)); non-ionic scaffolds (US Patent Nos. 5,386,023; 5,637,684; 5,602,240; No. 5216141; and No. 446963; Kiedrowski et al., Angew, Chem. Intl. Ed. English, 30: 423 (1991); Letsinger et al., J. Am. Chem. Soc., 110: 4470 (1988); Letsinger et al. Nucleosides & Nucleotides, 13: 1597 (1994); Chapters 2 and 3, ASC Symposium Series 580, "Carbohydrate Modifications in Antisense Research", YS Sanghui and P. Dan, Cook, Ed .; Mesmaeker et al., Bioorganic & Medicinal Chem. Lett., 4: 395 (1994); Jeffs et al., J. Biomolecular NMR, 34:17 (1994); Tetrahedron Lett., 37: 743 and US Pat. Nos. 5235033 and 5034506 and Chapters 6 and 7, ASC Symposium Series 580, "Carbohydrate Modifications in Antisense Research", Y. S. Sanghui and P.M. Dan. Nucleic acids having a non-ribose backbone, including those described in the Cook edition. Nucleic acids containing one or more carbocyclic sugars are also included within the definition of nucleic acid (see Jenkins et al., Chem. Soc. Rev., (1995) pp. 169-176). Several nucleic acid analogs are described in Rawls, C .; & E News, June 2, 1997, p. 35. All of these publications are incorporated herein by reference. These modifications can be made in the ribose-phosphate backbone to facilitate the addition of additional moieties, such as labels, or to increase the stability and half-life of such molecules in the physiological environment; preferable. In addition, mixtures of naturally occurring nucleic acids and analogs can be prepared. Also, mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs can be prepared. Nucleic acids may be single stranded or double stranded, if specified, or may contain portions of both double stranded or single stranded sequence. The nucleic acid may be DNA, both genomic DNA and cDNA, RNA or hybrid; wherein the nucleic acid is any combination of deoxyribo- and ribo-nucleotides, uracil, adenine, thymine, cytosine, guanine, inosine, xanthanine. , Hypoxantanine, isocytosine, isoguanine and base analogs such as nitropyrrole and nitroindole.
[0065]
In a preferred embodiment, the bioactive agent is a library of clonal nucleic acids, including DNA and RNA. In this embodiment, the individual nucleic acids are generally prepared using conventional methods (including, but not limited to, amplification in plasmid or phage vectors, amplification methods including PCR, etc.). . Preferably, the nucleic acids are arranged in a specific format, such as a microtiter plate format, and beads added to attach the library.
Chemical or affinity capture (including, for example, including derivatized nucleotides such as AminoLink or biotinylated nucleotides with which they can be used to attach nucleic acids to a surface, and affinity capture by hybridization), cross-linking, The attachment of the clone library (or any of the nucleic acids described herein) can be performed by various methods understood in the art, including, but not limited to, and electrostatic attachment.
[0066]
In a preferred embodiment, the clone nucleic acid is attached to the beads using affinity capture. For example, a cloned nucleic acid can be derivatized with one member of the binding pair and a bead with the other member of the binding pair. Suitable binding pairs are those described herein for the IBL / DBL pair. For example, cloned nucleic acids may be biotinylated (eg, using enzymatic incorporation of biotinylated nucleotides or by photoactivated cross-linking of biotin). The biotinylated nucleic acid can then be captured on streptavidin-coated beads, as is known in the art. Similarly, other hapten-receptor combinations can be used, such as digoxigenin and anti-digoxigenin antibodies. Alternatively, a chemical group can be added to the form of a derivatized nucleotide, thereby making the nucleic acid available for addition to a surface.
[0067]
The preferred attachment is a covalent bond, but if there are multiple attachment sites per nucleic acid molecule, relatively weak interactions (ie, non-covalent bonds) may be sufficient to attach the nucleic acid to the surface. It is possible. Thus, for example, by using beads having the opposite charge to the bioactive agent, electrostatic interactions can be used for attachment.
Similarly, cloned nucleic acids can be attached to beads using affinity capture using hybridization. For example, polyA-added RNA is routinely captured by hybridization to oligo-dT beads, as is known in the art. This may include oligo-dT capture and a subsequent crosslinking step (eg, psoralen crosslinking). If the nucleic acid of interest does not contain a polyA moiety, it can be attached by polymerization using terminal transferase or by ligation of an oligo A linker, as is known in the art.
[0068]
Alternatively, chemical cross-linking may be performed, for example, by photo-activated cross-linking of thymidine to a reactive group, as is known in the art.
Generally, special methods are required to decode an array of clones, as described in further detail below.
As already generally described for proteins, the nucleic acid bioactive agent may be a naturally occurring nucleic acid, a random nucleic acid, or a "biased" random nucleic acid. For example, digests of prokaryotic and eukaryotic genomes may be used, as described above for proteins.
Generally, the probes of the present invention are designed to be complementary to a target sequence (either the target analyte sequence of the sample or any of the other probe sequences described herein) so that the target Allow hybridization to occur with the probes of the present invention. This complementarity need not be perfect. There may be some number of base pair mismatches that interfere with hybridization between the target sequence and the single stranded nucleic acids of the invention. However, if the number of mutations is too high to allow hybridization to occur even under minimal stringency hybridization conditions, the sequence is a non-complementary target sequence. Thus, "substantially complementary" as used herein means that the probe is sufficiently complementary to the target sequence and hybridizes under the selected reaction conditions. High stringency conditions are known in the art and are described, for example, in Maniatis et al. , Molecular Cloning: A Laboratory Manual, 2d Edition, 1989, and Short Protocols in Molecular Biology, ed. Ausbel, et al. References (both are hereby incorporated by reference). Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. A further guide to the hybridization of nucleic acids is Tijssen, Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, is found in the "Overview of principles of Hybridization and the strategy of nucleic acid assays" (1993). Generally, stringent conditions are selected to be about 5-10 ° C below the melting point (Tm) of the specific sequence at a constant ionic strength and pH. Tm is the temperature (at a given ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize with the target sequence at equilibrium (at Tm because the target sequence is present in excess). , 50% of the probe is occupied at equilibrium). Stringent conditions are those wherein the salt concentration is less than about 1.0 M sodium ion, and typically about 0.01 to 1.0 M sodium ion concentration (or pH 7.0 to 8.3). Other salts), the temperature is at least about 30 ° C. for short probes (eg, 10 to 50 nucleotides) and at least about 60 ° C. for long probes (eg, longer than 50 nucleotides). is there. Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. In another embodiment, less stringent hybridization conditions are used. For example, moderate to low stringency conditions as known in the art may be used. See Maniatis and Ausbel, supra, and Tijssen, supra.
[0069]
As used herein, the term "target sequence" or grammatical equivalents means a nucleic acid sequence that is on one strand of a nucleic acid. The target sequence may be part of a gene, regulatory sequence, genomic DNA, cDNA, RNA, including mRNA and rRNA. It may be of any length, it being understood that longer sequences are more specific. As will be appreciated by those skilled in the art, a complementary target sequence can take many forms. For example, it may be contained within a larger nucleic acid sequence, ie, all or part of a gene or mRNA, a plasmid or a restriction fragment of genomic DNA, and the like. As described in detail below, a probe is prepared to hybridize to a target sequence to determine the presence or absence of the target sequence in a sample. Generally speaking, this term will be understood by those skilled in the art.
[0070]
In a preferred embodiment, the bioactive agent is an organic chemical moiety, and various organic chemical moieties are available in the literature.
In a preferred embodiment, each bead comprises a single type of bioactive agent, but preferably, a plurality of individual bioactive agents are attached to each bead. Similarly, a preferred embodiment is to use more than one microsphere containing one unique bioactive agent. That is, there is degeneracy built into the system by the use of a subpopulation of microspheres, and each microsphere in the subpopulation contains the same bioactive agent.
As will be appreciated by those skilled in the art, the bioactive agent may be synthesized directly on the beads, or may be attached to the beads after synthesis. In a preferred embodiment, a linker is used to attach the bioactive agent to the beads to allow for good attachment, sufficient flexibility to allow for good interaction with the target molecule, and undesired binding reactions. Try to avoid.
[0071]
In a preferred embodiment, the bioactive agent is synthesized directly on the beads. As is known in the art, many types of compounds, such as peptides, organic moieties and nucleic acids, have been synthesized on supports that include beads.
In a preferred embodiment, the bioactive agent is first synthesized and then covalently attached to the beads. This will be done depending on the composition of the bioactive agent and the beads, as will be appreciated by those skilled in the art. The functionalization of solid support surfaces, such as certain polymers, with chemically reactive groups, such as thiols, amines, carboxyls, etc., is widely known in the art. Thus, "blank" microspheres having surface chemical groups that facilitate the attachment of desired functional groups by the user may be used. Some examples of these surface chemical groups for blank microspheres include, but are not limited to, amino groups including aliphatic and aromatic amines, carboxylic acids, aldehydes, amides, chloromethyl groups, hydrazides, hydroxyl groups. Groups, sulfonates and sulfates.
[0072]
In general, any number of different candidate agents can be added to the beads using these functional groups using known chemical methods. For example, a carbohydrate-containing candidate agent can be attached to an amino-functionalized support. The carbohydrate aldehyde is made using standard methods, and then the aldehyde is reacted with amino groups on the surface. In another embodiment, a sulfhydryl linker may be used. There are many sulfhydryl-reactive linkers known in the art, such as SPDP, maleimide, α-haloacetyl, and pyridyl disulfide (see, for example, the 1994 Pierce Chemical Company catalog, technical section on cross-linkers, 15-pages, 200-pages). Cysteine-containing proteinaceous agents can be attached to a support using them. Alternatively, amino groups on the candidate agent may be used for attachment to amino groups on the surface. For example, many stable bifunctional linkers are well known in the art, and include homobifunctional and heterobifunctional linkers (see Pierce catalog and handbook pages 155-200). In a further embodiment, the carboxyl group (either from the surface or from the candidate agent) may be derivatized using a well-known linker (see Pierce catalog). For example, carbodiimides activate carboxyl groups to activate for attack by good nucleophiles such as amines (Torchillin et al., Critical Rev. Therapeutic Drug Carrier Systems, 7 (4): 275-308 (1991). Reference (herein incorporated by reference)). Proteinaceous candidate agents may be attached using other methods known in the art, for example, methods for attaching antibodies to polymers. See, for example, Slinkin et al. , Bioconj. Chem. 2: 342-348 (1991); Torchillin et al. , Supra; Truvetskoy et al. , Bioconj. Chem. King et al., 3: 323-327 (1992); , Cancer Res. 54: 6176-6185 (1994); and Wilbur et al. , Bioconjugate Chem. 5: 220-235 (1994) (herein incorporated by reference). It should be understood that the candidate agent may be attached in a variety of ways, including those described above. Preferably, the mode of attachment does not significantly alter the functionality of the candidate agent. That is, the candidate agent should be attached in a flexible manner to allow interaction with its target. Also, these types of chemical or biological functionalities may be used to attach arrays to assay locations, as well as individual sets of beads, as shown in FIG. 1F.
[0073]
Specific methods for immobilizing enzymes on microspheres are known in the art. In one example, NH<sub>2</sub>Surface chemical microspheres are used. Surface activation is performed using 2.5% glutaraldehyde in phosphate buffered saline (10 mM) (138 mM NaCl, 2.7 mM KCl) to bring the pH to 6.9. It is stirred for about 2 hours at room temperature on a stirred bed. The microspheres are then rinsed with ultrapure water with 0.01% Tween 20 (surfactant) -0.02% and rinsed again with PBS pH 7.7 with 0.01% Tween 20. Finally, the enzyme is added to the solution, preferably after pre-filtration using a 0.45 μm amicon micropure filter.
[0074]
In some embodiments, the microspheres further include an identity binding ligand for use in certain decoding systems. As used herein, “identifier binding ligand” or “IBL” specifically binds to the corresponding decoder binding ligand (DBL) to facilitate elucidation of the identity of the bioactive agent attached to the bead. Means a compound. That is, the IBL and the corresponding DBL form a binding partner pair. As used herein, "specifically binds" means that the IBL distinguishes between the corresponding DBL and another DBL (ie, a DBL to another IBL) or other component or contaminant component of the system. Binds to the DBL with sufficient specificity. The binding should be sufficient to continue binding under the conditions of the decoding step, including a washing step to remove non-specific binding. In some embodiments, for example, where the IBL and the corresponding DBL are proteins or nucleic acids, the dissociation constant of the IBL for that DBL is about 10<sup>-4</sup>-10<sup>-6</sup>M<sup>-1</sup>Less than about 10<sup>-5</sup>-10<sup>-9</sup>M<sup>-1</sup>Less than about 10<sup>-7</sup>-10<sup>-9</sup>M<sup>-1</sup>Is particularly preferred.
[0075]
IBL-DBL binding pairs are known or can be readily found using known techniques. For example, if the IBL is a protein, then the DBL includes a protein (especially, including an antibody or fragment thereof (such as a FAb)) or a small molecule, or vice versa (where the IBL is an antibody, DBL is a protein). Metal ion-metal ion ligand or chelator pairs are also useful. Antigen-antibody pairs, enzymes and substrates or inhibitors, other protein-protein interaction pairs, receptor-ligands, complementary nucleic acids (including triple-helix forming nucleic acid molecules), and also carbohydrates and their binding partners It is also a suitable binding pair. Also useful are nucleic acid-nucleic acid binding protein pairs, including single- or double-stranded nucleic acid binding proteins and small molecule nucleic acid binding agents. Similarly, as generally described in U.S. Pat. Nos. 5,270,163, 5,475,096, 5,567,588, 5,595,877, 5,637,459, 5,568,867, 5,705,337 and related patents (hereby incorporated by reference). ), Nucleic acids "aptamers" can be developed to bind to virtually any target; such aptamer-target pairs can be used as IBL-DBL pairs. Similarly, there is an extensive body of literature relating to the development of binding pairs based on combinatorial chemical methods.
[0076]
In a preferred embodiment, an IBL is a molecule whose color or luminescent properties change in the presence of selectively binding DBL.
In one embodiment, the DBL may be attached to a bead, ie, a "decoder bead," which may carry a label, such as a phosphor.
In a preferred embodiment, the IBL-DBL pair comprises a substantially complementary single-stranded nucleic acid. In this embodiment, the binding ligand may be referred to as an "identity probe" and a "decoder probe." Generally, the identifiers and decoder probes are between about 4 and about 1000 base pairs in length, with about 6 to about 100 being preferred, and about 8 to about 40 being particularly preferred. Importantly, the probe is long enough to be specific, that is, long enough to distinguish between different IBL-DBL pairs, and a) if necessary, under appropriate experimental conditions. Dissociation, and b) short enough to allow effective hybridization.
[0077]
In a preferred embodiment, IBL does not bind to DBL, as outlined in more detail below. Rather, IBL is used as the directly identified identity portion ("IM"), for example, through the use of mass spectroscopy.
Alternatively, in a preferred embodiment, the IBL and the bioactive agent are the same moiety; thus, for example, as outlined herein, especially where no optical signature is used, the bioactive agent is It can serve as both an identifier and the agent. For example, in the case of nucleic acids, a bead-bound probe (acting as a bioactive agent) can also bind to the decoder probe to identify the sequence of the probe on the bead. Thus, in this embodiment, the DBL binds to the bioactive agent. This is particularly useful because this embodiment can provide information about the array or assay in addition to decoding. For example, as described in more detail below, the use of DBL allows for array calibration and assay development. This is done even if the DBL is not used as is; for example, in non-random arrays, the use of these probe sets allows array calibration and assay development even when decoding is not required. be able to.
[0078]
In a preferred embodiment, the microsphere does not include an optical signature. That is, as outlined in U.S. patent application Ser. Nos. 08/818199 and 09/151877, conventional processing uses unique optical signatures used to identify unique bioactive agents in a sub-population of microspheres. Or each sub-population of microspheres containing an optical tag. That is, decoding takes advantage of the optical properties of the beads so that beads containing unique optical signatures are distinguished from beads at other locations that have different optical signatures. Thus, conventional processing assigns a unique optical signature to each bioactive agent, whereby microspheres containing the bioactive agent are identified based on the signature. These optical signatures contain dyes, usually chromophores or fluorophores, which are either incorporated or attached to the beads themselves. The variety of optical signatures utilized different fluorescent dyes, different ratios of fluorescent dye mixtures, and different concentrations (intensities) of fluorescent dye.
[0079]
Thus, the present invention does not rely solely on the use of optical properties (if any) to decode the array. However, as will be appreciated by those skilled in the art, in some embodiments, it is possible to utilize optical signatures as an additional coding method with the system of the present invention. Thus, for example, as described in greater detail below, the size of the array can be effectively increased, but with some methods using a set of decoding portions, one of the methods is Using beads in combination with optical signatures. Thus, for example, when using one "set" of decoding molecules, the use of two groups of beads, one with and without an optical signature, can effectively double the size of the array. The use of multiple optical signatures also increases the possible size of the array.
[0080]
In a preferred embodiment, each subpopulation of beads comprises a plurality of different IBLs. By using multiple different IBLs to encode each bioactive agent, the number of possible unique codes is substantially increased. That is, by using one unique IBL per bioactive agent, the size of the array will be the number of unique IBLs (assuming that "reuse" did not occur, as outlined below). Do). However, using the presence or absence of each IBL as an indicator, using multiple different IBLs per bead can reduce the size of the array by two.<sup>n</sup>Can be increased. For example, the application of 10 IBLs per bead results in a 10-bit binary code, where each bit is either "1" (IBL is present) or "0" (IBL is absent). Can be shown. The 10-bit binary code is 2<sup>10</sup>With one possible variant. However, as discussed in more detail below, if another parameter such as concentration or intensity is included, the size of the array can be further increased; thus, for example, using two different concentrations of IBL The size of the array<sup>n</sup>Increase. Thus, in this embodiment, each individual bioactive agent in the array is provided with a combination of IBLs that can be added to the beads before, after, or during the addition of the bioactive agent (ie, , IBL and bioactive agent components simultaneously).
[0081]
Alternatively, where the bioactive agent is a polymer of different residues, ie, where the bioactive agent is a protein or nucleic acid, different IBL combinations can be used to elucidate the sequence of the protein or nucleic acid.
Thus, for example, two different IBLs (IBL1 and IBL2) can be used to elucidate the first position of a nucleic acid. For example, adenosine can be indicated by the presence of both IBL1 and IBL2; thymidine can be indicated by the presence of IBL1 and the absence of IBL2, cytosine can be indicated by the presence of IBL2 and the absence of IBL1, and guanosine can be indicated by both. Can be indicated by the absence of The second position of the nucleic acid can be similarly elucidated using IBL3 and IBL4. Thus, the presence of IBL1, IBL2, IBL3 and IBL4 gives the sequence of AA; IBL1, IBL2 and IBL3 give the sequence AT; IBL1, IBL3 and IBL4 give the sequence TA. The third position utilizes IBL5 and IBL6 and the like. In this way, the use of 20 different identifiers can yield unique codes for all possible 10-mers.
[0082]
The system is similar for proteins, but requires a number of different IBLs to identify each position, depending on the diversity allowed at each position. Thus, for example, if every amino acid is allowed at every position, five different IBLs are needed for each position. However, as noted above, for example, when using random peptides as bioactive agents, there may be built-in bias in the system; not all amino acids can be present at all positions, and some Can be preset; therefore, it is possible to use four different IBLs for each amino acid.
In this way, a sort of "barcode" can be constructed for each sequence; the presence or absence of each separate IBL allows for the identification of each bioactive agent.
[0083]
In addition, the use of different concentrations or densities of IBLs allows for some "reuse". For example, if the beads containing the first drug have a 1 × concentration of IBL and the second beads containing the second drug have a 10 × concentration of IBL, then use the saturation concentration of the corresponding labeled DBL By doing so, the user can distinguish between the two beads.
Once the microspheres containing the candidate agent and the unique IBL have been created, they are added to a substrate to form an array. Most of the methods described herein add beads to the substrate prior to the assay, but the order of array creation, use and decoding can be varied. For example, an array can be made, decoded, and then assayed. Alternatively, arrays can be made, used in assays, and then decoded. This can be particularly useful if only a few beads need to be decoded. Alternatively, the beads can be added to the assay mixture, ie, the sample containing the target analyte, before the beads are added to the substrate; after addition and assay, the array can be decoded. This is particularly preferred when agitating or mixing the sample containing the beads; this can increase the amount of target analyte bound to the beads per unit time and thus (in the case of nucleic acid assays) The hybridization speed increases. This is particularly useful when the concentration of the target analyte in the sample is low; generally, at low concentrations, long binding times must be used.
[0084]
In addition, adding beads to the assay mixture can allow for sorting or selection. For example, a large library of beads may be added to the sample, or only beads that bind to the sample may be added to the substrate. For example, if the target analyte is fluorescently labeled (either directly (eg, by incorporation of the label into a nucleic acid amplification reaction) or indirectly (eg, by using a sandwich assay)), target analyte binding The beads that exhibit fluorescence as a result of can be sorted by fluorescence activated cell sorting (FACS), and only these beads can be added to the array and then decoded. Similarly, the selection may be performed by affinity techniques; an affinity column containing the target analyte can be created, and only the binding beads are used in the array. Similarly, two bead systems can be used; for example, magnetic beads containing a target analyte can be used to "pull" beads that bind to a target, followed by subsequent dissociation of the magnetic beads (eg, Can be added to the array.
[0085]
Generally, in order to maximize the number of different candidate agents that can be uniquely encoded, methods of making the array and decoding the array are performed. The compositions of the present invention may be made in various ways. Generally, arrays are made by adding a solution or slurry containing the beads to a surface containing sites for attachment of the beads. This may be done with various buffers, including aqueous and organic solvents, and mixtures. The solvent can be evaporated and excess beads are removed.
In a preferred embodiment, where the beads are bound to the array using a non-covalent method, a novel method of loading the beads onto the array is used. The method involves exposing the array to a solution of particles (including microspheres and cells) and then utilizing energy, for example, stirring or shaking the mixture. This results in an array with more tightly bound particles because it is agitated with enough energy to drop (or drop out, in the case of wells) the weakly bound beads. These sites can then be used to bind different beads. In this way, beads showing high affinity for the site are selected. Arrays made in this way have two main advantages over more static loads. First, a higher percentage of sites can be easily filled, and second, it shows that arrays loaded in this way substantially reduce the loss of beads in the assay. Thus, in a preferred embodiment, using these methods, an array is obtained that fills at least about 50%, preferably at least 75%, particularly preferably at least about 90% of the sites. Similarly, arrays obtained in this way preferably lose less than about 20%, preferably less than about 10%, particularly preferably less than about 5% of beads during the assay.
[0086]
In this embodiment, a substrate including a surface having separate sites is immersed in a solution containing particles (beads, cells, etc.). The surface may include wells, as described herein, or may include other types of sites on the patterned surface such that there is a differential affinity for the sites. It may be. Differential affinity results in a competitive process, so that more tightly bound particles are selected. Preferably, all surfaces to which the beads are to be "loaded" are in fluid contact with the solution. The solution is generally a slurry in the range of about 10000: 1 to 1: 1 bead: solution (volume: volume). Generally, solutions can include any number of reagents, including aqueous buffers, organic solvents, salts, other reagent components, and the like. In addition, the solution preferably contains an excess of beads; that is, there are more beads than sites on the array. Preferred embodiments use a 2- to 1-fold excess of beads.
Immersion can mimic the assay conditions; for example, if the array is "dipped" from above into a microtiter plate containing the sample, the arrangement can be repeated during loading, and thus gravity Minimize beads that probably fall out for.
[0087]
Once the surface has been immersed, the substrate, solution, or both, are subjected to a competitive process, thereby dissociating particles having a lower affinity from the substrate to particles having a higher affinity for the site. Can be replaced. The competitive process is carried out by introducing energy in the form of heat, sonication, stirring or mixing the solution or the substrate or both, and shaking or stirring.
Preferred embodiments use stirring or vibration. Generally, the amount of manipulation of the substrate is minimized to prevent damage to the array. Thus, the preferred embodiment employs agitation of the solution, rather than the array, whichever is preferred. As will be apparent to those skilled in the art, this stirring can be performed in any number of forms, and a preferred embodiment utilizes a microtiter plate containing a bead solution that is agitated using a microtiter plate shaker.
[0088]
Agitation is for a time sufficient to load the array to the desired amount. Depending on the size and concentration of the beads and the size of the array, this time may range from about 1 second to several days, with about 1 minute to about 24 hours being preferred.
It should be noted that not all sites in the array contain beads. That is, some sites on the substrate surface may be empty. In addition, although not preferred, there may be several sites containing more than one bead.
In some embodiments, for example, when performing chemical conjugation, it is possible to associate the beads in a non-random or ordered manner. For example, using a photoactivatable binding linker or a photoactivatable adhesive or masking agent, selected sites on the array are sequentially adapted for binding so that a predetermined population of beads is positioned. It may be.
[0089]
The array of the present invention `` decodes '' the random deposition of beads in fiber wells so that information about the identity of the candidate agent is incorporated into the array, allowing identification of the candidate agent at all locations. It is built to be. This may be done in a variety of ways, either before, during, or after the use of the array to detect the target molecule.
Thus, after creating the array, it is "decoded" to identify the location of each sub-population of one or more bioactive agents, ie, beads, on the surface of the substrate.
In a preferred embodiment, a selective decoding system is used. In this case, only those microspheres that show a change in the optical signal as a result of binding of the target analyte are decoded. This is typically done when the number of "hits", i.e., the number of sites to decode, is generally low. That is, the array is first scanned under experimental conditions in the absence of the target analyte. A sample containing the target analyte is added and only those locations that show a change in the optical signal are decoded. For example, beads at either the positive or negative signal location may be selectively labeled or dissociated from the array (eg, by a photocleavable linker) and then placed in a fluorescence activated cell sorter (FACS). It may be sorted or concentrated. That is, after all negative beads are dissociated, positive beads are dissociated or analyzed in situ. Alternatively, all positives are dissociated and analyzed. Alternatively, the label may comprise a halogenated aromatic compound, and detection of the label is performed using, for example, gas chromatography, chemical tags, isotope tags or mass spectral tags.
[0090]
As will be apparent to those skilled in the art, this may also be done in systems where the array is not decoded, ie, where the correlation between bead composition and location is never required. In this embodiment, the beads are loaded onto the array and the assay is performed. The beads that are "positive", that is, exhibit a change in optical signal as outlined generally below, are then "marked" to distinguish or separate them from "negative" beads. This can be done in several ways, preferably using fiber optic arrays. In a preferred embodiment, each bead contains a fluorescent dye. After the assay and identification of “positive” or “active beads”, only the positive fibers or only the negative fibers are exposed, generally in the presence of a photoactivation reagent (typically dissolved oxygen). In the former case, all active beads are photobleached. Thus, non-fluorescently active beads can be sorted out of fluorescent negative beads, for example, by non-selective release of the beads with subsequent sorting using a fluorescence activated cell sorter (FACS) machine. Alternatively, if light is directed to the negative fiber, all negatives are non-fluorescent and positives are fluorescent, and the selection can proceed. Characterization of the bound bioactive agent may be performed directly, for example, using a mass spectrometer.
[0091]
Alternatively, identification may be by use of an identifier moiety ("IM") that is similar to IBL but does not necessarily need to bind to DBL. That is, rather than directly elucidating the structure of the bioactive agent, the composition of the IM acts as an identifier. Thus, for example, certain combinations of IMs can serve to encode beads, which can be used to dissociate from the beads and subsequently analyzed, for example, using gas chromatography or mass spectroscopy. Substances on the beads can be identified.
Alternatively, rather than having each bead contain a fluorescent dye, each bead includes a non-fluorescent precursor of the fluorescent dye. For example, photoactivation of a fluorescent dye can be performed using a photocleavable protecting group such as certain ortho-nitrobenzyl groups on the fluorescent molecule. After the assay, light is again directed to either the "positive" or "negative" fibers to distinguish these populations. The illuminated precursor is then chemically converted to a fluorescent dye. All the beads are then dissociated from the array using sorting to form a population of fluorescent and non-fluorescent beads (either positive and negative or vice versa).
[0092]
In another preferred embodiment, the binding sites (eg, wells) of the beads comprise a photopolymerizable reagent, or are added to an array in which the photopolymerizable agents have been constructed. After the test assay is performed, light is re-illuminated on either the "positive" or "negative" fibers to distinguish these populations. As a result of the irradiation, either all positives or all negatives polymerize and are captured or bound to the site, while other populations of beads can be dissociated from the array.
In a preferred embodiment, the location of any bioactive agent is determined using a decoder binding ligand (DBL). As noted above, the DBL is a binding ligand that binds the identity binding ligand, if present, or, preferably, the bioactive agent itself when the bioactive agent is a nucleic acid or protein.
In a preferred embodiment, as described above, DBL binds to IBL.
[0093]
In a preferred embodiment, the bioactive agent is a single-stranded nucleic acid and the DBL is substantially a complementary single-stranded nucleic acid that binds (hybridizes) to the bioactive agent (referred to herein as a decoder probe). ). A decoder probe substantially complementary to each candidate probe is created and used to decode the array. In this embodiment, the candidate and decoder probes should be of sufficient length to provide specificity (and a decoding step performed under appropriate conditions); Binds to its corresponding decoder probe with sufficient specificity to allow discrimination of the probe.
In a preferred embodiment, the DBL is directly or indirectly labeled. By the term "labeled" herein is meant that the compound has at least one element, isotope or compound attached to it, to allow detection of the compound. In general, labels are classified into three classes: a) isotopic labels, which may be radioisotopes or heavy isotopes; b) magnetic, electrical, thermal; and c) colored or luminescent dyes. Include enzymes as well as particles such as magnetic particles. Preferred labels include luminescent labels. In a preferred embodiment, the DBL is directly labeled. That is, the DBL includes a label. In another embodiment, the DBL is indirectly labeled. That is, a labeled binding ligand (LBL) that will bind to DBL is used. In this embodiment, the label binding ligand-DBL pair can be as described above for the IBL-DBL pair. Suitable labels are fluorescent lanthanide complexes, including but not limited to europium and terbium complexes, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarin, pyrene, malachite green, stilbene, Lucifer Yellow (Lucifer Yellow), Cascade Blue (Cascade Blue)^TM), Texas Red, FITC, PE, cy3, cy5 and Richard P.C. Includes other labels described in Molecular Probes Handbook, 6th Edition by Haugland (particularly incorporated herein by reference).
[0094]
In one example, the label is a molecule whose color or luminescent property changes in the presence of IBL due to a change in the local environment. For example, the label may be (1) a fluorescent pH indicator whose luminescence intensity changes with pH; (2) a fluorescent ion indicator whose luminescence changes with ion concentration; or (3) a fluorescent intensity indicator in a hydrophobic environment. Or a fluorescent molecule such as an ethidium salt which increases in the amount.
Thus, the location of individual beads (or a sub-population of beads) can be determined by the binding between the labeled DBL and the IBL or bioactive agent (ie, if the bioactive agent is a nucleic acid, candidate and decoder probes). The hybridization is performed using one or more decoding steps including hybridization. After decoding, the DBL can be removed and the array can be used, but in some situations, for example, if the DBL binds to the IBL and does not bind to the bioactive agent, DBL removal is not required (some It is desirable in some situations). Further, as outlined herein, decoding may occur before, during, or after the array is used in the assay.
[0095]
In one example, a single decoding step is performed. In this embodiment, each DBL is labeled with a unique label such that the number of unique labels is equal to or greater than the number of bioactive agents (in some cases, as described herein). As such, "re-use" of unique labels is possible; similarly, if variants are encoded in another dimension, i.e., in bead size or label, fewer variants of the candidate probe can share the same decoder. ). For each bioactive agent or IBL, a DBL is created that specifically binds to it and contains a unique label, eg, one or more fluorescent dyes. Thus, both the identity of each DBL, its composition (ie, its sequence, if it is a nucleic acid) and its label are known. The array containing the bioactive agent is then placed under conditions that allow the formation of a complex of DBL with either the bioactive agent or IBL (called a hybridization complex if the component is a nucleic acid). By adding DBLs, the location of each DBL can be ascertained. This allows the location of each bioactive agent to be identified; a random array is decoded. The DBL can then be removed if necessary and the target sample is applied.
[0096]
In a preferred embodiment, the number of unique labels is less than the number of unique bioactive agents, so a series of decoding steps is used. This example is illustrated for nucleic acids, although other types of bioactive agents and DBL are similarly useful for ease of discussion. In this embodiment, the decoder probe is divided into n sets for decoding. The number of sets corresponds to the number of unique tags. Each decoder probe is labeled in n separate reactions using n separate tags. All decoder probes share the same n tags. Each pool of decoders contains only one of the n tag versions of each decoder, and there are no two decoder probes with the same sequence of tags across the pool. The number of pools needed to achieve this is determined by the number of decoder probes and n. Hybridization to an array of each pool generates a signal at any address, including the IBL. Successive hybridization of each pool in turn will yield a unique sequence-specific code for each candidate probe. This identifies a candidate probe at each address in the array. For example, if 4 tags are used, 4 × n consecutive hybridizations would ideally require more steps in some cases, but 4 × nⁿCan be identified. After hybridization of each pool, the hybrids are denatured and the decoder probe is removed so that the probe is single-stranded for the next hybridization (but the target is not saturated so that the available probes are saturated). It is also possible to carry out hybridization with a limited amount of tandem. Continuous hybridization can be performed and analyzed by subtracting the pre-existing signal from the previous hybridization).
[0097]
As will be appreciated by those skilled in the art, hybridization or incubation times will vary. Generally, hybridization or incubation times last from seconds to minutes or hours or days or more. When utilizing a hybridization chamber as described herein, hybridization or incubation times may be increased as compared to incubation times without a hybridization chamber.
An example is shown. Assume an array of 16 probe nucleic acids (numbers 1 to 16) and 4 unique tags (4 different fluors, eg, labels AD). Create a decoder probe 1-16 corresponding to the probe on the bead. The first step is to label the decoder probe 1-4 with the tag A, the decoder probe 5-8 with the tag B, the decoder probe 9-12 with the tag C, and the decoder probe 13-16 with the tag D. The probes are mixed and the pool is contacted with an array containing beads to which candidate probes have been bound. The location of each tag (and thus each decoder and candidate probe pair) is then determined. Then, the first set of decoder probes is removed. This time, decoder probes 1, 5, 9, and 13 are labeled with tag A, decoder probes 2, 6, 10, and 14 are labeled with tag B, and decoder probes 3, 7, 11, and 15 are labeled with tag C, Add a second set of decoder probes 4, 8, 12 and 16 labeled with tag D. Thus, beads containing tag A in both decoding steps contain candidate probe 1; tag A in the first decoding step and tag B in the second decoding step contain candidate probe 2; Tag A in the decoding step and Tag C in the second step contain candidate probe 3, and so on. As will be apparent to those skilled in the art, the decoder probes can be made in any order and can be added in any order.
[0098]
In one example, the decoder probe is labeled in situ. That is, they do not need to be labeled before the decoding reaction. In this embodiment, the input decoder probe is shorter than the candidate probe, creating a 5 '"overhang" on the decoding probe. The addition of labeled ddNTPs (each labeled with a unique tag) and polymerase allows for the addition of the tag in a sequence-specific manner, thus creating a sequence-specific pattern of signal. Similarly, other modifications can be made, including ligation and the like.
Furthermore, since the size of the array is set by the number of unique decoding binding ligands, it is possible to "reuse" a unique set of DBLs to take into account a larger number of test sites It is. This may be done in several ways, for example by using several subpopulations containing optical signatures. Similarly, the use of a positional coding scheme within the array; different sub-bundles may reuse the set of DBLs. Similarly, one embodiment utilizes bead size as a coding modality, thus allowing the reuse of a unique set of DBLs for each bead size. Alternatively, continuous partial loading of the array with beads also allows for DBL reuse. Further, "code sharing" can occur as well.
[0099]
In a preferred embodiment, DBL can be reused by including an optical signature in some subpopulations of beads. In a preferred embodiment, the optical signature is generally a mixture of a reporter dye, preferably fluorescent. By varying both the composition of the mixture (ie, the ratio of one dye to another) and the concentration of the dye (leading to differences in signal intensity), a matrix of unique optical signatures can be created. This may be done by covalently attaching the dye to the surface of the bead, or by incorporating the dye into the bead. The dye may be a chromophore or a fluorophore, but is preferably a fluorescent dye, which provides a good signal to noise ratio for decoding due to its strong signal. Dyes suitable for use in the present invention include those enumerated above for labeled DBL.
[0100]
In a preferred embodiment, the encoding may be achieved in a ratio of at least two dyes, for example, but may add more encoding dimensions in bead size. Further, the labels are distinguishable from each other; thus, the two different labels may comprise different molecules (ie, two different fluorophores) or, alternatively, may be one label at two different concentrations or intensities. Is also good.
In a preferred embodiment, the dye is covalently attached to the surface of the bead. This may be done using functional groups on the surface of the beads, as generally outlined for the attachment of bioactive agents. As will be apparent to those skilled in the art, these linkages are made to minimize the effect on the dye.
In a preferred embodiment, the dye is non-covalently associated with the bead, generally by trapping the dye in the pores of the bead.
In addition, encoding in a ratio of two or more dyes, rather than a single dye concentration, is preferred because it is insensitive to the light intensity used to determine the signature and detection sensitivity of the reporter dye.
[0101]
In a preferred embodiment, a spatial or positional coding system is provided. In this embodiment, there are sub-bundles or sub-arrays that are utilized (ie, part of the entire array). Telephone System For example, each sub-array is a "region code" and can have the same indicia (ie, telephone number) of other sub-arrays separated by the location of the sub-array. Thus, for example, the same unique indicator can be reused for each bundle. Thus, the use of 50 unique labels in combination with 100 different subarrays can form an array of 5000 different bioactive agents. In this embodiment, it is important that one bundle be able to be identified from the other bundles; in general, this can be by hand or using marker beads (these can be beads containing a unique tag for each subarray). ) Or by using the same marker beads in different amounts, or by using two or more marker beads in different ratios.
[0102]
In another embodiment, additional encoding parameters, such as microsphere size, can be added. For example, the use of different sized beads may also allow for reuse of a set of DBLs. That is, it is possible to use microspheres of different sizes to increase the encoding dimension of the microsphere. Optical fiber arrays containing pixels with different fiber diameters or cross-sections can be made; alternatively, two or more optical fiber bundles (each having a different cross-section of an individual fiber) to form a larger bundle Can be added together; or a fiber optic bundle with fibers of the same size cross section but with different size beads can be used. With different diameters, the largest well can be filled with the largest microspheres, and then smaller microspheres are moved in progressively smaller wells until all sized wells are filled. In this way, the same dye ratio can be used to encode microspheres of different sizes, thereby expanding the number of different oligonucleotide sequences or chemical functionalities present in the array. Although outlined for fiber optic substrates, this method, as well as the other methods outlined herein, can be similarly performed using other substrates and other bonding modes.
[0103]
In a preferred embodiment, coding and decoding is achieved by continuous loading of the microspheres into an array. In this embodiment, the optical signature can be "re-used" as outlined above for spatial coding. In this embodiment, the library of microspheres, each containing a different bioactive agent (or each subpopulation containing a different bioactive agent) is divided into a plurality of sublibraries; for example, the desired array size and Depending on the number of unique tags, ten sub-libraries may be generated, each containing about 10% of the total library, with each sub-library containing approximately the same unique tag. The first sub-library is then added to the fiber optic bundle containing the wells, and the location of each bioactive agent is determined, generally by use of DBL. Then a second sub-library is added and the location of each bioactive agent is determined again. In this case, the signals will include the signals from the "first" DBL and the "second" DBL. By comparing the two matrices, the location of each bead in each sublibrary can be determined. Similarly, the array could be filled by successively adding third, fourth, etc. sublibraries.
[0104]
In a preferred embodiment, the code can be "shared" in several ways. In a first embodiment, a single code (i.e., an IBL / DBL pair) can be given to two or more agents for target analytes with sufficiently different avidities. For example, the two nucleic acid probes used in the mRNA quantification assay can share the same code if their hybridization signal intensity ranges do not overlap. This can occur, for example, if one of the target sequences is always present at a much higher concentration than the other. Alternatively, the two target sequences may always be present at similar concentrations, but differ in hybridization efficiency.
Alternatively, a single code could be given to multiple drugs if the drugs are functionally equivalent. For example, if a set of oligonucleotide probes is designed for the common purpose of detecting the presence of a particular gene, the probes are functionally equivalent, even if the sequences are different. Similarly, if a class or "family" of analytes was desired, all probes for different members of the class, such as kinases or G-protein coupled receptors, could share the code. Similarly, this type of array could be used to detect homologs of known genes. In the embodiment, each gene is represented by a heterogeneous set of probes that hybridize (and thus differ in sequence) to different gene regions. The sets of probes share a common code. If homologues are present, they may hybridize to some, but not all, of the probes. The level of homology is indicated by the fraction of hybridizing probes as well as the average hybridization intensity. Similarly, multiple antibodies to the same protein could all share the same code.
[0105]
In a preferred embodiment, decoding of a self-assembled random array is performed based on pH titration. In such embodiments, in addition to the bioactive agent, the beads include an optical signature, wherein the optical signature is a pH-responsive dye, such as a luminophore (sometimes referred to herein as a "pH dye"). ). The embodiment is similar to the above, except that when the solution pH is adjusted from below pKa to above pKa (or vice versa), the dyes used in the present invention exhibit a change in fluorescence intensity (or other property). Similar to those outlined in PCT US98 / 05025 and US Patent No. 09/151877, both of which are expressly incorporated herein by reference. In a preferred embodiment, a set of pH dyes, each having a different pKa, is used, preferably distinguished by at least 0.5 pH units. Preferred specific examples are pKa 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7. PH dyes of 0.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11 and 11.5 are used. Each bead can contain any subset of the pH dye, thus providing a unique code for the bioactive agent. Thus, decoding of the array is achieved by titrating the array from pH 1 to pH 13 and measuring the fluorescence from each bead as a function of solution pH.
[0106]
In a preferred embodiment, there are additional ways to increase the number of unique or distinct tags. That is, the number of codes can be increased using separate attributes on each bead. In addition, continuous decoding allows for code reuse in a novel way. These attributes are independent of each other, and thus the number of codes can be increased exponentially as a function of the number of decoding steps and the number of attributes (eg, separate codes). However, by increasing the amount of decoding information obtained in a single decoding step, the number of decoding steps is significantly reduced. Alternatively, the number of distinct codes increases significantly. By increasing the number of attributes per decoding step, fewer decoding steps are required for a given number of codes. Thus, in a preferred embodiment, various methods are used to generate a large number of codes useful in the process of decoding the array, while at the same time minimizing the required decoding steps. For example, various different coding strategies can be combined. Thus, different "colors", combinations of colors ("hues"), different intensities of colors and / or hues, etc., can all be combined.
[0107]
In a preferred embodiment, the DBL relies on attaching or embedding a quantitative or distinct set of physical attributes to the beads, ie, labeling the beads. Preferred physical attributes of the beads include, but are not limited to, "smoothness" or "roughness" of the surface, color (fluorescence and other), color intensity, size, detectable chemical moiety, chemical Reactivity, magnetism, pH sensitivity, energy transfer efficiency between existing dyes, hydrophobicity, hydrophilicity, absorption, charge, pH sensitivity, and the like.
The bead decoding scheme involves imparting / soaking a single quantifiable attribute to each bead type, where each bead type differs in the quantifiable value of the attribute. For example, a predetermined number of phosphors can be attached to the beads, and the number of phosphors attached during the decoding process can be quantified. Accurate measurement may be problematic. Generally, the goal is to reduce the coefficient of variation (CV). By the coefficient of variation is meant the variability in labeling the beads in a continuous labeling. The CV can be determined by labeling the beads with a predetermined number of labels (eg, fluorophores) in a plurality of tests and measuring the resulting signal emitted by the beads. Large CVs limit the number of "levels" available and resolvable for any given attribute.
[0108]
More robust decoding schemes use ratio rather than absolute measurements to separate quantitative attributes into codes. By ratiometric decoding, it means labeling the beads with a certain ratio of labels (ie, 1:10, 1: 1 and 10: 1). In theory, any ratio can be used as long as the signal difference between the ratios is detectable. The process results in a smaller CV and allows for more attribute splits within a given dynamic range. Thus, in a preferred embodiment, the use of ratio meter decoding reduces the coefficient of variation.
Further, as will be apparent to those skilled in the art, ratio meter decoding can be accomplished in different ways. In this embodiment, a predetermined number of DBLs having a first dye (or dye combination) intensity in a first decoding reaction and a second number having a second dye intensity in a continuous second decoding reaction. Rather than adding, the ratiometric analysis may be performed with a ratio of labeled: unlabeled DBL. That is, given a set of saturating concentrations of decoding beads, eg, 100,000 DBL / reaction, the first intensity decoding step may be performed by adding 100,000 labeled DBLs, and the second step is This can be done by adding 10,000 labeled DBL and 90000 unlabeled DBL. Equilibrium indicates that the second step gives a tenth signal intensity.
[0109]
Due to the diffusion of quantitatively measured attribute values, the number of distinct codes is actually limited to less than 12 codes. However, by successively "painting" (i.e., temporarily bonding the attribute level to the bead) and "peeling" (removing the attribute level) beads having different attribute values, the potential code The number increases exponentially with the number of successive steps in the decoding process.
An example is shown. For example, nine different bead types and three distinguishable attribute distributions (Table 1). By "painting" (labeling) the beads in two different stages with different attribute values in combinatorially distinct patterns, a unique code is generated for each bead type. That is, nine distinct codes result. Thus, in a preferred embodiment, the beads are labeled in multiple stages with different attributes in combinatorially distinct patterns. This results in a unique code for each bead type. Examples of different attributes are described above. Labeling of the beads with different attributes is performed by methods known in the art.
[0110]
[Table 1]

[0111]
Fluorescent color is a particularly convenient attribute for use in decoding schemes. The fluorescent color can be attached to any drug that recognizes the IBL to form a labeled DBL. Discussion is directed to oligonucleotides as DBLs, including nucleic acid analogs. Fluorescently labeled oligonucleotides can specifically and reversibly target any desired subset of beads in a particular color through the process of hybridization and dehybridization (ie, to a DBL having a complementary sequence). It is a particularly useful DBL because it can simply be "painted" (marked). In addition, fluorescence is easily imaged and quantified using standard optical hardware and software. In order to "paint" a given bead type with a particular color, the bead type must be labeled with a unique hybridizable DNA sequence (IBL) and the decoding solution is labeled with that sequence color. Must contain complement.
[0112]
One consideration in implementing a decoding scheme is to minimize the number of images collected. In a color-based scheme, the number of images collected is a product of the number of colors and the number of steps. The number of images can be reduced by "painting" the beads with multiple colors for each given step. Providing multiple colors to the beads increases the number of effective codes. For example, in a 24-bit three-color scheme (eg, red, green, blue) coloring process used by a computer, a total of 256^*256^*256 = 16700000 different "hues" can arise from only three colors (red, green, blue).
Thus, in a preferred embodiment, the DBL is labeled with a combination of colored phosphors. As such, the method is useful in increasing the number of codes available for labeling DBL with only a few different dyes (colors). Increasing the number of codes available at each decoding step will greatly reduce the number of decoding steps required in a given decoding step.
[0113]
In one example, a population of oligonucleotides encoding a single DBL is defined such that each bead to which the DBL binds is identified based on a characteristic "hue" formulated from a combination of colored fluorophores. It is labeled with the ratio color. In a preferred embodiment, two distinct colors are used. In a preferred embodiment, three or more distinct dyes (colors) are available. In this example, the number of distinguishable codes generated by labeling a population of oligonucleotides encoding a single DBL with any given color is three. However, considering the combination of colors and color levels in labeling yields more codes.
For decoding by hybridization, the preferred number of distinguishable shades is 2-2000, the more preferred number of distinguishable shades is 2-200, and the most preferred number of distinguishable shades is 2-20. . With three different tones (intensities) and three colors, the number of different hues is three.⁴= 81. The combination of hue and continuous decoding can produce a virtually unlimited number of codes.
[0114]
As previously described, DBL can be any agent that binds to IBL. In a preferred embodiment, a single DBL is labeled with a predetermined ratio of colors. The ratio is varied for each DBL, thus allowing a unique "hue" for each DBL itself to be labeled. After treatment of the beads with DBL, the beads are analyzed to determine the "hue" associated with each bead, thereby identifying the beads with the associated bioactive agent.
For example, with four primary colors and two intensity levels (presence or absence of color), fifteen different hues / stages are possible. With four dyes and three different intensity levels (absent, half present, completely present), 73 different hues / stages are possible. In this case, the acquisition of an image of only four colors is sufficient to obtain information on the hues of the 73 different codings.
[0115]
In a preferred embodiment, the present invention provides an array composition comprising a first substrate having a surface comprising a discrete site. A preferred embodiment utilizes a population of microspheres distributed at the site, the population comprising at least a first and a second subpopulation. Each sub-population comprises a bioactive agent and, in addition, at least one optical dye having a predetermined pKa. The pKa of different optical dyes is different.
In a preferred embodiment, for example, where the array contains cloned nucleic acids, there are several methods that can be used to decode the array. In a preferred embodiment, if some sequence information is known about the cloned nucleic acid, certain decoding probes can be generated, as broadly outlined herein.
[0116]
In a preferred embodiment, "random" decoding probes can be generated. As outlined previously, the use of a series of hybridizations or multiple labels can result in a unique hybridization pattern for each sensor element. As a result, all of the beads representing a given clone can be considered to belong to the same group. Generally, this is done with random or partially degenerate decoding probes that bind in a sequence-dependent but not highly sequence-specific manner. The process can be repeated many times, each time with a different labeling substance, and can produce different signal patterns based in a sense on specific interactions. In this way, a unique optical signature is finally established for each sensor element. By applying pattern recognition or clustering algorithms to optical signatures, beads can be grouped into sets that share the same signature (ie, have the same probe).
[0117]
Additional methods are required to identify the actual sequence of the clone itself, for example, direct sequencing can be performed. By using an ordered array containing clones, such as a spotted cDNA, the "key" relating the hybridization pattern to a particular clone whose position within the set is known is known. Can occur. In this way, clones can be recovered and further characterized.
Alternatively, clonal arrays can be decoded using binary decoding with vector tags. For example, a partially randomized oligo is cloned into a nucleic acid vector (eg, a plasmid, phage, etc.). Each oligonucleotide sequence consists of a subset of a limited set of sequences. For example, if the limited set contains 10 sequences, each oligonucleotide may have some subset (or all 10) sequences. That is, each of the ten sequences may or may not be present in the oligonucleotide. Therefore, 2¹⁰Or there are 1024 possible combinations. The sequences may overlap and may show minor variants (eg, A, C, T and G substitutions) to increase the number of possible combinations. The nucleic acid library is cloned into a vector containing a random coding sequence. Alternatively, tags can be added using other methods, such as PCR. In this way, a small number of oligo decoding probes can be used to decode an array of clones.
[0118]
In a preferred embodiment, discriminant analysis and cluster algorithms and computer equipment are used to analyze the decoding data from the arrays of the invention. The potentially large number of codes used in the present invention, combined with the use of different intensity and "hue" phosphors in a multi-stage decoding process, requires good classification of the data. The data, especially the intensity data, indicates that at each stage the beads are reversibly labeled with a different color or mixture of colors ("hues") (eg, dye-labeled complementary decoding oligonucleotides are labeled on the IBL on the beads). (Either by hybridizing to a probe or by forming a binding ligand pair for a non-nucleic acid IBL-DBL pair). The challenge is to correctly classify the beads according to which color was painted at each step. The closer the markers are to one another (as determined by the optical imaging system), the more difficult the classification.
[0119]
The proximity of the dye as indicated by the imaging system is determined by the spectral characteristics of the decoding dye and the spectral channel separation of the imaging system. Better color separation is achieved by using fluorescent dyes with a narrow emission spectrum and by narrow bandpass excitation and emission designed to excite the dye "at the peak" and measure its emission "at the peak" This is achieved by using an optical system having a filter. The process of optically imaging the dye on the beads resembles the human visual process in which the brain sees color by measuring the ratio of excitation in three different cone types in the eye. However, with optical imaging systems, the actual number of color channels is much greater than the three present in the human eye. An imaging system based on a CCD can "see" colors from 350 nm to 850 nm, but the eye cone is tuned to the visible spectrum of 500-600 nm.
[0120]
The problem of decoding a bead array is essentially a discriminant analysis classification problem. Thus, in a preferred embodiment, the analysis of the variation in the hyperspectral α-space is performed on a known set of bead colors or hues. The center of the bead cluster in α space is called the cluster's center of gravity, and the scattering of points within the cluster determines the cluster's diffusion. A robust classification scheme requires that the distance between the centers of gravity of different bead classes (hues) be much greater than the diffusion of either cluster class. Further, the location of the center of gravity should remain unchanged from fiber to fiber and from experiment to experiment.
Thus, in a preferred embodiment, a hue "zone" is defined as an area in the alpha space that extends around the centroid of the hue and beyond the diffusion radius of the cluster. Using a control set of hue centroids and diffusion radii, as determined empirically, the classification of a new set of data is based on whether a given bead point falls near or into a `` zone '' of a hue cluster. Can be achieved by seeking This is achieved by calculating the Mahalanobis distance of the bead points from the centroids of the different hue classes, in this case it is simply a Euclidean distance metric. For the data shown in FIG. 3, the locations of the centers of gravity and their distances from each other are shown in Table 2.
[0121]
[Table 2]

[0122]
A Euclidean distance cutoff of 0.3 was chosen to classify different beads into specific hue classes. The two nearest centroids, Bod-R6G and Bod-564 (distance = 0.55), overlap slightly in the decoding zone when using a Euclidean or Mahalanobis distance of 0.3. Improvements in classification are achieved by reducing this distance and weighting different coordinate axes appropriately.
Thus, the present invention provides a computer method for analyzing and classifying bead color. Classification of bead colors is performed by examining the beads in the hyperspectral "α" space (a₁= I₁/ SI_i, A₂= I₂/ SI_i, A₃= I₃/ SI_i, Etc.), where each coordinate axis represents a fraction of the bead intensity within a given image channel. For example, if beads are imaged using four image channels, the color or hue of the beads can be indicated by a point in the 3-Dα space (Sa_i= 1, so no fourth dimension is needed). If different sets of primary dyes are used to label the beads, the number of hues that can be generated from these dyes is unlimited, as the dyes can be combined in changing ratios and changing combination patterns. The actual number of hues is determined experimentally by the separation of different hue clusters in the hyperspectral α space.
[0123]
FIG. 3 shows a hyperspectral α plot of beads labeled with four different hues imaged in four separate image channels. Note that the beads form four distinct clusters. The fact that these four clusters are well separated allows a robust decoding classification scheme to be performed.
In a preferred embodiment, a quality control analysis of the decoding process is performed. This is achieved by performing a cluster analysis of α space for each decoding stage. The number of clusters determined will be fixed by the expected number of hues. The location of the cluster centroid will be monitored and any deviation from the expected location will be noted.
[0124]
Accordingly, the present invention provides an apparatus for decoding the array of the present invention. In addition to the compositions outlined herein, the device includes a central processing unit that communicates via a bus with a memory and a set of input / output devices (eg, keyboard, mouse, monitor, printer, etc.). The general interaction between a central processing unit, memory, input / output devices and a bus is known in the art. One aspect of the present invention is directed to a hyperspectral “α” space classification system stored in memory.
The classification system program includes a data acquisition module that receives data from an optical reader or confocal microscope (or other imaging system). In general, the classification program will also be able to analyze changes in hyperspectral alpha space, calculate cluster centroids, calculate cluster scattering (diffusion), and identify hue zones and distance cutoffs. Is included. In general, the analysis module will further determine whether the data point falls within the hue zone by calculating the Mahalanobis distance.
[0125]
Ultimately, the analysis module will analyze a different set of decoding information to ultimately provide the bioactive agent to the bead location.
Thus, each step performs a series of decoding steps using discriminant analysis calculations, each giving each bead in the array to a hue cluster. The accumulation of a series of decoding information gives the correlation between the location of the beads and the chemistry contained therein.
Once made, the compositions of the present invention can be used in many applications. In a preferred embodiment, the composition is used to probe the sample solution for the presence or absence of the target analyte, including quantifying the amount of the target analyte present. By "target analyte" or "analyte" or grammatical equivalents herein is meant any atom, molecule, ion, molecular ion, compound or particle that is detected or evaluated for a binding partner. As will be apparent to those skilled in the art, many analytes can be used in the present invention, and basically any analyte that binds a bioactive agent or binding partner (ie, drug candidate) Any analyte for which is required can be used.
[0126]
Suitable analytes include organic and inorganic molecules, including biological molecules. When detecting target analytes, suitable target analytes include, but are not limited to, environmental pollutants (including pesticides, pesticides, toxins, etc.) and chemicals (solvents, polymers, organic substances, etc.). ), Therapeutic molecules (including therapeutic and abusive drugs, antibiotics, etc.), biological molecules (hormones, cytokines, proteins, nucleic acids, lipids, carbohydrates, cell membrane antigens and receptors (nerves, hormones, nutrients and cell surface receptors) Or their ligands), whole cells (including prokaryotic cells (eg, pathogenic bacteria) and eukaryotic cells, mammalian tumor cells), viruses (including retroviruses, herpesviruses, adenoviruses, lentiviruses, etc.) ) And spores. Particularly preferred analytes are nucleic acids and proteins.
[0127]
In a preferred embodiment, the target analyte is a protein. As will be apparent to those skilled in the art, there are a number of potential proteinaceous target analytes that can be detected or evaluated for binding partners using the present invention. Suitable protein target analytes include, but are not limited to, (1) immunoglobulins; (2) enzymes (and other proteins); (3) hormones and cytokines, many of which serve as ligands for cellular receptors. ) And (4) other proteins.
In a preferred embodiment, the target analyte is a nucleic acid. As outlined broadly in U.S. Patent Nos. 60/160027; 60/161148; 09/425633 and 60/160917, all of which are expressly incorporated herein by reference, these assays include: Used for a wide range of applications.
[0128]
In a preferred embodiment, the probe is used for genetic diagnosis. For example, probes were made using the methods disclosed herein to produce non-polyposis colon cancer, BRCA1 breast cancer gene, P53, a gene associated with various cancers, ApoE4 showing greater risk of Alzheimer's disease A target sequence such as a gene can be detected, and the patient, cystic fibrosis gene, cytochrome p450s or any other mutation well known to those skilled in the art can be easily presymptomically screened. It becomes.
In a further embodiment, the detection of viruses and bacteria is performed using the complexes of the present invention. In this embodiment, probes are designed to detect target sequences from various bacteria and viruses. For example, modern blood screening techniques rely on the detection of anti-HIV antibodies. The methods disclosed herein allow for direct screening of clinical samples to detect HIV nucleic acid sequences, particularly highly conserved HIV sequences. In addition, as with improved methods of assessing the efficacy of antiviral therapy, this allows for direct measurement of the virus circulating in the patient's body. Similarly, viruses associated with leukemia, HTLV-I and HTLV-II can be detected in this way. Bacterial infections such as tuberculosis, chlamydia and other sexually transmitted diseases can also be detected.
[0129]
In a preferred embodiment, the nucleic acids of the invention are used as probes for toxic bacteria in screening water and food samples. For example, a sample may be processed to lyse bacteria and release its nucleic acids, and then salmonella, Campylobacter, Vibrio cholerae, Leishmania, e. Probes can be designed that recognize bacterial strains, including, but not limited to, pathogenic strains such as E. coli enterotoxin strains and Veterans Affairs bacteria. Similarly, bioremediation methods can be evaluated using the compositions of the present invention.
In a further embodiment, the probe is used for forensic "DNA fingerprinting" to match criminal-scene DNA to samples taken from victims and suspects.
In a further embodiment, the probes in the array are used for sequencing by hybridization.
[0130]
The present invention is also used as a method for detecting a mutation or mismatch in a target nucleic acid sequence. For example, the use of polymorphic DNA markers to analyze the relationship between genetic variation and phenotype has recently attracted attention. Previous studies have utilized short tandem repeats (STRs) as polymorphic position markers, but recently the use of single nucleotide polymorphisms (SNPs) has received attention. Common SNPs occur at an average frequency of 1 or more per kilobase of human genomic DNA. Some SNPs, especially within and near the coding sequence, appear to be the direct cause of therapeutically relevant phenotypic variants. There are many known polymorphisms that cause a clinically significant phenotype, for example, the apoE2 / 3/4 variant is associated with different relative risks of Alzheimer's disease and other diseases (Cordor et al., Science 261 (1993) )). Multiplex PCR amplification of SNP positions using subsequent hybridization to an oligonucleotide array has been shown to be a rapid and reliable method of genotyping at least hundreds of SNPs simultaneously: Wang et al. , Science, 280: 1077 (1998); see also Schaffer et al. , Nature Biotechnology 16: 33-39 (1998). The compositions of the present invention can easily replace prior art arrays. In particular, single base extension (SBE) and pyrosequencing techniques are particularly useful for the compositions of the present invention.
[0131]
In a preferred embodiment, the compositions of the invention are used to screen for bioactive agents to find agents that bind to the target molecule and preferably modify its function. As mentioned above, a wide variety of different assay formats may be operated, as will be apparent to those skilled in the art. Generally, the binding partner labels the desired target analyte, binds the target analyte to the bioactive agent and recruits the label to the beads, followed by detection.
In a preferred embodiment, the binding of the bioactive agent to the target analyte is specific; that is, the bioactive agent specifically binds to the target analyte. As used herein, "specific binding" means that the substance binds to the analyte sufficiently specifically to distinguish the analyte from other components or contaminants of the test sample. However, as would be apparent to one of skill in the art, less specific binding could be used to detect the analyte. For example, the system may use an array of different binding ligands, eg, different ligands, and any detection of a particular analyte is due to its “signature” binding to a panel of binding ligands, and an “electronic nose”. ) ”Is similar to the way it works. This has particular utility in the detection of chemical analytes. Binding includes a washing step to remove non-specific binding (although in some embodiments, a washing step is not required) to maintain binding under the conditions of the assay, ie, to bind the low affinity binding partner. Should be sufficient to detect. In some embodiments, such as the detection of certain biomolecules, the separation constant of the analyte to bound ligand is about 10^-4-10^{− 6}M^-1Smaller, about 10^{− 5}-10^{− 9}M^{− 1}Preferably smaller than 10^-7-10^-9M^-1Particularly preferred is smaller.
[0132]
Generally, a sample containing a target analyte (for detection of the target analyte or for screening for a binding partner of the target analyte) is subjected to conditions suitable for binding of the target analyte to at least one bioactive agent. It is added to the array below, ie, generally under physiological conditions. The presence or absence of the target analyte is then detected. As will be appreciated by those skilled in the art, this can be done in a variety of ways, generally using changes in the optical signal. This change can occur via many different mechanisms. A few examples include binding of dye-tagged analytes to beads, production of dye species on or near beads, disruption of existing dye species, and optical interaction in analyte interactions with dye on beads. Includes changes in the signature or other optically responsive signals.
[0133]
In a preferred example, the change in the optical signal results from the binding of a target analyte labeled with a directly or indirectly detectable label, preferably an optical label such as a fluorescent dye. Thus, for example, when using a proteinaceous target analyte, it can be labeled directly or indirectly, for example, by the use of labeled antibodies. Similarly, nucleic acids are readily labeled with fluorescent dyes as is known in the art, for example, through PCR amplification. Alternatively, hybridization indicators can be used as labels, based on the binding of the target sequence. Hybridization indicators typically bind reversibly and preferentially to double-stranded nucleic acids. Hybridization indicators include intercalators and minor and / or major groove junctions. In a preferred embodiment, an intercalator can be used; the intercalation usually only occurs in the presence of double-stranded nucleic acid, so that the label becomes bright only in the presence of target hybridization. Thus, based on the binding of the target analyte to the bioactive agent, a new optical signal is generated at this site and can be detected.
[0134]
Alternatively, in some cases, as described above, the target analyte, such as an enzyme, results in a directly or indirectly optically detectable species.
Further, in some embodiments, the change in the optical signature may be based on the optical signal. For example, the interaction of certain chemical target analytes with certain fluorescent dyes on the beads can change the optical signature, thus producing a different optical signal.
As will be appreciated by those skilled in the art, in some embodiments, the presence or absence of the target analyte may include other optical or non-optical signals (including surface enhanced Raman spectroscopy, surface plasmon resonance, radioactivity, etc.). , But is not limited to this).
[0135]
As will be appreciated by those skilled in the art, the assays can be performed under various experimental conditions. In screening assays, various other reagents may be included. These include salts, neutral proteins (eg, albumin, detergents, etc.) that can be used to promote optimal protein-protein binding and / or reduce non-specific or background interactions. Various reagents. Also, reagents that improve the efficiency of the assay in other ways, such as protease inhibitors, nuclease inhibitors, antimicrobial agents, etc. can be used. The mixture of components can be added in any order that provides for the requisite binding. Various blocking and washing steps can be used, as known to those skilled in the art.
[0136]
In a preferred example, a two-color competitive hybridization assay is performed. These assays can be based on conventional sandwich assays. The beads include a capture sequence located on one side (upstream or downstream) of the SNP and can capture the target sequence. Two SNP allele-specific probes, each labeled with a different fluorophore, hybridize to the target sequence. Usually, the genotype can be obtained from the ratio of the two signals using a calibration sequence that shows better binding. This is advantageous in the target sequence itself, which does not need to be labeled. Furthermore, since the probes are antagonistic, this means that the binding conditions need not be optimized. Under conditions where the mismatched probe stably binds, the matched probe can be further replaced. Thus, competitive assays can give better discrimination under those conditions. Since many assays are performed in parallel, the conditions cannot be optimized for all probes simultaneously. Thus, a competitive assay system can be used to help compensate for non-optimal conditions for mismatch discrimination.
[0137]
In one preferred embodiment, the compositions of the invention are used to perform dideoxynucleotide chain termination sequencing. In this embodiment, the primer is extended using ddNTPs that have been fluorescently labeled with a DNA polymerase. The 3 'end of the primer is adjacent to the SNP site. In this method, the single base extension is complementary to the sequence at the SNP site. Using four different fluorophores, one for each base, the sequence of the SNP can be derived by comparing the four base-specific signals. This can be done in several ways. In the first embodiment, the capture probe can be extended; in this approach, the probe is synthesized 5'-3 'on the bead or attached to the 5' end to provide a free 3 'end for polymerase extension. Must be given. Alternatively, a sandwich-type assay can be used; in this embodiment, the target is captured on a bead by a probe, then the primer is annealed and extended. In the latter case, the target sequence may not be labeled. Furthermore, since the sandwich assay requires two specific interactions, this increases the stringency which is particularly useful for analyzing complex samples.
[0138]
Furthermore, if both the target analyte and the DBL bind to the drug, detection of the unlabeled target analyte via antagonism of decoding is also possible. In one preferred embodiment, the method of the present invention is useful in array quality control. Prior to the present invention, no method was disclosed to provide a positive test of the performance of any probe on any array. Array decoding not only provides this test, but also by utilizing the data generated during the decoding process itself. Therefore, no further experimental work is required. The present invention requires only one set of data analysis algorithms that can be coded into software.
[0139]
Quality control measures can identify a wide range of systematic and random issues in the array. For example, random spots of dust or other contaminants can cause some sensors to give an incorrect signal-which can be detected during decoding. Leaks of one or more drugs from multiple arrays can also be detected. The advantage of this quality control procedure is that it can be performed just before the assay itself and is a true functional test of the individual sensors. Thus, any problems that may arise between the array assembly and actual use can be detected. Decoding and quality control can be performed both before and after actual sample analysis in applications where a very high level of reliability is required and / or there is a significant potential for sensor failure during experimental procedures. .
[0140]
In one preferred embodiment, reagent quality control can be performed using an array. In many instances, biological macromolecules are used as reagents and must be quality controlled. For example, a large set of oligonucleotide probes can be provided as reagents. It is usually difficult to perform quality control on a large number of different biological macromolecules. Using the approach described herein, this can be done by treating a variable reagent (formulated as DBL) instead of an array.
In a preferred embodiment, the methods described herein are used for array calibration. For many applications, such as mRNA quantification, having a signal that is a linear response to the concentration of the target analyte, or, alternatively, in a non-linear case, determining the relationship between concentration and signal to determine the target analyte It is desirable to be able to evaluate the density of light. Accordingly, the present invention provides a method for generating a calibration curve for a plurality of beads in an array in parallel. Calibration curves can be generated under conditions that simulate the complexity of the sample being analyzed. Each curve can be constructed independently of the other curves (eg, for different concentration ranges) and simultaneously with all other curves for the array. Thus, in this embodiment, a series of decoding schemes are implemented using different concentrations, rather than different fluorophores, as code "labels". In this way, the signal as a response to concentration can be measured for each bead. The calibration can be performed immediately before use of the array, so that every probe on every array is individually calibrated as needed.
[0141]
In a preferred embodiment, the method of the invention can be used for assay development as well. Thus, for example, the method allows for the identification of good and bad probes; as will be appreciated by those skilled in the art, some probes will not hybridize well or will cross-hybridize with one or more sequences, Does not work well. These problems are easily detected during decoding. The ability to quickly assess the performance of a probe has the potential to greatly reduce assay development time and costs.
Similarly, in one preferred embodiment, the methods of the invention are useful in quantification in assay development. The major challenge in many assays is the ability to detect differences in analyte concentration between samples, the ability to quantify these differences, and the absolute concentration of the analyte, all in the presence of a complex mixture of the analytes of interest. Is the measurement. An example of this task is the quantification of specific mRNA in the presence of total cellular mRNA. One approach developed as the basis for mRNA quantification utilizes multiple matched and mismatched probe pairs (Lockhart et al., 1996), the contents of which are hereby incorporated by reference. This approach is simple, but requires a relatively large number of probes. In this approach, a quantitative response to concentration is obtained by averaging the signals from a set of different probes for the gene or sequence of interest. This is essential since only some probes respond quantitatively and these probes cannot be reliably predicted. Without prior knowledge, only the average response of a properly selected collection of probes is quantitative. However, the present invention has general application to nucleic acid-based arrays as well as other assays. In short, the approach is to identify probes that respond quantitatively in a particular assay, not the average with other probes. This is done by the array calibration scheme described above using a concentration based code. The advantages of this approach are that each and all sequences can be tested in an efficient manner; fewer probes are required; the accuracy of the measurement is less dependent on the number of probes used; and the response of the sensor is Sometimes known for a high degree of certainty. It is important to note that well-functioning probes are selected empirically, avoiding the difficulty and uncertainty of predicting probe performance, especially in complex sequence mixtures. In contrast, in the experiments described previously using order arrays, a relatively small number of sequences are checked by performing quantitative spiking experiments that add known mRNA to the mixture.
[0142]
In a preferred embodiment, a cDNA array is generated for RNA expression profiling. In this embodiment, individual cDNA clones are amplified (eg, using PCR) from a cDNA library grown in a host vector system. Each amplified DNA is bound to a population of beads. The different populations are mixed together to create a collection of beads representing the cDNA library. The beads are arrayed, decoded as described above, and used in the assay (described herein, but decoding may also occur after the assay). The assay is performed using RNA samples (whole cells or mRNA) that have been extracted, optionally labeled, and hybridized to the array. Competition analysis allows the detection of differences in the expression levels of individual RNAs. Comparison with an appropriate set of calibration standards allows quantification of the absolute amount of RNA.
[0143]
Also, cDNA arrays can be used for mapping, for example, to map deletions / insertions or, for example, to map copy number changes in genomes from tumors or other tissue samples. This can be done by hybridizing genomic DNA. Instead of cDNA (or EST, etc.), other STSs (sequence tag sites) containing random genomic fragments can be arrayed for this purpose.
All references cited herein are hereby incorporated by reference in their entirety.
[Brief description of the drawings]
FIG. 1A, 1B, 1C, 1D and 1E show examples of several different “two-component” systems of the present invention.
FIGS. 2A and 2B depict two different “one-component” systems.
FIG. 3 represents aggregation in hyperspectral alpha space (α₁= I₁/ ΣI_I, Α₂= I₂/ ΣI_i, Α₃= I₃/ ΣI_iSuch).
FIG. 4 shows a two-color database that uses either FAM-labeled or Cy3-labeled oligo complement to “paint” (label) different bead types on the array. Show the coding process.
FIG. 5 shows decoding of 128 different bead types in 4 colors and 4 decoding stages (inset shows a single decoding stage decoding 16 bead types with 4 different dyes) ).
FIG. 6 depicts grayscale decoding of 16 different beads.
FIG. 7 schematically represents a lid and a base plate.
FIG. 8 schematically depicts a hybridization chamber including a lid 10 and a base plate 60.
FIG. 9 shows a base plate with holes 105.
FIG. 10 depicts the changing solution volume and localization on a membrane caused by pressure and / or vacuum.
FIG. 11 depicts a flowchart of an exemplary assay scheme that is useful for a hybridization chamber.

Claims (28)

a) a substrate having a surface comprising a plurality of assay locations in a hybridization chamber, wherein each assay location comprises a plurality of independent sites; and
b) a population of microspheres comprising at least first and second subpopulations, each subpopulation comprising a bioactive agent;
The device comprising: wherein the microspheres are distributed at each assay location. The device of claim 1, wherein each of the assay locations comprises a substantially similar combination of bioactive agents. 3. The device according to claim 1, wherein the substrate is a microtiter plate and each assay site is a microtiter well. 4. The device of claim 1, 2 or 3, wherein each independent site is one bead well. The device of claim 1, 2, 3, or 4, wherein the subpopulations each further comprise an optical signature that can identify the bioactive agent. 6. The device of claim 1, 2, 3, 4, or 5, wherein each of said subpopulations further comprises an identifier binding ligand that will bind to a decoder binding ligand so that the identity of the bioactive agent can be ascertained. a) a first substrate having a surface including a plurality of assay locations;
b) a second substrate comprising a plurality of array locations, wherein each array location comprises an independent site;
c) a population of microspheres comprising at least a first and a second subpopulation, wherein each subpopulation comprises a bioactive agent;
Wherein the microspheres are distributed at each of the array locations;
d) a hybridization chamber configured to receive the second substrate;
An apparatus, including: The apparatus of claim 7, wherein said first substrate is a microtiter plate. 9. The apparatus of claim 7, wherein the second substrate comprises a plurality of fiber optic bundles comprising a plurality of individual fibers, each bundle including one array location, each individual fiber comprising a bead well. 10. The device according to claim 7, 8 or 9, wherein said hybridization chamber further comprises at least one component port. 11. The device according to claim 7, 8, 9 or 10, wherein each of said subpopulations further comprises an optical signature capable of identifying said bioactive agent. 12. The device of claim 7, 8, 9, 10, or 11, wherein each of the subpopulations further comprises an identifier binding ligand that binds to a decoder binding ligand such that the identity of the bioactive agent can be ascertained. a) a base plate having a base cavity formed therein for holding a first array component;
b) a lid including at least one component port for immobilizing the second array component;
c) a sealant between the base plate and the lid;
A hybridization chamber. 14. The chamber of claim 13, wherein said second array component is a fiber optic bundle. 15. The chamber of claim 13 or claim 14, further comprising at least one alignment mechanism. 16. The chamber of claim 15, wherein at least one alignment mechanism is a male-female fit. 17. The chamber of claim 13, 14, 15 or 16, wherein said first array component is a microtiter plate. 18. The chamber according to claim 13, 14, 15, 16 or 17, further comprising at least one liquid handling device. A method of decoding an array composition, comprising:
a) supplying an array composition to a hybridization chamber, wherein the array composition comprises:
i) a substrate having a surface including a plurality of assay locations, wherein each assay location includes an independent site;
ii) a population of microspheres comprising at least first and second subpopulations, wherein each subpopulation comprises a bioactive agent;
Including;
Wherein the microspheres are distributed at the site;
b) A method comprising adding a plurality of decoding binding ligands to the array composition to identify at least a plurality of bioactive agents. A method of decoding an array composition, comprising:
a) supplying an array composition to a hybridization chamber, wherein the array composition comprises:
i) a substrate having a surface including a plurality of array locations, wherein each array location includes an independent site;
ii) a population of microspheres comprising at least first and second subpopulations, wherein each subpopulation comprises a bioactive agent;
Including;
Wherein the microspheres are distributed at the site;
b) A method comprising adding a plurality of decoding binding ligands to the array composition to identify at least a plurality of bioactive agents. 21. The method of claim 19 or 20, wherein at least one subpopulation of microspheres comprises an identifier binding ligand capable of binding a decoding binding ligand. 21. The method of claim 19 or claim 20, wherein the decoding binding ligand binds the bioactive agent. 23. The method of claim 19, 20, 21 or 22, wherein said decoding binding ligand is labeled. 24. The method of claim 19, 20, 21, 22, or 23, wherein the position of each subpopulation is determined. A method for determining the presence of one or more target analytes in one or more samples, comprising:
a) said sample;
i) a substrate having a surface including a plurality of array locations, wherein each array location includes an independent site;
ii) a population of microspheres comprising at least a first and a second subpopulation, wherein each subpopulation comprises a bioactive agent, wherein the microspheres have independent sites containing microspheres. Is distributed at the site).
Contacting with the composition, comprising:
b) Incubate in hybridization chamber;
c) determining the presence or absence of the target analyte. A method for determining the presence of one or more target analytes in one or more samples, comprising:
a) adding a sample to a first substrate containing a plurality of assay locations to cause the sample to be contained in the plurality of assay locations;
b) the sample is
i) a surface comprising a plurality of array locations, each array location comprising an independent site;
ii) a population of microspheres, each comprising a bioactive agent, comprising at least a first and a second sub-population, wherein the microspheres are distributed at said sites such that their independent sites contain microspheres. Contacting with a second substrate comprising:
b) Incubate in hybridization chamber;
c) A method comprising determining the presence or absence of a target analyte. A method of mixing solutions in an array format,
a)
i) a base plate including holes at least two of which are connected by channels;
ii) a membrane;
iii) a lid including at least one component port for immobilizing the array components;
iv) a sealant between the base plate and the lid;
Providing a hybridization chamber comprising:
b) applying a vacuum to the membrane, thereby forming a well in the membrane;
c) providing a solution to the membrane, thereby allowing the solution to penetrate into at least one well;
d) a method comprising intermittently applying a vacuum to the membrane, thereby mixing the solution. 28. The method of claim 27, wherein said solution is allowed to penetrate a plurality of wells.

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