JP3955952B2 - Analytical method of specimen by molecular array using micro infrared spectroscopy - Google Patents

Description

【００５４】
以上のことから、本検出システムにおいてタンパク質の立体構造についての情報を得ることができるかを確認することを目的として、立体構造の異なる（２次構造含有率の異なる）３種のタンパク質について金基板上での顕微ＦＴ−ＩＲ測定を行った。
（１）方法
金基板をピランハ溶液で５分間前処理を行い、水で洗浄後、スピンドライヤーで乾燥した。マイクロアレイヤ−を用いて測定サンプルを金基板にスポットした。測定サンプルとしては、ＢＳＡ（αへリックスのみ）、Ａｖｉｄｉｎｅ（βシートのみ）、RＮアーゼＳ−タンパク質（αへリックスとβシート）をそれぞれ１．５ｐｍｏｌ用いた。真空デシケーターで金基板を乾燥させた後、顕微ＦＴ−ＩＲによる測定を行った。測定条件は、分解能を２ｃｍ^−１、スキャンの回数を５００回、アパチャ−の大きさを１００μｍ×１００μｍとした。
（２）結果
測定結果を図９に示す。各蛋白質の二次構造含有率の違いをＩＲスペクトルパターンの違いとして検出できたことが分かる。
【００５５】
実施例６
［顕微ＦＴ−ＩＲによる蛋白質濃度変化の観察］
（１）方法
金基板をピランハ溶液で５分間前処理を行い、水で洗浄後、スピンドライヤーで乾燥した。金基板にマイクロアレイヤーを用いてＢＳＡを２．５、２．０、１．５、１．０、０．５、０（ｃｏｎｔｒｏｌ）ｐｍｏｌそれぞれスポットした。１サンプルあたり１０スポット）。真空デシケーターで金基板を乾燥させた後、顕微ＦＴ−ＩＲによる測定を行った。測定結果よりアミド結合の特性吸収である１７００−１５００ｃｍ^−１の吸収を抽出し、イメージ画像（ＣｈｅｍｉＭａｐ）を構築した。
（２）結果
測定結果を図１０に示す。図１０は１７００−１５００ｃｍ^−１のケミマップであり、（ａ）は2次元のケミマップ、（ｂ）は３次元のケミマップを表している。サンプル量の減少に伴う吸光度の減少が観察された。１０スポットの平均吸光度及び標準偏差を算出し、サンプル量に対してプロットした結果を図１１に示す。また、各サンプル量の平均吸光度及び標準偏差を表２に示す。各スポット間の吸光度のばらつきも少なく、サンプル量に対して線形的な相関があり、定量的な分析ができることが分かった。
【００５６】
【表２】

【００５７】
実施例７
［金基板上のＤＮＡ検出］
（１）方法
まず、前処理として金基板をピランハ溶液に５分間浸し、水で洗浄後、遠心乾燥を行った。次に、ＤＮＡマイクロアレイヤーを用いて、金基板上に３９．７ｐｍｏｌ（ｂａｓｅ当たり）の仔牛胸腺ＤＮＡを４つスポットし、ＩＲイメージング測定を行った。測定条件は、ピクセルの数を１２０×１１４、ピクセルサイズを６．２５μｍ×６．２５μｍ、測定範囲を２０００〜８００ｃｍ^−１、分解能を８ｃｍ^−１、スキャンの回数を４とした。
（２）結果
測定結果を図１２に示す。図１２（ａ）は、ＤＮＡのＩＲスペクトルを表し、図１２（ｂ）は、１１０５−１０７５ｃｍ^−１のケミマップ（False Color Image）を表し、図１２（ｃ）は、１１０５−１０７５ｃｍ^−１のケミマップ（３D surface Projection）を表している。顕微ＦＴ−ＩＲ法による金基板上のＤＮＡの検出が可能であることが分かった。また、４本のピークの高さは同程度であった。
【００５８】
実施例８
［顕微ＩＲ法によるＤＮＡ濃度変化の観察］
（１）方法
まず、前処理として金基板をピランハ溶液に５分間浸し、水で洗浄後遠心処理を行った。次に、ＤＮＡのマイクロアレイヤーを用いて、金基板上に１９．８５、９．９３、４．９６、２．４８、１．２４ｐｍｏｌ（ｂａｓｅ当たり）の仔牛胸腺ＤＮＡを４つスポットし、ＩＲイメージング測定を行った。測定条件は、ピクセルの数を５２６×５１２、ピクセルサイズを６．２５μｍ×６．２５μｍ、測定範囲を２０００〜８００ｃｍ^−１、分解能を３２ｃｍ^−１，スキャンの数を４回とした。
（２）結果
測定結果を図１３示す。図１３（ａ）は、１１０５−１０７５ｃｍ^−１のケミマップ（False Color Image）を表し、図１３（ｂ）は、１１０５−１０７５ｃｍ^−１のケミマップ（３D surface Projection）を表している。また図中、ＡからＥの符号はサンプルの濃度を表し、Ａが１９．８５ｍM、Ｂが９．９３ｍM、Ｃが４．９６ｍM、Ｄが２．４８ｍM、Ｅが１．２４ｍMである。この測定結果より、１スポット当たりの検出限界は絶対量で約２．５ｐｍｏｌ（リン酸ジエステル基当たり）であることが分かった。
また、図１３のデータから書くスポットの平均吸光度・標準偏差を算出し、サンプル量に対してプロットを行った。結果を表３及び図１４に示す。この結果より、ＤＮＡの量と吸光度との間に線形的な相関があることが分かった。
【００５９】
【表３】

【００６０】
実施例９
［１本鎖・２本鎖ＤＮＡの検出］
（１）方法
まず、前処理として金基板をピランハ用絵師に５分間浸し、水で洗浄後、遠心乾燥を行った。次に、ＤＮＡマイクロアレイヤーを用いて、金基板上に０．４ｍＭの１本鎖ＤＮＡ（２０ｍｅｒのプローブ）及び２本鎖ＤＮＡ（２０ｍｅｒのプローブと５０ｍｅｒのターゲットをハイブリダイズさせたもの）をスポットし、ＩＲイメージング測定を行った。測定条件は、ピクセルの数を１１３×１０６、ピクセルサイズを２５μｍ×２５μｍ、測定範囲を２０００〜８００ｃｍ^−１、分解能を３２ｃｍ^−１、スキャンの回数を１６回とした。
（２）結果
結果を図１５に示す。図１５（ａ）は、１１０５−１０７５ｃｍ^−１のケミマップ（False Color Image）を表し、図１５（ｂ）は、１１０５−１０７５ｃｍ^−１のケミマップ（３D surface Projection）を表している。また、図中Ａは、０．４ｍM（2本鎖ＤＮＡあたり）の2本鎖ＤＮＡ（２０ｍｅｒ−５０ｍｅｒ）を表し、Ｂは０．４ｍM（1本鎖ＤＮＡあたり）の1本鎖ＤＮＡを表している。これにより、１本鎖及び２本鎖のＤＮＡの識別が可能であることが分かった。
【００６１】
実施例１０
［金基板へのＤＮＡの固定化］
（１）方法
まず、前処理として金基板をピランハ溶液に５分間浸し、水で洗浄後、遠心乾燥を行った。次に、ＤＮＡマイクロアレイヤーを用いて、金基板上に０．４ｍMの１本鎖ＤＮＡ（２０ｍｅｒのプローブ）及び２本鎖ＤＮＡ（２０ｍｅｒのプローブと５０ｍｅｒのターゲットをハイブリダイズさせたもの）をスポットした。これを高温の密閉容器に入れ、３７℃で約２時間放置した。基板を取り出し、ＳＳＣで２回洗浄した後、ＩＲイメージング測定を行った。測定条件は、ピクセルの数を１２０×１１１、ピクセルサイズを２５μｍ×２５μｍ、測定範囲を２０００〜８００ｃｍ^−１、分解能を３２ｃｍ^−１、スキャンの回数を１６回とした。
（２）結果
結果を図１６に示す。図１６（ａ）は、１１０５−１０７５ｃｍ^−１のケミマップ（False Color Image）を表し、図１６（ｂ）は、１１０５−１０７５ｃｍ^−１のケミマップ（３D surface Projection）を表している。また、図中Ａは、０．４ｍM（2本鎖ＤＮＡあたり）の2本鎖ＤＮＡ（２０ｍｅｒ−５０ｍｅｒ）を表し、Ｂは０．４ｍM（1本鎖ＤＮＡあたり）の1本鎖ＤＮＡを表している。この測定結果より、固定化・洗浄後の２本鎖ＤＮＡの検出が可能であることが分かった。１本鎖ＤＮＡについては、２スポットの内一方は検出でき、もう一方は検出できなかった。
【００６２】
実施例１１
［アミド結合数の異なるタンパク質の顕微ＦＴ−ＩＲによる観察］
（１）方法
まず、前処理として金基板をピランハ溶液で処理した（５分間、２回）。次いで、マイクロアレイヤーを用いて、測定サンプルを基板上にスポットした（Cartesian Technologies社、PixSys PS5000 (Pin 方式)。測定サンプルとしては、ＲＮアーゼＳタンパク質、ＢＳＡ、Ａｖｉｄｉｎｅのそれぞれ１．５ｐｍｏｌを用いた。これらのサンプルの分子内タンパク質アミド結合の数は、順に１０３、６０６、１２８であり、ＢＳＡが最も多くＲＮアーゼＳタンパク質とＡｖｉｄｉｎｅが同程度である。そして、基板を真空乾燥し、顕微ＦＴ−ＩＲ測定を行った。測定条件は、ピクセルサイズを２５μｍ×２５μｍ、分解能を１６ｃｍ^−１、スキャンの回数を１６回、測定モードを反射イメージングモードとした。そして、アミド結合に由来する１７００〜１５００ｃｍ^−１のデータを抽出し、イメージング画像として処理した。
（２）結果
測定結果より得られたイメージング画像を図１７に示す。図１７（ａ）は２次元イメージング画像を表し、図１７（ｂ）は、３次元イメージング画像を表している。また、両図中ＡはＲＮアーゼＳタンパク質を、ＢはＢＳＡを、CはＡｖｉｄｉｎｅを表している。分子内アミド結合の数の多いＢＳＡがその数の少ないＲＮアーゼＳタンパク質やＡｖｉｄｉｎｅより吸収が大きくなっている。これによって、アミド結合数の差を赤外吸収強度に差として捉えることができたことが分かる。
【００６３】
実施例１２
［ＦＴ−ＩＲＲＡＳ測定によるＣＥＡ−ＡｎｔｉＣＥＡ Ａｎｔｉｂｏｄｙ相互作用の検出］
（１）方法
金基板をピランハ溶液で処理した（５分間、２回）。この基板をシャーレに置き、１ｍM dithiodipropionic acidを添加し、室温で溶液を２０時間攪拌した。次いで、これをエタノール中で５分間超音波照射した。この基板を蒸留水で洗浄後、１００ｍｇ／ｍｌ ＥＤＣａｑ．と１００ｍｇ／ｍｌ ＮＨＳａｑ．を等量混合し、室温で１０時間攪拌した。次に、１ｍM ＨＥＰＥＳ buffer （ｐH８．０）で洗浄後、０．１ｍｇ／ｍｌの抗ＣＥＡ抗体１ｍM ＨＥＰＥＳ buffer 溶液を基板にのせて、室温で３時間反応させた。次いで、基板を蒸留水中で１時間洗浄後、１ｍMの３−Aminopropanol を加え、室温で３０分攪拌した。次いで、基板を蒸留水で洗浄後、真空デシケーターで１時間乾燥させ、ＦＴ−ＩＲ測定を行った。次いで、１ｍｇ／ｍｌのＣＥＡ ＰＢＳ ｂｕｆｆｅｒ（ｐＨ７．４）溶液を基板に添加し、室温で３時間静置した。次いで、基板を純水中で１０分間洗浄後、真空デシケーターで１時間乾燥させ、ＦＴ−ＩＲＲＡＳ測定を行った。
（２）結果
測定結果を図１８に示す。両スペクトルの１６００ｃｍ^−１付近にある鋭いピークはアミド基由来のものを表し、ＣＥＡのスペクトルの３３００ｃｍ^−１付近にあるピークは糖鎖由来のものを表している。測定の結果、ＣＥＡを添加・洗浄後のスペクトルにおいてアミド結合の吸収強度が若干上昇することが分かった。このように、ＣＥＡをノンラベル、二次抗体の使用なしに検出することに成功した。また、この方法ではラベル化が必要でないため、ＣＥＡの情報について誤って検出するという問題がないという利点を有する。
【００６４】
実施例１３
［金基板への抗ＣＥＡ抗体の固定化］
（１）方法
金基板をピランハ処理した（５分間、２回）。ついで、この基板に１０ｍM 3,3’-dithiodipropionic acid dihydroxysuccimide ester/DMSO溶液を滴下し、２４時間静置した。その後、基板をＤＭＳＯ中で超音波処理し、水洗し、スピンドライヤーで乾燥し、真空デシケーターで乾燥した。次いで、０．１ｍｇ／ｍｌ抗ＣＥＡ抗体１０ｍM HEPES ｂｕｆｆｅｒ（ｐＨ８．０）溶液を基板に滴下し、２４時間静置した。そして、基板を水洗し、スピンドライヤーで乾燥し、真空デシケーターで乾燥した。そして、基板をＦＴ−ＩＲＲＡＳ測定した。
（２）結果
測定結果を図１９に示す。抗ＣＥＡ抗体のアミド結合に由来する吸収スペクトルを検出することができた。
【００６５】
【発明の効果】
以上に詳細に説明したように、本発明による顕微フーリエ変換赤外分光法を利用して分子アレイにより検体を分析する方法は、次のような種々の優れた利点を有する。即ち、１）検出感度が高いので、極めて微量である多数の検体を一度に分析できる、２）原理的にラベル化操作を必要とせず、もしラベル化を行うなら、さらに高感度化が期待できる、３）ラベル化操作を必要としないため、全体の測定手続きが大幅に簡素化される、４）検体はラベル化による化学修飾を受けないため、検体分子が本来有する特性・機能を誤りなく評価できる、５）検体の分子種（核酸、タンパク質、糖質、脂質、人工合成分子等）を問わず全く同一の観測手段、即ち、顕微フーリエ変換赤外分光法によってあらゆる分子アレイを観測できる、６）アレイの基板には金属及びガラス、セラミックス、フッ化カルシウム等の多様な素材が利用可能であり、プローブ分子を基板に固定する手法について極めて多様な選択が可能である、７）検体分子についての定量性も優れている。
【図面の簡単な説明】
【図１】 図１は、本発明の検体の分析方法に係り、リンカーを介して生体物質を基板上に固定化する方法の一例を示す図である。
【図２】 図２は、本発明の検体の分析方法に係り、リンカーを介して生体物質を基板上に固定化し、それと共にマスキング剤を基板上に固定化する方法の一例を示す図である。
【図３】 図３は、本発明の検体の分析方法に係り、リンカーを介して生体物質を基板上に固定化し、それと共にマスキング剤を基板上に固定化する方法の図２と同様の一例を示す図である。
【図４】 図４は、本発明の検体の分析方法に係り、リンカーを介して生体物質を基板上に固定化し、それと共にマスキング剤を基板上に固定化する方法のもう一つの例を示す図である。
【図５】 図５は、DNAマイクロアレイの顕微フーリエ変換赤外分区測定装置により、2本鎖DNAである仔牛胸腺DNAのリン酸エステル基の1086 cm^-1の赤外吸収の強度を二次元に画像化したものである。
【図６】 図６は、顕微フーリエ変換赤外分光法により、DNAマイクロアレイの基板上の1600 個の仔牛胸腺DNA（30 pmol）のスポットが画像化できたことを示すものである。
【図７】 図７は、金蒸着ガラスプレートを用いたDNAチップを用いて顕微フーリエ変換赤外分光法で画像化することによりSNP解析が可能であることを示すものである。
【図８】 図８は、ペプチドチップを顕微フーリエ変換赤外分光測定装置により画像化したものである。
【図９】 図９は、２次構造含有率の異なるタンパク質の顕微ＩＲ測定により、各蛋白質の二次構造含有率の違いをＩＲスペクトルパターンの違いとして検出できたことを示すものである。
【図１０】 図１０は、ＢＳＡを２．５、２．０、１．５、１．０、０．５、０（ｃｏｎｔｒｏｌ）ｐｍｏｌそれぞれスポットし、顕微ＦＴ−ＩＲによる測定を行った時の、アミド結合の特性吸収である１７００−１５００ｃｍ^−１のケミマップを示す。
【図１１】 図１１は、ＢＳＡサンプルの１０スポットの平均吸光度をサンプル量に対してプロットした結果を示す。
【図１２】 図１２は、顕微IR法による金基板上でのＤＮＡ検出の状態を示す。
【図１３】 図１３は、顕微IR法によるＤＮＡの濃度変化の状態を示す。
【図１４】 図１４は、サンプルのＤＮA濃度と平均吸光度との関係を示す。
【図１５】 図１５は、１本鎖ＤＮＡと２本鎖ＤＮＡの顕微ＦＴ−ＩＲによる測定を行った時の、リン酸エステルの特性吸収である１１０５−１０７５ｃｍ^−１のケミマップを示す。
【図１６】 図１６は、固定化ＤＮＡの検出の状態を示す１１０５−１０７５ｃｍ^−１のケミマップである。
【図１７】 図１７は、アミド結合数の異なるタンパク質を顕微ＦＴ−ＩＲにより測定した時の、アミド結合の特性吸収である１７００−１５００ｃｍ^−１のケミマップを示す。
【図１８】 図１８は、ＦＴ−ＩＲＲＡＳ測定による抗ＣＥＡ抗体とＣＥＡ添加後の抗ＣＥＡ抗体のスペクトルを示す。
【図１９】 図１９は、抗ＣＥＡ抗体のＦＴ−ＩＲＲＡＳ測定によるスペクトルを示す。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a molecular array analysis method by spectroscopy. More specifically, a specimen is allowed to act on a molecular array in which probes are fixedly arranged on a substrate, and probe microspots are formed.Microscopic Fourier transform infraredSpectroscopy scans for molecules in the specimen that specifically bind to the probeinfraredThe present invention relates to a method of analyzing a specimen by detecting by absorption measurement. The analysis method of the present invention is basically applicable to the analysis of all molecules and compounds, but is particularly useful in the analysis of biomolecules and related molecules that require high sensitivity, high speed, and large amount of sample processing.
[0002]
[Prior art]
The current state of genetic research has begun the post-genomic era with the draft decoding of the entire human genome sequence completed. Future research will focus on functional genomics related to gene expression, mutation, single nucleotide polymorphism (SNP), and proteomics related to protein structure and function. Gene expression is complexly influenced by factors such as cell cycle, tissue type, species and group type, and interactions with other proteins and genes. Therefore, the combination of these elements is an astronomical number in conducting research. For this reason, in future research or clinical examination, a high-throughput tool that can process and detect a large number of genes or proteins at once in a short time is strongly demanded. Under such circumstances, molecular array technology (array technology) is expected as one of the most effective tools.
[0003]
In array technology, first, an array is prepared in which probe molecules such as known nucleic acids and proteins are modified in a spot shape at a high density as many points on a substrate. Next, the sample is reacted with nucleic acid, protein, etc. as a sample, spots that specifically interact with the nucleic acid or protein are detected on the array, and the type and amount of substances in the sample are determined from the intensity of the detection signal of each spot. Get information about. In addition, the same information as described above can be obtained by arranging the specimens on the substrate in the same manner and operating the probe molecules.
[0004]
Many problems remain to be solved in current array technology. Of particular concern is the method of detecting nucleic acids and proteins bound to individual spots on the array. Until now, it has been generally performed to label a sample substance (biomolecule) that acts on an array with a fluorescent dye or an electrochemically active compound. For example, in the case of a fluorescent dye, after the sample substance is allowed to act, the presence and amount of the chemical species bound to the probe are estimated based on the presence and intensity of fluorescence indicated by the spots on the array. However, it takes a lot of time to label the analyte molecule with a fluorescent dye, and it is generally difficult to control the location and number of label molecules bound to a huge molecule such as a nucleic acid or protein molecule. In particular, in the case of a protein, when a fluorescent label is introduced into the molecular recognition site or reaction active site of the protein, the function / property that the original protein should originally show changes and information about the original protein may not be obtained. There is also sex. Therefore, a detection method that does not require labeling is desired.
[0005]
Until now, as a method for detecting a biological substance that does not use chemical modification such as a fluorescent label, there are a method using a crystal oscillator (for example, Non-Patent Document 1) and a method using surface plasmon resonance (for example, Non-Patent Document 2). is there. These are detection methods based on a change in frequency due to a change in mass of the surface due to a biological substance bound to a nucleic acid or protein immobilized on a crystal oscillator or a gold thin film, or a change in refractive index due to plasmons. However, these detection limits are several ng, and the detection sensitivity is not always sufficient.
[0006]
[Non-Patent Document 1]
Y.Okahata, Y. Matsunobu, K. Ijiro, M. Mukai, A. Murakami, K. Makino, J. Am. Chem. Soc., 114, 8299-8300 (1992)
[Non-Patent Document 2]
Kazuhiro Nagata, Hiroshi Handa "Real-time analysis experiment of biological material interaction" Springer Fairlark Tokyo Co., Ltd. (1998)
[0007]
[Problems to be solved by the invention]
Accordingly, it is an object of the present invention to provide a method for analyzing a sample that can be detected with high sensitivity using an unlabeled molecular array without particularly chemically modifying the molecules of the sample.
[0008]
[Means for Solving the Problems]
As a result of intensive research aimed at finding a highly sensitive detection method on a molecular array without labeling, the inventors reacted with a probe on the molecular array. Specimen moleculesMicroscopic Fourier transform infraredBy spectroscopyinfraredThe inventors have found that the above object can be achieved by measuring and detecting absorption, and have completed the present invention.
[0009]
It has been well known that information about chemical functional groups of a measurement target substance can be obtained by measurement using spectroscopy. However, with conventional spectroscopy, only one substance to be measured can be measured at a time. Therefore, if the spectroscopic method can be arrayed, there is an effect that a large number of substances to be measured can be measured simultaneously.
On the other hand, the method of arraying spectroscopic methods has an advantage not found in conventional microarray methods. That is, the conventional microarray method can only obtain information via the label used, and as a result, there is a problem that it is easy to obtain erroneous information about the measurement target substance. On the other hand, when the spectroscopic method is arrayed, it is not necessary to label the measurement target substance, so that information on the measurement target substance itself can be obtained. Information on structural changes and quantitative changes can be obtained using functional groups as indices. Such an application becomes possible only by arraying the spectroscopy.
[0010]
As described above, applying spectroscopy to a molecular array has advantages not found in the past. However, simply combining the spectroscopic method and the molecular array has caused the specimen detection sensitivity to be too low to measure the specimen.
The present inventor has made it possible to detect a specimen with high sensitivity by selecting a linker when immobilizing a biological substance on a substrate and examining the orientation of the immobilized substance from the substrate by an immobilization method. Here, the immobilized orientation of the biological material was achieved by selection of a masking agent. Since the masking agent reduces non-specific adsorption, the background can be lowered and the detection sensitivity of the specimen can be increased.
[0011]
That is, the first invention of the present invention isMicroscopic Fourier transform infraredThis is a method for analyzing a sample by a molecular array using spectroscopy, in which a sample is allowed to act on a molecular array in which probes are fixedly arranged on a substrate, and probe microspots are formed.Microscopic Fourier transform infraredSpectroscopy scans for molecules in the specimen that specifically bind to the probeinfraredA method for analyzing a sample, wherein the detection is performed by measuring absorption.
[0012]
The second invention of the present invention is:Microscopic Fourier transform infraredThis is a method of analyzing a sample by a molecular array using spectroscopy, in which a probe is applied to a molecular array in which a sample is fixedly arranged on a substrate, and a minute spot of the sample is formed.Microscopic Fourier transform infraredSpectroscopy scans for molecules in the specimen that specifically bind to the probeinfraredA method for analyzing a sample, wherein the detection is performed by measuring absorption.
[0013]
The third invention of the present invention is:Microscopic Fourier transform infraredThis is a method for analyzing a sample by a molecular array using spectroscopy, in which a sample and a probe are allowed to interact and then immobilized on a substrate to analyze a minute spot of the sample.Microscopic Fourier transform infraredMolecules in the sample that have been scanned by light and specifically bound to the probeinfraredA method for analyzing a sample, wherein the detection is performed by measuring absorption.
[0014]
According to a fourth aspect of the present invention, there is provided the sample analysis method according to any one of claims 1 to 3, wherein the probe or the sample is immobilized on a substrate via a linker.
[0015]
According to a fifth aspect of the present invention, there is provided the sample analysis method according to any one of claims 1 to 4, wherein a masking agent is immobilized on a substrate.
[0016]
The present inventionMicroscopic Fourier transform in infraredLight used for spectroscopyIs infrared light.
[0017]
AndThe present inventionInMicroscopic Fourier transform infrared spectroscopy as spectroscopyIt is the feature to use.
[0018]
First of the present invention6The invention ofMicroscopic Fourier transform infraredObtained by scanning the entire substrate of a molecular array with spectroscopyinfraredThe absorption measurement result is imaged and displayed, and a molecule specifically bound to the probe is detected.5The method for analyzing a specimen according to any one of the above.
[0019]
First of the present invention7According to the invention, the molecular array is selected from a nucleic acid chip, a nucleic acid microarray, a protein chip, a sugar chip, a cell chip, a peptide chip, an antibody chip, an enzyme activity detection chip, an environmental hormone chip, a gene expression chip, and a drug discovery chip The method for analyzing a specimen according to any one of claims 1 to 8, wherein the method is one.
[0020]
The present inventionIsOn a molecular array substrateInfraredMeasuring the reflected lightOf the specimenIt is an analysis method.
[0021]
First of the present invention8The invention has a wavelength specific to the molecules in the specimen.infraredThe absorption is measured to detect a molecule specifically bound to the probe.7The method for analyzing a specimen according to any one of the above.
[0022]
First of the present invention9The invention of the present invention has a wavelength specific to a functional group of a molecule in a specimen or a functional group introduced as a label on the molecule.infraredThe absorption is measured to detect a molecule specifically bound to the probe.8The method for analyzing a specimen according to any one of the above.
[0023]
First of the present invention10The invention has a wavelength specific to a phosphate ester group or a carbonyl group derived from a nucleic acid, an amide group or a carbonyl group derived from a peptide bond, or a hydroxyl group, a cyano group, an isocyanate, or a thioisocyanate.infraredThe absorption is measured to detect nucleic acid, protein or peptide in the sample specifically bound to the probe.9The method for analyzing a specimen according to any one of the above.
[0024]
First of the present invention11The invention according to claim 1 is characterized in that a nucleic acid as a specimen and a DNA probe are allowed to act and analyzed by a dot blot method.10The method for analyzing a specimen according to any one of the above.
[0025]
DETAILED DESCRIPTION OF THE INVENTION
The present invention particularly emphasizes that a probe including a probe containing a nucleic acid such as unknown DNA or a biomolecule such as protein is allowed to act on a molecular array in which molecules such as nucleic acid or protein are fixedly arranged on the substrate as a probe. This is an applied technique for analyzing a sample by detecting biomolecules such as nucleic acids and proteins in the specifically bound sample by measuring light absorption by spectroscopy.
[0026]
Further, as another embodiment of the present invention, a probe such as a nucleic acid or protein is allowed to act on a molecular array in which a specimen containing a nucleic acid such as unknown DNA or a biomolecule such as protein is fixed on a substrate. It is also possible to analyze a specimen by detecting biomolecules such as nucleic acids and proteins in the specimen specifically bound to the probe by measuring light absorption by spectroscopic methods.
[0027]
Furthermore, as another embodiment of the present invention, the specimen can be detected by spectroscopic methods by fixing both the specimen and the probe to the substrate without allowing the specimen and the probe to interact with each other and then immobilizing the specimen and the probe on the board. According to this method, multistage interactions can be detected by the array. For example, the state of intermolecular interaction can be known by a technique in which a hormone and a hormone receptor are allowed to act and then both are bound to a DNA double helix on the substrate.
[0028]
The molecular array used in the analysis method of the present invention is a substrate in which biomolecules such as DNA and RNA such as nucleic acids and proteins are fixedly arranged as probes. Typical examples include nucleic acid chips, nucleic acid microarrays, and proteins. A chip etc. are mentioned. A DNA chip, which is one of nucleic acid chips, is synthesized by applying optical lithography technology for manufacturing semiconductor integrated circuits using a plurality of types of oligonucleotides of about 20 bases as probes on a silicon substrate. A DNA microarray, which is one of nucleic acid microarrays, is obtained by mechanically arranging and immobilizing DNA prepared in advance on a substrate such as a slide glass as a probe. DNA chips and DNA microarrays detect nucleic acid molecules such as DNA in a specimen by utilizing the specific binding of complementary DNA molecules to each other. Widely used for analysis of types (SNP). Similarly, RNA chips and RNA microarrays using RNA instead of DNA are also widely used. The protein chip is, for example, one in which various proteins are immobilized as probes on a substrate, and is used for analysis of proteins in a specimen by interaction between proteins. For example, a protein chip that searches for receptor proteins in which various peptides are immobilized on a substrate, a protein chip in which various peptides or proteins are immobilized as ligands, and the like can be mentioned.
The present invention is not limited to those exemplified above, and other various nucleic acid chips, nucleic acid microarrays, protein chips and the like can be used.
[0029]
Furthermore, in the present invention, various other arrays of biomolecules can be used. For example, a sugar chip used for immobilizing various sugars on a substrate and analyzing nucleic acids and proteins, a cell chip used for immobilizing various cells on a substrate and analyzing interactions with cells, and various DNA-binding peptides immobilized on the substrate Peptide chip for analyzing purified DNA, antibody chip for searching for antigenic proteins with various antibodies immobilized on the substrate, enzyme activity detection chip with various peptides having protease inhibitory activity immobilized on the substrate, environmental hormone substances An environmental hormone chip that monitors the change in gene function of the cell by reacting the gene extracted from the exposed cells with the chip gene, a gene expression chip that can simultaneously monitor thousands of genes, and various candidates Immobilize natural and artificial drugs on the substrate to detect drugs that interact specifically with the target protein. Such as drug discovery chips and the like. Furthermore, with respect to nucleic acid chips, a DNA probe method is used to examine the presence or abundance of specific genes in many types of specimens by immobilizing nucleic acids, which are many different specimens, on a substrate and interacting with DNA probes. Application to the dot blot method (Experimental Medicine Vol. 5, No. 11, 84-86, 1987) is also possible.
[0030]
These molecular arrays are obtained by immobilizing nucleic acids, peptides, proteins, etc. on silicon substrates, slide glasses, etc. In addition to these, other various substrates can be used as substrates for the molecular array of the present invention. Can do. For example, a single crystal plate of calcium fluoride can be used. Nucleic acids and the like can be immobilized by surface treatment such as treatment of the plate with an aminosilane-based polymer or coating with polylysine. It is also possible to immobilize proteins or nucleic acids on a glass substrate coated with gold by utilizing a bond between a thiol group and gold. Furthermore, it is also possible to use a substrate obtained by extending a functional group from a surface modified with artificial diamond or diamond-like carbon (DLC) on quartz glass. Moreover, ceramics etc. can also be used as a substrate.
[0031]
Of the present inventionMicroscopic Fourier transform infraredSince the specimen analysis method using the spectroscopic method has high specimen detection sensitivity, it is possible to analyze a very large number of specimens at once by using this analysis method.
[0032]
As described above, the specimen analysis method using the molecular array using the spectroscopic method of the present invention is characterized by high specimen detection sensitivity. This is a method of immobilizing a biological substance on the substrate of the molecular array. It was realized by improving. That is, 1) a linker for immobilizing a biological substance on the substrate was selected, and 2) the orientation of the immobilized substance from the substrate by the immobilization method was examined, thereby enabling highly sensitive detection of the specimen. Here, the immobilized orientation of the biological material was achieved by selection of a masking agent.
[0033]
In the analysis of the specimen using the spectroscopic method, the absorption intensity (signal) changes depending on the molecular orientation of the biological substance that is the specimen. Therefore, it is necessary to align the orientation of the biological substance so as to increase the signal. In the present invention, the linker plays a role of connecting the biological material and the substrate, and also has an effect of aligning the orientation of the biological material to be immobilized, so that the detection sensitivity of the specimen can be improved by using the linker.
An example of immobilization of a biological material using a linker is shown in FIG. In this example, an anti-CEA antibody is used as a biological material immobilized on the substrate. A self-assembled film is formed on the gold substrate with 3,3′-Dimercaptopropionic acid, and the terminal carboxylic acid is activated with WSCI and N-Hydroxysuccimide. Next, the antibody CEA antibody is immobilized on a gold substrate via a peptide bond. Other linkers for immobilizing the anti-CEA antibody include dithiodipropionic acid and dithiodipropionic acid N-hydroxysuccimide ester.
[0034]
In addition to this, in the present invention, the immobilization orientation of the biological material can be further improved by using a masking agent together with the linker. The masking agent has a function of preventing nonspecific adsorption of biomolecules to the substrate by filling the portion other than the biological material immobilized on the substrate via the linker, and also aligns the biomaterial. Has the effect of aligning. As the masking agent, for example, as an ethylene glycol type, HS (CH₂)₁₁(OCH₂CH₂)₅A substance represented by OH can be used.
[0035]
An example of immobilizing a biological material using a linker and a masking agent is shown in FIGS. In this example, HS (CH₂)₁₁(OCH₂CH₂)₅OCH₂Using a substance represented by COOH, HS (CH₂)₁₁(OCH₂CH₂)₅A substance represented by OH is used. After the linker and the masking agent are mixed and immobilized on the gold substrate, the protein binding portion of the linker is activated to immobilize the protein. The linker and the masking agent are immobilized on gold by Au—S bond at the thiol moiety. The linker and the masking agent form a monomolecular film (SAM) with the alkyl chain portion, and an ethylene glycol layer is formed thereon. Then, an environment in which there is a water layer in the ethylene glycol layer portion is created, and the protein is fixed to the linker so that the protein floats there.
[0036]
FIG. 4 shows another example of immobilizing a biological material using a linker and a masking agent. In this example, HS- (CH₂)₆The substance represented by -OPi (Pi represents phosphoric acid), a DNA double strand, and a peptide-DNA linking molecule are used. As a masking agent, mercaptohexanol (HS- (CH₂)₆-OH), but HS (CH₂)₁₁(OCH₂CH₂)₅A substance represented by OH may be used. In FIG. 4 (a), HS- (CH₂)₆A substance represented by -OPi and a DNA double strand connected to each other is immobilized on a gold substrate, and the peptide is connected to the DNA double strand via a peptide-DNA linking molecule. In this example, where the linker and the masking agent are immobilized on gold by Au-S bonds at the thiol moiety, the double-stranded DNA is firmly immobilized on the substrate, so that the peptide can stand vertically from the substrate. . Thereby, the orientation on the substrate of the specimen acting with this peptide is improved, and the specimen can be detected with high sensitivity. In FIG. 4B, HS- (CH₂)₆The substance represented by -OPi and DNA double strands are immobilized on a gold substrate, and the peptide is connected to the DNA double strand via a peptide-DNA linking molecule, as shown in Fig. 4 (c). The point that the thread type intercalator is bonded to the DNA double-stranded region is different from FIG. FIG. 4 (c) shows three examples of the sewing type intercalator. In these intercalators, the substituent group jumps out from the DNA double strand, so that the ethylene glycol part, polyol part and sugar chain part of the intercalator hydrate with water. Accordingly, in the example of FIG. 4B, the environment where the peptide is immobilized can be made an environment such as an aqueous solution. In the examples of FIGS. 4 (a) and (b), the adsorption of the peptide onto the substrate can be suppressed. However, in the example of FIG. 4 (b), the double strand of DNA is stabilized by the stitched intercalator and the binding of the peptide is prevented. Further, it has an effect of strengthening and suppressing the peeling of the peptide probe after the protein interaction.
[0037]
In the present invention, the biomolecule in the specimen specifically bound to the probe can be detected by measuring light absorption at a wavelength specific to the biomolecule. Further, by measuring light absorption at a wavelength specific to the functional group in the biomolecule, it is possible to detect the biomolecule in the specimen specifically bound to the probe. For example, a nucleic acid has a functional group that characteristically absorbs light of a specific wavelength such as a phosphate group or a carbonyl group derived from a nucleobase, and a protein has a specific wavelength such as an amide group or a carbonyl group derived from a peptide bond. It has a functional group that characteristically absorbs light. Therefore, after the sample is allowed to act on the molecular array, the probe's fine spot on the array is scanned by spectroscopy, and light absorption at a specific wavelength for a phosphate ester group or amide group is measured. Highly sensitive detection of a minute amount of nucleic acid, protein, etc. in a minute spot specifically bound is possible.
Further, in the present invention, in addition to the above-described functional groups, a sample that specifically binds to the probe is measured by measuring light absorption at a wavelength specific to a functional group such as a hydroxyl group, a cyano group, an isocyanate, or a thioisocyanate. It is also possible to detect nucleic acids, proteins, peptides, etc. therein.
[0038]
Specifically, a specimen sample containing unknown molecules is reacted to an array in which thousands or tens of thousands of probes are regularly arranged and fixed on a solid substrate as minute spots having a diameter of about 0.5 to 0.05 mm. Are scanned with a spectroscopic instrument. Scanning refers to irradiating a substrate with a very fine beam of light and carefully measuring the reflected or transmitted light over the entire surface of the substrate at each location. Upon reflection, molecules in the specimen that are bound to the probe spot on the substrate surface absorb light of a wavelength specific to the molecules. For example, since nucleic acids and proteins have functional groups such as phosphate ester groups and amide groups, they absorb light having a specific wavelength specific to these functional groups. For example, the phosphate group is 1086 cm^-1The amide group is 1500-1700 cm^-1Absorbs light. Therefore, if the presence / absence and intensity of light of these specific wavelengths are measured for each spot on the substrate, the presence / absence / amount of molecules such as nucleic acids and proteins in the specimen, the binding characteristics of unknown molecules, the chemical structure, etc. are known. be able to. Further, the location can be confirmed even if the presence or absence of the spot is unknown in advance. You may measure the light which permeate | transmits a board | substrate instead of reflection of the light on a board | substrate. In this case, as in the case of reflected light, chemical information such as the presence / absence, amount, and binding characteristics of molecules in the specimen can be obtained. For example, when the above-described calcium fluoride is used as the substrate of the molecular array, the transmitted light is measured. When the above-described glass substrate coated with gold, quartz glass or the like is used, the reflected light is measured.
[0039]
Further, in the present invention, not only the light absorption of the wavelength specific to the functional group of the molecule in the specimen is measured, but also the light absorption of the wavelength specific to the functional group of the molecule introduced as a label to the molecule is measured. Thus, the specimen can be analyzed by detecting a molecule specifically bound to the probe.
[0040]
In the present invention, a specimen containing a biological molecule such as nucleic acid or protein is allowed to act on these molecular arrays, and the biological molecule such as nucleic acid or protein in the specimen that is specifically bound to the probe,Microscopic Fourier transform infraredDetect by spectroscopy. As the light used for the spectroscopic method, infrared light is used because it has excellent sensitivity, can detect dyes other than in vivo dyes, and does not require labeling at all.Is appropriate.In addition, as a spectroscopic method used in the present invention, microscopic Fourier transform infrared spectroscopic method is used because it can measure an absorption spectrum in a minute region with high sensitivity and high speed.Is appropriate.
[0041]
Microscopic Fourier transform infrared spectroscopy is an analytical method called microinfrared spectroscopy, microscopic FT-IR method, or microscopic IR method. It is a method for measuring the absorption of infrared rays based on vibration of the molecular skeleton, and is present in the molecule. This is based on the phenomenon that a biomolecule or the like absorbs an infrared ray having a specific wavelength with a characteristic intensity depending on a functional group such as a carbonyl group or a phosphate group. Microscopic Fourier transform infrared spectroscopy can measure an infrared absorption spectrum in a minute region with high sensitivity and high speed. Microscopic Fourier transform infrared spectrometers are commercially available. In the present invention, these commercially available apparatuses can be used as they are.
[0042]
In the present invention, when measuring the light absorption of molecules in a specimen with a spectroscopic measurement device, it is preferable to display the measurement result as a normal image. Imaging is usually performed as follows. For example, after the sample is allowed to act on the molecular array, the entire substrate of the molecular array is scanned with a spectroscopic measurement device, and the light absorption at a specific wavelength of each probe micro spot on the substrate is measured. Images can be imaged by color-coding according to the intensity and displaying the color-coded results in a two-dimensional manner corresponding to the entire substrate. Further, the measured light absorption intensity can be made to correspond to the height of the mountain, and the result can be displayed in three dimensions in a form corresponding to the entire substrate. These imagings are usually achieved by computer processing the data obtained by the spectrometer. In this way, the measurement result is imaged and displayed over the entire substrate of the molecular array, whereby the molecules in the specimen can be analyzed very easily and with high sensitivity.
[0043]
According to the present invention, the difference in the secondary structure content in the protein can be detected as the difference in the spectrum pattern, so that the difference in the protein can be distinguished, the expression state of the protein is examined, the structure of the protein Change can be examined. This method can also be applied to diagnosis using proteins.
[0044]
According to the present invention, a difference in light absorption intensity occurs between a protein that interacts with a capture molecule immobilized on a substrate and a protein that does not interact with the captured molecule on the substrate. The protein can be observed directly.
According to the present invention, the presence or absence of sugar or phosphate can be detected by measuring the intensity of light absorption in the course of protein interaction. In addition, in the course of protein interaction, changes in the amount of sugar or phosphate can also be detected by measuring changes in the intensity of light absorption. It is also possible to examine whether a protein is phosphorylated by using absorption of phosphoric acid.
[0045]
Hereinafter, an example of the analysis method of the present invention will be described more specifically by taking as an example the case of using a DNA chip and a protein chip as a molecular array.
In the case of a DNA chip, various DNA probes immobilized on a substrate are hybridized with DNA or RNA in a specimen sample. Infrared absorption of nucleobase-derived carbonyl groups and phosphate ester groups using a microscopic Fourier transform infrared spectrometer to determine where DNA and RNA in the sample sample formed a double strand with the DNA probe on the substrate Measure and image. This makes it possible to know what genes are present in the specimen sample. The abundance can be known from the infrared absorption intensity. The DNA probe present first also absorbs infrared rays and can be detected by intensity change before and after hybridization. In addition, a DNA-binding peptide (an artificial peptide having characteristics similar to DNA; called Peptide Nucleic Acid, PNA. J. Wang et al., Anal. Chem., 69, 5200-5202 (1997)) is used as a DNA probe, If the infrared absorption of the phosphate ester group is measured, detection is possible without considering the background absorption of the probe.
In the case of a protein chip, various peptides immobilized on a substrate are allowed to interact with a protein from a biological sample as a specimen, and then the infrared absorption of a carbonyl group is used as a guideline with a microscopic Fourier transform infrared spectrometer. Detection is possible. It is also possible to examine whether a protein is phosphorylated by using infrared absorption of phosphoric acid.
[0046]
Furthermore, the analysis method of the present invention can be carried out by using other various molecular arrays. For example, a sugar chip used for analysis of nucleic acids and proteins by immobilizing various sugars on a substrate, a cell chip used for analyzing interactions with cells by immobilizing various cells on the substrate, and various DNA-binding peptides on the substrate A peptide chip for analyzing the immobilized DNA, an antibody chip for searching for an antigen protein in which various antibodies are immobilized on a substrate, an enzyme activity detection chip in which various peptides having protease inhibitory activity or the like or a synthetic drug are immobilized on the substrate, A gene expression chip capable of monitoring thousands of genes at the same time, a candidate for an environmental hormone chip that monitors the changes in the gene's function by reacting a gene extracted from a cell exposed to an environmental hormone substance with the gene of the chip, candidate Drugs that interact specifically with the target protein by immobilizing various drugs And search for drug discovery chips can be cited as examples. Search and analyze nucleic acids, peptides, proteins, etc. in specimens specifically bound to various probe molecules immobilized on the substrate of each chip, based on the infrared absorption of functional groups that they have And can be quantified.
[0047]
As described above, according to the analysis method of the present invention, it is possible to easily analyze a sample without the need to specifically label component substances and contained molecules of the sample with a fluorescent dye or the like. However, in order to further improve the detection sensitivity as necessary, it is also possible to use a technique in which a functional group having a large light absorption is introduced into a biomolecule in the specimen for detection. For example, by introducing a nitrile group or the like into the sample component, the component in the sample can be labeled with a functional group that does not overlap with the light absorption of normal molecules and is rare in the sample component. Such chemical modification is easier to react than chemical modification with fluorescent dyes and the like, and since the functional group itself is small, it has little influence on the original chemical function of the biomolecule. Therefore, by introducing such a functional group, it is possible to analyze the specimen with higher sensitivity.
[0048]
【Example】
EXAMPLES The present invention will be described in more detail with reference to examples below, but the present invention is not limited to these examples.
Example 1
[Detection of double-stranded DNA on calcium fluoride plate]
(1) Method
Calcium fluoride (CaF₂On the single crystal substrate of), 25 calf thymus DNA concentrations were spotted with a DNA arrayer. From the left, 3 μM, 30 μM, 0.3 mM, 3 mM, and 30 mM calf thymus DNA (all concentrations per base pair) were spotted in 20 nl units. The absolute amount spotted is 60 fmol, 0.6 pmol, 6 pmol, 60 pmol, 0.6 nmol. These DNAs were measured with a microscopic Fourier transform infrared spectrometer. The conditions are as follows.
Transmission imaging, resolution: 8 cm^-1, Measuring range: 4000 ~ 700 cm^-1, Aperture size: 100 μm × 100 μm, area: 5.8 mm × 5.8 mm, measurement time: about 30 minutes.
Infrared characteristic absorption of phosphate ester group 1086 cm^-1The chemimap at was measured.
(2) Results
The measurement results are shown in FIG. FIG. 5 shows a 1086 cm of phosphate group of calf thymus DNA, which is a double-stranded DNA, using a microscopic Fourier transform infrared spectrometer of a DNA microarray.^-1This is a two-dimensional image of the intensity of infrared absorption. The part in a circular ring is the part where double-stranded DNA is present. A-E's vertical circular rings are all double-stranded DNA of the same concentration. From FIG. 5, B could barely be detected, but all of C could be detected with a circular ring visible. From this, it was clarified that double-stranded DNA at several picomolar level can be detected by micro fourier transform infrared spectroscopy.
[0049]
Example 2
[Detection of double-stranded DNA on calcium fluoride plate]
(1) Method
CaF₂1600 calf thymus DNA solutions were spotted on a single crystal substrate using a DNA array. 30 mM calf thymus DNA (all concentrations per base pair) was spotted in 1 nl increments. The absolute amount spotted is 30 pmol. This was measured with a microscopic Fourier transform infrared spectrometer. The conditions are as follows.
Transmission imaging, resolution: 64 cm^-1, Measuring range: 2000-800 cm^-1, Pixel size: 25 μm × 25 μm, Area: 2 cm × 2 cm, Measurement time: About 60 minutes.
Infrared characteristic absorption of phosphate ester group 1086 cm^-1The chemimap at was measured.
(2) Results
The measurement results are shown in FIG. FIG. 6A shows an infrared absorption spectrum of a portion where the double-stranded DNA concentration is high when imaged. FIG. 6B is a two-dimensional image of the integrated value of the peak appearing in the wave number region based on the absorption of the phosphate group in the gray portion. A part of the image obtained by reflection imaging is shown in FIG. The conditions at this time are as follows.
Resolution: 16 cm^-1, Measuring range: 4000 ~ 700 cm^-1, Pixel size: 25 μm × 25 μm, Area: 1.3 cm × 1.3 cm, Measurement time: About 60 minutes.
FIG. 6 (b) shows 1600 calf thymus DNA (30 pmol) spots. (C) of FIG. 6 is an infrared absorption spectrum when a part of (b) is measured by reflection imaging. It is an infrared absorption spectrum of a portion with a plus mark on the lower left side of (b) (the upper right plus mark is subtracted as a background). FIG. 6D shows an image of a part of the array with the integrated value of infrared absorption based on the amide group in the gray part of FIG.
From the results in FIG. 6, it was revealed that a large number of DNA spots on the substrate of the DNA microarray can be imaged by microscopic Fourier transform infrared spectroscopy.
[0050]
Example 3
[SNP analysis on gold-deposited glass plate]
(1) Method
The piranha solution was dropped onto a gold-deposited glass plate, left for 3 minutes, and washed with distilled water. This was hybridized with an LPL arita WT probe introduced with a thiol group at the 5′-end and arita WT or arita MT, and then spotted on this substrate (2 × SSC buffer, DNA concentration was 0.5 in terms of double strand). 0.5 nmol with 1 nl mM). LPL is an abbreviation for lipoprotein lipase and triggers hyperlipidemia by arita mutation. Left in a humidity-controlled container overnight. This was washed with a 2 × SSC solution and then measured with a microscopic Fourier transform infrared spectrometer. The conditions are as follows.
Transmission imaging, resolution: 8 cm^-1, Measuring range: 2000-800 cm^-1, Pixel size: 25 μm × 25 μm, Area: 0.8 cm × 0.8 cm, Measurement time: About 20 minutes.
Infrared characteristic absorption of phosphate ester, 1086 cm^-1The chemimap was measured by reflection imaging.
The base sequences of various probes are as follows.
LPL arita WT probe: 5'-GGATAGCTTCTCC-3 '
arita WT: 3'-CCTATCGAAGAGG-5 '
arita MT: 3'-CCTATC-AAGAGG-5 '(G → deficient)
(2) Results
The measurement results are shown in FIG. In FIG. 7, the upper left and lower right of the left figure are images of the same wild-type (WT) DNA probe made to act on a wild-type (WT) gene using a microscopic Fourier transform infrared spectrometer. . The lower left and upper right of the figure on the left are the wild type (WT) DNA probes that have been mutated (MT). In the figure on the left, which shows the results of measuring the gene sample before washing, all the spots could be imaged by this technique (the presence of double-stranded DNA was confirmed). However, as shown in the figure on the right, which shows the result of measuring the gene sample after washing, the spots in the lower left and upper right of the left figure disappeared by washing, but the upper left and lower right spots in the left figure remain. It was. This result indicates that only the portion where the DNA probe and the target gene form a fully matched double strand can be detected by the analysis method of the present invention. In other words, gene mutations could be identified by the analysis method of the present invention.
[0051]
Example 4
[Detection of proteins that interact with specific peptides using peptide chips]
(1) Method
Calcium fluoride (CaF₂) Spotted 1 nl each of a 1 mM aqueous solution of S-protein (amino acid residue 104, amide bond number 103) and S-peptide (amino acid residue 20, amide bond number 19) using an arrayer on a single crystal plate . There is 1 pmol of S-peptide and S-protein per spot. This was imaged with a microscopic Fourier transform infrared spectrometer.
(2) Results
The measurement results are shown in FIG. FIG. 8 (a) is a two-dimensional image from 1500 to 1700 cm.^-1Infrared absorption intensity changes are imaged as color differences. (B) represents the difference in absorption intensity as a three-dimensional diagram. 1500 to 1700 cm^-1Since the infrared absorption of the protein corresponds to the amide bond of protein or peptide, it should be proportional to the amount of peptide or protein present there. FIG. 8 shows the intensity of the spot depending on the amount of amide bond as expected. As a result, it was found that when the protein interacts with a specific spot on the peptide chip, the amount of amide bond on the spot increases, and the change can be detected with a microscopic Fourier transform infrared spectroscopy system. In other words, the application of peptide chips to imaging could be proved. In addition, high sensitivity detection of protein at picomolar level was achieved by this method.
[0052]
Example 5
[Detect differences in protein structure]
A protein chip is a tool for analyzing a function of a protein. A protein chip that can follow the three-dimensional structural change in the functional expression of a protein is very useful from the viewpoint of functional proteomics research. Although this detection system is based on FT-IR measurement, it is possible to read three-dimensional structure information from the IR spectrum of a protein.
It has been found that infrared absorption derived from protein amide bonds gives a spectrum reflecting its three-dimensional structure, particularly secondary structures such as α-helix and β-sheet. This is because the hydrogen bond pattern of the main chain amide bond in the α helix or β sheet is different. Table 1 shows the secondary structure of the protein and the vibration frequencies of the amide I and amide II bands.
[0053]
[Table 1]

[0054]
From the above, for the purpose of confirming whether or not information about the three-dimensional structure of the protein can be obtained in this detection system, gold substrates for three types of proteins having different three-dimensional structures (different secondary structure contents) are used. The above microscopic FT-IR measurement was performed.
(1) Method
The gold substrate was pretreated with a piranha solution for 5 minutes, washed with water, and then dried with a spin dryer. A measurement sample was spotted on a gold substrate using a microarrayer. As measurement samples, 1.5 pmol each of BSA (α-helix only), Avidine (β-sheet only), and RNase S-protein (α-helix and β-sheet) were used. After the gold substrate was dried with a vacuum desiccator, measurement by microscopic FT-IR was performed. Measurement condition is 2cm resolution.^-1The number of scans was 500, and the aperture size was 100 μm × 100 μm.
(2) Results
The measurement results are shown in FIG. It can be seen that the difference in the secondary structure content of each protein could be detected as the difference in the IR spectrum pattern.
[0055]
Example 6
[Observation of protein concentration change by microscopic FT-IR]
(1) Method
The gold substrate was pretreated with a piranha solution for 5 minutes, washed with water, and then dried with a spin dryer. BSA was spotted at 2.5, 2.0, 1.5, 1.0, 0.5, and 0 (control) pmol using a microarrayer on a gold substrate. 10 spots per sample). After the gold substrate was dried with a vacuum desiccator, measurement by microscopic FT-IR was performed. 1700-1500cm which is characteristic absorption of amide bond from the measurement result^-1The absorption of γ was extracted and an image (ChemiMap) was constructed.
(2) Results
The measurement results are shown in FIG. 10 is 1700-1500cm^-1(A) represents a two-dimensional chemimap, and (b) represents a three-dimensional chemimap. A decrease in absorbance with decreasing sample volume was observed. FIG. 11 shows the results of calculating the average absorbance and standard deviation of 10 spots and plotting them against the sample amount. Table 2 shows the average absorbance and standard deviation of each sample amount. It was found that there was little variation in absorbance between the spots, and there was a linear correlation with the sample amount, and quantitative analysis was possible.
[0056]
[Table 2]

[0057]
Example 7
[Detection of DNA on gold substrate]
(1) Method
First, as a pretreatment, the gold substrate was immersed in a piranha solution for 5 minutes, washed with water, and then centrifugally dried. Next, 4 calf thymus DNAs of 39.7 pmol (per base) were spotted on a gold substrate using a DNA microarrayer, and IR imaging measurement was performed. The measurement conditions are 120 × 114 for the number of pixels, 6.25 μm × 6.25 μm for the pixel size, and 2000 to 800 cm for the measurement range.^-1, Resolution is 8cm^-1The number of scans was 4.
(2) Results
The measurement results are shown in FIG. Fig. 12 (a) shows the IR spectrum of DNA, and Fig. 12 (b) shows 1105-1075 cm.^-1Fig. 12 (c) shows 1105-1075 cm.^-1Represents a 3D surface projection. It was found that the DNA on the gold substrate could be detected by the microscopic FT-IR method. Moreover, the height of four peaks was comparable.
[0058]
Example 8
[Observation of DNA concentration change by microscopic IR method]
(1) Method
First, as a pretreatment, the gold substrate was immersed in a piranha solution for 5 minutes, washed with water, and then centrifuged. Next, using a DNA microarrayer, four calf thymus DNAs of 19.85, 9.93, 4.96, 2.48, and 1.24 pmol (per base) were spotted on a gold substrate, and IR imaging was performed. Measurements were made. The measurement conditions are: the number of pixels is 526 × 512, the pixel size is 6.25 μm × 6.25 μm, and the measurement range is 2000 to 800 cm.^-1, Resolution is 32cm^-1The number of scans was 4 times.
(2) Results
The measurement results are shown in FIG. FIG. 13A shows 1105-1075 cm.^-1Fig. 13 (b) shows 1105-1075 cm.^-1Represents a 3D surface projection. In the figure, the symbols A to E represent the concentration of the sample, and A is 19.85 mM, B is 9.93 mM, C is 4.96 mM, D is 2.48 mM, and E is 1.24 mM. From this measurement result, it was found that the detection limit per spot was about 2.5 pmol (per phosphodiester group) in absolute amount.
Further, the average absorbance / standard deviation of the spot written from the data of FIG. 13 was calculated and plotted against the sample amount. The results are shown in Table 3 and FIG. From this result, it was found that there was a linear correlation between the amount of DNA and the absorbance.
[0059]
[Table 3]

[0060]
Example 9
[Detection of single-stranded and double-stranded DNA]
(1) Method
First, as a pretreatment, the gold substrate was immersed in a Piranha painter for 5 minutes, washed with water, and then centrifuged. Next, using a DNA microarrayer, 0.4 mM single-stranded DNA (20-mer probe) and double-stranded DNA (20-mer probe and 50-mer target hybridized) are spotted on a gold substrate. IR imaging measurement was performed. The measurement conditions are 113 × 106 pixels, 25 μm × 25 μm pixel size, and 2000-800 cm measurement range.^-1, Resolution is 32cm^-1The number of scans was 16 times.
(2) Results
The results are shown in FIG. FIG. 15A is 1105-1075 cm.^-1Fig. 15 (b) shows 1105-1075 cm.^-1Represents a 3D surface projection. In the figure, A represents 0.4 mM (per double-stranded DNA) double-stranded DNA (20 mer-50 mer), and B represents 0.4 mM (per single-stranded DNA) single-stranded DNA. Yes. Thus, it was found that single-stranded and double-stranded DNA can be distinguished.
[0061]
Example 10
[Immobilization of DNA on a gold substrate]
(1) Method
First, as a pretreatment, the gold substrate was immersed in a piranha solution for 5 minutes, washed with water, and then centrifugally dried. Next, using a DNA microarrayer, 0.4 mM single-stranded DNA (20-mer probe) and double-stranded DNA (20-mer probe and 50-mer target hybridized) were spotted on a gold substrate. . This was put into a high temperature sealed container and left at 37 ° C. for about 2 hours. The substrate was taken out and washed twice with SSC, and then IR imaging measurement was performed. The measurement conditions are 120 × 111 for the number of pixels, 25 μm × 25 μm for the pixel size, and 2000 to 800 cm for the measurement range.^-1, Resolution is 32cm^-1The number of scans was 16 times.
(2) Results
The results are shown in FIG. FIG. 16A shows 1105-1075 cm.^-1Represents a chemimap (False Color Image), and FIG. 16B is 1105-1075 cm.^-1Represents a 3D surface projection. In the figure, A represents 0.4 mM (per double-stranded DNA) double-stranded DNA (20 mer-50 mer), and B represents 0.4 mM (per single-stranded DNA) single-stranded DNA. Yes. From this measurement result, it was found that double-stranded DNA after immobilization and washing can be detected. For single-stranded DNA, one of the two spots could be detected and the other could not be detected.
[0062]
Example 11
[Observation of proteins with different numbers of amide bonds by microscopic FT-IR]
(1) Method
First, as a pretreatment, the gold substrate was treated with a Piranha solution (twice for 5 minutes). Next, a measurement sample was spotted on a substrate using a microarrayer (Cartesian Technologies, PixSys PS5000 (Pin method). As the measurement sample, 1.5 pmol of RNase S protein, BSA, and Avidine were used. The number of intramolecular protein amide bonds in these samples is 103, 606, 128 in order, BSA is the most, RNase S protein and Avidine are comparable, and the substrate is vacuum dried and microscopic FT-IR The measurement conditions were as follows: pixel size 25 μm × 25 μm, resolution 16 cm^-1The number of scans was 16, and the measurement mode was the reflection imaging mode. And 1700-1500cm derived from amide bond^-1Were extracted and processed as imaging images.
(2) Results
An imaging image obtained from the measurement result is shown in FIG. FIG. 17A shows a two-dimensional imaging image, and FIG. 17B shows a three-dimensional imaging image. In both figures, A represents RNase S protein, B represents BSA, and C represents Avidine. BSA with a large number of intramolecular amide bonds is more absorbed than RNase S protein and Avidine with a small number of them. Thus, it can be seen that the difference in the number of amide bonds could be regarded as a difference in the infrared absorption intensity.
[0063]
Example 12
[Detection of CEA-AntiCEA Antibody Interaction by FT-IRRAS Measurement]
(1) Method
The gold substrate was treated with a piranha solution (5 minutes, 2 times). This substrate was placed in a petri dish, 1 mM dithiodipropionic acid was added, and the solution was stirred at room temperature for 20 hours. This was then sonicated in ethanol for 5 minutes. After washing this substrate with distilled water, 100 mg / ml EDCaq. And 100 mg / ml NHSaq. Were mixed in equal amounts and stirred at room temperature for 10 hours. Next, after washing with 1 mM HEPES buffer (pH 8.0), 0.1 mg / ml anti-CEA antibody 1 mM HEPES buffer solution was placed on the substrate and reacted at room temperature for 3 hours. Subsequently, after wash | cleaning a board | substrate in distilled water for 1 hour, 1 mM 3-Aminopropanol was added and it stirred for 30 minutes at room temperature. Subsequently, after wash | cleaning a board | substrate with distilled water, it was dried with the vacuum desiccator for 1 hour, and the FT-IR measurement was performed. Next, a 1 mg / ml CEA PBS buffer (pH 7.4) solution was added to the substrate and allowed to stand at room temperature for 3 hours. Next, the substrate was washed in pure water for 10 minutes and then dried in a vacuum desiccator for 1 hour, and FT-IRRAS measurement was performed.
(2) Results
The measurement results are shown in FIG. 1600cm of both spectra^-1The sharp peak in the vicinity is derived from an amide group and is 3300 cm in the CEA spectrum.^-1The peak in the vicinity represents a sugar chain-derived peak. As a result of the measurement, it was found that the absorption intensity of the amide bond slightly increased in the spectrum after addition and washing of CEA. Thus, CEA was successfully detected without the use of a non-labeled secondary antibody. In addition, since this method does not require labeling, there is an advantage that there is no problem of erroneously detecting CEA information.
[0064]
Example 13
[Immobilization of anti-CEA antibody on gold substrate]
(1) Method
The gold substrate was subjected to piranha treatment (5 minutes, twice). Subsequently, 10 mM 3,3′-dithiodipropionic acid dihydroxysuccimide ester / DMSO solution was dropped onto the substrate and allowed to stand for 24 hours. Thereafter, the substrate was sonicated in DMSO, washed with water, dried with a spin dryer, and dried with a vacuum desiccator. Next, a 0.1 mg / ml anti-CEA antibody 10 mM HEPES buffer (pH 8.0) solution was dropped onto the substrate and allowed to stand for 24 hours. The substrate was washed with water, dried with a spin dryer, and dried with a vacuum desiccator. Then, the substrate was subjected to FT-IRRAS measurement.
(2) Results
The measurement results are shown in FIG. An absorption spectrum derived from the amide bond of the anti-CEA antibody could be detected.
[0065]
【The invention's effect】
As explained in detail above, according to the present inventionMicroscopic Fourier transform infraredThe method of analyzing a specimen by a molecular array using spectroscopy has various excellent advantages as follows. In other words, 1) Since the detection sensitivity is high, it is possible to analyze a very large number of samples at once. 2) In principle, no labeling operation is required, and if labeling is performed, higher sensitivity can be expected. 3) Since the labeling operation is not required, the entire measurement procedure is greatly simplified. 4) Since the sample is not chemically modified by labeling, the characteristics and functions inherent to the sample molecule can be evaluated without error. 5) Exactly the same observation means regardless of the molecular species of the specimen (nucleic acid, protein, carbohydrate, lipid, artificially synthesized molecule, etc.)That is,All molecular arrays can be observed by microscopic Fourier transform infrared spectroscopy.6) ArrayVarious materials such as metal, glass, ceramics, calcium fluoride, etc. can be used for the substrate, and there are a great variety of methods for immobilizing probe molecules on the substrate. 7) Quantitative analysis of analyte molecules Is also excellent.
[Brief description of the drawings]
FIG. 1 is a diagram showing an example of a method for immobilizing a biological material on a substrate via a linker, according to a sample analysis method of the present invention.
FIG. 2 is a diagram showing an example of a method for immobilizing a biological material on a substrate via a linker and immobilizing a masking agent on the substrate together with a linker according to the sample analysis method of the present invention. .
3 is an example similar to FIG. 2 of a method for immobilizing a biological material on a substrate via a linker and immobilizing a masking agent on the substrate together with a linker according to the sample analysis method of the present invention. FIG.
FIG. 4 shows another example of a method for immobilizing a biological material on a substrate via a linker and immobilizing a masking agent on the substrate together with a linker, according to the sample analysis method of the present invention. FIG.
FIG. 5 shows 1086 cm of the phosphate group of calf thymus DNA, which is a double-stranded DNA, by means of a microscopic Fourier transform infrared segmentation measuring device of a DNA microarray.^-1This is a two-dimensional image of the intensity of infrared absorption.
FIG. 6 shows that 1600 calf thymus DNA (30 pmol) spots on the substrate of the DNA microarray could be imaged by microscopic Fourier transform infrared spectroscopy.
FIG. 7 shows that SNP analysis is possible by imaging with microscopic Fourier transform infrared spectroscopy using a DNA chip using a gold-deposited glass plate.
FIG. 8 shows a peptide chip imaged with a microscopic Fourier transform infrared spectrometer.
FIG. 9 shows that a difference in secondary structure content of each protein was detected as a difference in IR spectrum pattern by microscopic IR measurement of proteins having different secondary structure contents.
FIG. 10 shows the results when BSA was spotted at 2.5, 2.0, 1.5, 1.0, 0.5, 0 (control) pmol and measured by microscopic FT-IR. The characteristic absorption of amide bond is 1700-1500cm^-1The chemimap of is shown.
FIG. 11 shows the result of plotting the average absorbance of 10 spots of a BSA sample against the sample amount.
FIG. 12 shows the state of DNA detection on a gold substrate by the microscopic IR method.
FIG. 13 shows the state of DNA concentration change by the microscopic IR method.
FIG. 14 shows the relationship between the DNA concentration of a sample and the average absorbance.
FIG. 15 shows the characteristic absorption of phosphate ester when measured by microscopic FT-IR of single-stranded DNA and double-stranded DNA, 1105-1075 cm.^-1The chemimap of is shown.
FIG. 16 shows the state of detection of immobilized DNA 1105-1075 cm^-1It is a chemimap.
FIG. 17 is a graph showing 1700-1500 cm, which is a characteristic absorption of an amide bond when proteins having different numbers of amide bonds are measured by microscopic FT-IR.^-1The chemimap of is shown.
FIG. 18 shows spectra of anti-CEA antibody and anti-CEA antibody after addition of CEA by FT-IRRAS measurement.
FIG. 19 shows a spectrum obtained by FT-IRRAS measurement of an anti-CEA antibody.

Claims (1)

This is a method for analyzing a sample by a molecular array using microscopic Fourier transform infrared spectroscopy. (1) A sample is allowed to act on a molecular array in which probes are fixedly arranged on a substrate, and microscopic Fourier transform infrared is applied to a probe microspot. Scan by spectroscopy, (2) Probes act on a molecular array in which a specimen is fixedly arranged on a substrate, and scan microscopic spots of the specimen by microscopic Fourier transform infrared spectroscopy, or (3) specimen and probe Is fixed on the substrate after the interaction, and by scanning the microscopic spot of the specimen by microscopic Fourier transform infrared spectroscopy, the molecules in the specimen specifically bound to the probe are detected by measuring the infrared absorption. in the analysis method of the specimen, the probe or analyte, as a linker _{(a) HS (CH 2)} 11 (OCH 2 CH 2) 5 CH ₂ COOH, _{or, (B) HS- (CH 2} ) 6 -OPi (Pi represents phosphate.) Which was connected to three-way material and DNA2 stranded and Peptide-DNA linking molecule represented by, A method for analyzing a specimen characterized by being immobilized on a substrate via a substrate .

Images