JP4217476B2 - Probe medium and probe fixing method on substrate - Google Patents

Description

【０１９６】
【外２】

化合物Ｉ
【０１９７】
（３）プローブ媒体の調整
上記実施例１と全く同様にしてプローブ媒体を調整した。
【０１９８】
（４）基板洗浄
上記実施例１と全く同様にして基板洗浄を行ない、ガラス基板を用意した。
【０１９９】
（５）プローブ媒体のスポッティング
上記実施例１と全く同様にしてプローブ固定基材を作製した。
【０２００】
（６）ブロッキング処理
上記実施例１と全く同様にしてブロッキング処理を全く行ない、１Ｍ ＮａＣｌ／５０ｍＭリン酸緩衝液（ｐＨ７．０）溶液で洗浄し、プローブ固定基材上を濡らし、均一なハイブリダイゼーション処理ができるようにした。
【０２０１】
（７）ハイブリダイゼーション処理
上記実施例１と全く同様にしてハイブリダイゼーション処理を行なった。その後、プローブアレイを１Ｍ ＮａＣｌ／５０ｍＭリン酸緩衝液（ｐＨ７．０）溶液で洗浄してプローブ核酸とハイブリダイズしなかった一本鎖ＤＮＡプローブを洗い流した。ついで、純水で余分な塩分を除去した後、窒素ブローによりプローブ固定基材を乾燥させた。次に該プローブ固定基材のスポットの蛍光を、蛍光スキャナ（商品名：ＧｅｎｅＰｉｘ４０００Ｂ；ＡｘｏｎＩｎｓｔｒｕｍｅｎｔｓ，Ｉｎｃ．製）を用いて蛍光強度を評価した。評価するにあたり、レーザーパワーを１００％に設定し、ＰＭＴを４００Ｖに設定した。
【０２０２】
（８）結果
上記（７）での蛍光スキャナ評価結果を解析したところ、標識化一本鎖ＤＮＡプローブと完全マッチである配列番号１のＤＮＡプローブのスポットでは、５３２ｎｍでの蛍光強度は２３４００であった。また、ＤＮＡプローブのスポット部以外の蛍光強度を観察したところ６５８あった。各ＤＮＡプローブのスポットを蛍光で観察した状態では、各々のスポット形状がほぼ円形であり、同じプローブ媒体をスポッティングしたスポット間においては蛍光強度の差異は殆ど認められなかった。ブロッキング処理を行なわないことによるスポット部以外での蛍光強度の増加は殆ど認められなかった。また、隣接するスポットとの間隔はほぼ均等であり、約２００μｍの間隔で格子状にスポットが配置されていることが観察された。
【０２０３】
（実施例１８）
（１）プローブの合成
実施例１７と全く同様にして配列番号５の一本鎖核酸を合成した。続いて通常の脱保護を行ない、ＤＮＡを回収し、高速液体クロマトグラフィーにて精製し、以下の実験に用いた。
【０２０４】
（２）プローブ固定物質
プローブを基材上に固定する物質の調整としてスクシイミドエステル基を有するシランカップリング剤（スクシイミジルトリエトキシシリルプロピオネート：化合物II）を含むシランカップリング剤を以下にスキームを示した方法により合成した。合成の確認は^１ＨＮＭＲ、^１３ＣＮＭＲによって行った。このシランカップリング剤を以下の実験に用いた。
【０２０５】
【外３】

【０２０６】
【外４】

化合物II
【０２０７】
（３）プローブ媒体の調整
上記実施例１７と全く同様にしてプローブ媒体を調整した。
【０２０８】
（４）基板洗浄
上記実施例１７と全く同様にして基板洗浄を行ない、ガラス基板を用意した。
【０２０９】
（５）プローブ媒体のスポッティング
上記実施例１７と全く同様にしてプローブ固定基材を作製した。
【０２１０】
（６）ブロッキング処理
上記実施例１７と全く同様にしてブロッキング処理を全く行ない、１Ｍ ＮａＣｌ／５０ｍＭリン酸緩衝液（ｐＨ７．０）溶液で洗浄し、プローブ固定基材上を濡らし、均一なハイブリダイゼーション処理ができるようにした。
【０２１１】
（７）ハイブリダイゼーション処理
上記実施例１７と全く同様にしてハイブリダイゼーション処理を行なった。その後、プローブアレイを１Ｍ ＮａＣｌ／５０ｍＭリン酸緩衝液（ｐＨ７．０）溶液で洗浄してプローブ核酸とハイブリダイズしなかった一本鎖ＤＮＡプローブを洗い流した。ついで、純水で余分な塩分を除去した後、窒素ブローによりプローブ固定基材を乾燥させた。次に該プローブ固定基材のスポットの蛍光を、蛍光スキャナ（商品名：ＧｅｎｅＰｉｘ４０００Ｂ；ＡｘｏｎＩｎｓｔｒｕｍｅｎｔｓ，Ｉｎｃ．製）を用いて蛍光強度を評価した。評価するにあたり、レーザーパワーを１００％に設定し、ＰＭＴを４００Ｖに設定した。
【０２１２】
（８）結果
上記（７）での蛍光スキャナ評価結果を解析したところ、標識化一本鎖ＤＮＡプローブと完全マッチである配列番号１のＤＮＡプローブのスポットでは、５３２ｎｍでの蛍光強度は２３４００であった。また、ＤＮＡプローブのスポット部以外の蛍光強度を観察したところ６５８であった。各ＤＮＡプローブのスポットを蛍光で観察した状態では、各々のスポット形状がほぼ円形であり、同じプローブ媒体をスポッティングしたスポット間においては蛍光強度の差異は殆ど認められなかった。ブロッキング処理を行なわないことによるスポット部以外での蛍光強度の増加は殆ど認められなかった。また、隣接するスポットとの間隔はほぼ均等であり、約２００μｍの間隔で格子状にスポットが配置されていることが観察された。
【０２１３】
（比較例）
（１）プローブの合成
標的物質に対して特異的に結合可能なプローブとして一本鎖ＤＮＡプローブを用いた。ＤＮＡ自動合成機を用いて配列番号１０の一本鎖核酸を合成した。なお配列番号１の一本鎖ＤＮＡ末端にはＤＮＡ自動合成機での合成時にチオールモディファイア（Ｔｈｉｏｌ−Ｍｏｄｉｆｉｅｒ）（グレンリサーチ（ＧｌｅｎＲｅｓｅａｒｃｈ）社製）を用いる事によってメルカプト（ＳＨ）基を導入した。続いて通常の脱保護を行ない、ＤＮＡを回収し、高速液体クロマトグラフィーにて精製し、以下の実験に用いた。
配列番号：１０
5’HS-(CH2)6-O-PO2-O-ACTGGCCGTCGTTTTACA3’
【０２１４】
（２）プローブ媒体の調整
上記実施例１と全く同様にして、配列番号１０の一本鎖ＤＮＡプローブ溶液を調製した（正確な濃度は吸収強度から算出）。調整したプローブ溶液を以下の実験に用いた。
【０２１５】
上記のとおり調整したプローブ溶液を１００μｌ用意し、これに、グリセリン７．５ｗｔ％、尿素７．５ｗｔ％、及びチオジグリコール７．５ｗｔ％を含む水溶液を９００μｌ滴下した。５分間混合した後、３０分間放置、プローブ媒体を調整した。
【０２１６】
（３）基板洗浄
上記実施例１と同様にして基板洗浄を行ない、ガラス基板を準備した。
【０２１７】
（４）プローブ固定化処理
アミノ基を有するシラン化合物（Ｎ−β（アミノエチル）γ−アミノプロピルトリメトキシシラン）を含むシランカップリング剤（商品名：ＫＢＭ−６０３；信越化学工業（株）社製）の１ｗｔ％水溶液を室温下で３０分間攪拌し、シランカップリング剤水溶液を調整した。次いでこの水溶液に上記（３）で洗浄したガラス基板を室温（２５℃）で２０分間浸した後、流水で十分にすすぎ、余分なシランカップリング剤水溶液を除去した。すすいだ基板に窒素ガスを基板両面に吹き付けて乾燥させた。次に基板を１２０℃に加熱したオーブン中で１時間ベークしてシランカップリング処理を完結させた。次いでＮ−マレイミドカプロイロキシスクシンイミド Ｎ−（６−Ｍａｌｅｉｍｉｄｏｃａｐｒｏｙｌｏｘｙ）ｓｕｌｆｏｓｕｃｃｉｎｉｍｉｄｅ （商品名：ＥＭＣＳ；（株）同仁化学研究所社製）を２．７ｍｇ秤量し、ジメチルスルホキシド（ＤＭＳＯ）／エタノールの１：１溶液に最終濃度が０．３ｍｇ／ｍｌとなる様に溶解させ、ＥＭＣＳ溶液を用意した。シランカップリング処理を行った基板をこのＥＭＣＳ溶液に室温で３０分間浸漬した。ＥＭＣＳ溶液から引き上げた基板はＤＭＳＯ及びエタノールの混合溶媒及びエタノールで順次洗浄した後、窒素ガス雰囲気下で乾燥させた。このようにしてプローブ固定化処理を完了させた。プローブ固定化処理の効果を確認するために純水接触角を測定した。その結果、基板上における接触角は２５〜３０°であった。この結果から、基板上へのプローブ固定化処理が適切に実施できていることが確認できた。
【０２１８】
（５）プローブ媒体のスポッティング
上記（２）で調整したプローブ媒体を上記実施例１と全く同様にしてスポッティングを行ない、プローブ固定基材を作製した。スポッティング終了後、ガラス基板を顕微鏡により観察したところ、ガラス基板表面にマトリックス状のスポット配列が形成されていることが確認された。また、プローブ固定化処理の効果を確認するために純水接触角を測定した。その結果、基板上における接触角は２５〜３０°であった。この結果から、スポット部位以外にはプローブ媒体等による汚染などは発生していないことが確認できた。スポッティング終了後のガラス基板は１Ｍ ＮａＣｌ／５０ｍＭリン酸緩衝液（ｐＨ７．０）溶液を満たした容器中に浸漬し、容器ごと冷蔵庫に入れ冷蔵保管（４℃）した。このようにしてプローブ固定基材を作製した。
【０２１９】
（６）ブロッキング処理
上記実施例１と全く同様にしてブロッキング処理を行なった。
【０２２０】
（７）ハイブリダイゼーション処理
上記（１）に記載の配列番号１０の一本鎖ＤＮＡプローブと相補的な塩基配列を有する一本鎖ＤＮＡプローブをＤＮＡ自動合成機で合成し、５’末端にローダミンを結合させて標識化した一本鎖ＤＮＡプローブを得た。この標識化一本鎖ＤＮＡを１Ｍ ＮａＣｌ／５０ｍＭリン酸緩衝液（ｐＨ７．０）に最終濃度１μＭとなるように溶解し、この溶液中に上記（６）で得たブロッキング処理したプローブ固定基材を浸漬し、室温（２５℃）で３時間ハイブリダイゼーション処理を行なった。その後、プローブアレイを１Ｍ ＮａＣｌ／５０ｍＭリン酸緩衝液（ｐＨ７．０）溶液で洗浄してプローブ核酸とハイブリダイズしなかった一本鎖ＤＮＡプローブを洗い流した。ついで、純水で余分な塩分を除去した後、窒素ブローによりプローブ固定基材を乾燥させた。次に該プローブ固定基材のスポットの蛍光を、蛍光スキャナ（商品名：ＧｅｎｅＰｉｘ４０００Ｂ；Ａｘｏｎ Ｉｎｓｔｒｕｍｅｎｔｓ，Ｉｎｃ．製）を用いて蛍光強度を評価した。評価するにあたり、レーザーパワーを１００％に設定し、ＰＭＴを４００Ｖに設定した。
【０２２１】
（８）結果
上記（７）での蛍光スキャナ評価結果を解析したところ、標識化一本鎖ＤＮＡプローブと完全マッチである配列番号１０のＤＮＡプローブのスポットでは、５３２ｎｍでの蛍光強度は１２７９０であった。また、ＤＮＡプローブのスポット部以外の蛍光強度を観察したところ７９２であった。各ＤＮＡプローブのスポットを蛍光で観察した状態では、各々のスポット形状がほぼ円形であり、同じプローブ媒体をスポッティングしたスポット間においては蛍光強度の差異は殆ど認められなかった。また、隣接するスポットとの間隔はほぼ均等であり、約２００μｍの間隔で格子状にスポットが配置されていることが観察された。
【０２２２】
【発明の効果】
以上説明した様に、本発明によればプローブおよびプローブと結合しうる物質、基材上と結合しうる物質をプローブ媒体中に含ませることにより、プローブ媒体を基材上に付与するだけで、プローブを効率良く、安定的に基材上に固定させることができる。また、この方法を用いることによってプローブ固定基材を製造することにより、少ない工程でプローブ固定基材を製造できるようになるばかりではなく、プローブ固定基材を高品質で尚且つ、簡略的に製造できる。
【０２２３】
また本発明によれば、多種のプローブを一つの基材上に固定する場合、各プローブ種に対応して結合物質種，濃度を選択することが可能となる。さらにはプローブおよびプローブと結合しうる物質、基材上と結合しうる物質間の結合反応条件の各プローブ種に応じて調整可能となる。このため、一つの基材上において多種プローブをプローブ種毎に最適な条件で結合させることができる。
【０２２４】
更に本発明によれば少量の検体からでも標的物質に関するより多くの情報を、より正確に検査可能なプローブアレイを得ることができ、またそれを用いることでサンプル中に標的物質が存在するか否かをより正確、且つ迅速に判定できる。同様にこのプローブアレイを用いることでサンプル中の標的物質の構造をより正確に、且つ迅速に特定することができる。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a probe medium for fixing a probe on a substrate and a fixing method thereof. The present invention also relates to a detection method for detecting a target substance using a probe-fixing base material obtained by fixing a probe medium on the base material.
[0002]
[Prior art]
Various methods are known as methods for fixing the probe on the substrate. Specifically, there are a method of fixing the probe on the substrate by synthesizing the probe on the substrate, and a method of fixing the probe by applying a probe prepared in advance on the substrate with a pin or a stamp. Specifically, various arrangements on the substrate can be achieved by repeatedly removing protecting groups from selected regions of the substrate with an activator and attaching monomers having removable protecting groups to the regions. There is known a method for synthesizing a polymer having R (see, for example, Patent Document 1). In addition, fixing is performed by bringing a fixing material composed of a base material and a polymer compound having a carbodiimide group carried on the base material into contact with a biologically active substance having reactivity with the carbodiimide group. A method is known (see, for example, Patent Document 2). Similarly, in the detection of a biologically active substance, a detection method using immobilization via a carbodiimide group on a compound having carbodiimide is known (see, for example, Patent Document 3). Furthermore, a solid phase carrier having a DNA fragment having a thiol group at the terminal and a chain molecule having a reactive substituent capable of forming a covalent bond by reacting with the thiol group, fixed on the surface at one terminal; There is known a method of immobilizing a DNA fragment on the surface of a solid phase carrier by forming a covalent bond between the DNA fragment and a chain molecule by bringing the DNA fragment into contact with each other in a liquid phase (for example, Patent Document 4). reference).
[0003]
In addition, many methods are known as methods for detecting biological components. There has been described a method for easily measuring a substance such as RNA, cDNA, oligonucleotide, etc., such as biological components, with high sensitivity and high accuracy while eliminating the influence of background noise (see, for example, Patent Document 5). ). Furthermore, a method is known in which a reactive substance is fixed to a substrate by discharging a solution containing the reactive substance from a nozzle to a desired position on the substrate to be reacted (see, for example, Patent Document 6). The substrate is made of glass or silicon, and the surface thereof is surface-treated for imparting affinity. The reactive substance is DNA fragment, cDNA, RNA, enzyme, antigen, antibody, epitope, protein, polynucleotide. , At least one selected from the group consisting of peptides.
[0004]
[Patent Document 1]
US Pat. No. 5,138,545
[Patent Document 2]
JP-A-8-23975
[Patent Document 3]
JP-A-8-334509
[Patent Document 4]
JP 2001-178442 A
[Patent Document 5]
JP 2001-116699 A
[Patent Document 6]
JP 2001-116750 A
[0005]
[Problems to be solved by the invention]
However, as a probe medium including a probe that can specifically bind to a target substance that has been conventionally used, a probe, a site that can bind to the probe, and a site that can bind to the substrate are combined into one medium. There was nothing to include. In addition, as a method for immobilizing a probe that can specifically bind to a target substance, which has been conventionally used, on a substrate, a material including a portion that can bind to the probe is coated on the substrate, or After forming a binding layer or the like on the base material by a method such as forming a portion capable of binding to the probe by treating the top, the probe is fixed by applying a medium containing at least the probe. For this reason, before applying a probe medium on a base material, it was necessary to perform the process for forming the site | part which can couple | bond with a probe beforehand on a base material. Furthermore, a compound having a site capable of binding to the probe and a base material are used as a probe fixing material, and the probe is fixed by treating the base material in advance with the probe fixing material. In this method, a substrate treatment with a probe fixing material is required before the probe is fixed.
[0006]
[Means for Solving the Problems]
The above problems have been solved by the present invention described below.
(1) a single-stranded nucleic acid probe capable of specifically binding to a target substance and having a functional group modified at the end, a functional group capable of binding to the end of the single-stranded nucleic acid probe, and Silanol group A silane coupling agent having Dissolved in aqueous solution Probe medium. Or A single-stranded nucleic acid probe capable of specifically binding to a target substance and having a functional group modified at the end, and a functional group capable of binding to the end of the single-stranded nucleic acid probe and a substrate. A silane coupling agent having a site that can be obtained in the presence of water, A probe medium further comprising a bivalent reagent for binding the single-stranded nucleic acid probe and the silane coupling agent. The site capable of binding to the probe and the substrate may be a functional group. This bonding method may be any one of covalent bonding, ionic bonding, and physical adsorption including electrostatic adsorption. This probe may be a DNA probe.
(2) A probe fixing method for fixing the probe medium by applying the probe medium onto a substrate by a spotting method. A DNA array in which the probes on the probe-fixing substrate prepared by this probe-fixing method are DNA probes. A detection method in which a probe medium is fixed by being applied onto a substrate by a spotting method, and a target substance is detected using the obtained probe-fixed substrate.
(3) A probe medium in which the probe and the substance for fixing the probe on the base material are respectively housed in a container and mixed when fixed on the base material.
The DNA array according to the present invention binds specifically to a target substance and binds to a single-stranded nucleic acid probe having a functional group modified at the end and the end of the single-stranded nucleic acid probe. Possible functional groups and Silanol group And a silane coupling agent having In aqueous solution A DNA array, characterized in that the probe medium is disposed in a spot-like manner on the substrate.
[0007]
DETAILED DESCRIPTION OF THE INVENTION
The probe medium and the method for immobilizing a probe on a substrate according to the present invention comprise a medium including a probe, a portion that can bind to the probe, and a portion that can bind to the substrate.
[0008]
Examples of the probe include a single-stranded nucleic acid probe, a single-stranded DNA probe, a single-stranded RNA probe, and a single-stranded PNA probe. As the amount of the probe contained in the medium, considering the stability of the nucleic acid probe as the probe medium, for example, 2 mer to 500 mer, particularly 2 mer to 80 mer nucleic acid probe, 0.05 to 500 μmol / l, In particular, it is preferable to adjust to a concentration of 0.5 to 50 μmol / l. These probes may be single probes, but those having linker modifications at the probe ends are preferred. Examples of the linker modification include biotinylation and functional group modification. Examples of the functional group include functional groups such as an amino (NH 2) group, a carbonyl (COOH) group, a mercapto (SH) group, and a hydroxyl group (OH) group. In particular, the probe functional group is preferably an amino group in consideration of the ease of probe functional group synthesis, and a mercapto group is preferable in consideration of the reactivity of the probe functional group.
[0009]
The site capable of binding to the probe is not particularly limited, but is preferably one that binds to the linker at the end of the probe. There are various forms of bonding with the linker at the end of the probe, but chemical bonding is preferred. Examples of the site capable of binding to the probe linker include avidin and functional groups. Examples of the functional group include an epoxy (CHOCH2) group, an amino (NH2) group, a mercapto (SH) group, a maleimide group, a succinimide ester group, a chloropropyl (C3H6Cl) group, and an isocyanate (NCO) group. In particular, the functional group that can be bonded to the probe is preferably an epoxy group, a maleimide group, a succinimide ester group, a chloropropyl group, or an isocyanate group in consideration of bonding with the probe functional group. The amount of the site capable of binding to the probe contained in the medium is not particularly limited because it varies depending on the site capable of binding to the probe, but considering the binding property with the probe, it is 100 to 100,000 with respect to the probe amount. It is preferable to adjust so that it may become equivalent.
[0010]
The site that can be bonded on the substrate is not particularly limited, but those that bond firmly on the substrate are preferable. The site capable of binding may be a functional group, and preferably forms a chemical bond with the substrate. Functional groups for bonding on the substrate include alkoxysilyl (SiOR) groups, silanol (SiOH) groups, alkoxy (OR) groups, and the like. In particular, an alkoxysilyl group and a silanol group are preferable from the viewpoint of easiness of fixing on the substrate, but when an amino group and a carbonyl group are formed on the substrate, those having reactivity with each other are preferable. The silanol group may be formed by hydrolysis of an alkoxysilyl group. The probe and the substance for immobilizing the probe on the substrate may be bonded by reacting in a medium, and the reaction is preferably a covalent bond by a chemical reaction between functional groups.
[0011]
There are various combinations of these probes, sites that can bind to the probe, and sites that can bind to the substrate. If a specific combination as a functional group is given, for example, the probe functionality A combination of the group is an amino group, the functional group at the site capable of binding to the probe is an epoxy group, a succinimide ester group, and the functional group at the site capable of binding on the substrate is a silanol group. In addition, the probe functional group is a mercapto group, the functional group at the site capable of binding to the probe is a combination of maleimide groups, the probe functional group is a carbonyl group, and the functional group capable of binding to the probe is an amino group on the substrate. The functional group at the site that can be bonded to a combination of silanol groups, the functional group at the probe is an amino group, the functional group at the site that can be bonded to the probe is an isocyanate group, and the functional group at the site that can be bonded to the substrate is a silanol group Combination. Of these, if considering the ease of synthesis of the probe functional group and the reactivity of the functional group at the site capable of binding to the probe, the functionality of the site where the probe functional group is an amino group and can be bound to the probe. Those in which the group is an isocyanate group are preferred. These combinations are preferably reacted by mixing in a medium, and may be processed under predetermined conditions for reaction.
[0012]
A single substance has a functional group at a site capable of binding to the probe and a functional group at a site capable of binding on the substrate, and the functional group at the site capable of binding on the substrate is an alkoxysilyl group or a silanol group. In some cases, a silane coupling agent can be mentioned if a specific substance is given as a single substance. In the silane coupling agent, silanol groups are formed by hydrolysis of alkoxysilyl groups. When the other functional group different from the alkoxysilyl group in the silane coupling agent is an epoxy group, an isocyanate group, a mercapto group, a chloropropyl group, a maleimide group, or a succinimide ester group mentioned above, The silane coupling agents are epoxy silane, isocyanate silane, mercapto silane, chloropropyl silane, maleimide silane, and succinimide ester silane, respectively. These silane coupling agents are hydrolyzed as necessary and mixed with the probe.
[0013]
Regarding maleimide silane and succinimide silane, Japanese Patent Publication 2001-72690 and US Pat. No. 5277813 have some related descriptions, but no detailed description of the portions related to the present invention is found. .
[0014]
The functional group at the site capable of binding to the probe and the functional group at the site capable of binding to the substrate are in different substances, and the functional group at the site capable of binding to the probe is a maleimide group, In the case where the functional group at the site that can be bonded is an alkoxysilyl group or a silanol group, a bivalent reagent and a silane coupling agent can be used if specific examples of combinations of the respective substances are given. If a specific substance name is given as a bivalent reagent, N- (4-Maleimidocaproyloxy) succinimide, N- (6-Maleimidocaproxylo) succinimide, N- (8-Maleimidocaproxilomoidoproxilomoxydeoxymoxyproxyloxymidoproxilomoxy) 11 succinimide. In particular, if it is preferable that these substances are water-soluble when handled as a probe medium, N- (4-Maleimidocapropyloxy) sulfosuccinimide, sodium salt, N- (6-Maleimidocaproxixy) sulfosuccinimicin, which made them water-soluble Sodium salt, N- (8-Maleimidocaproxy), sulfosuccinimide, sodium salt, N- (11-Maleimidocaproxy), sulfosuccinimide, sodium salt. Moreover, about a silane coupling agent, a silanol group may be formed by hydrolysis of an alkoxysilyl group. There are various silane coupling agents that can react with the divalent reagent, and aminosilanes having an amino group are preferred. These silane coupling agents are hydrolyzed as necessary and mixed with the probe.
[0015]
As the medium of the probe medium comprising the probe and the substance for fixing the probe on the substrate, a medium containing water is preferable. What is further contained in the medium in addition to water includes organic solvents such as ethylene glycol, thiodiglycol, glycerin, isopropyl alcohol, and ethanol. Specific examples of the medium include water containing 70 to 95 wt%, thiodiglycol 0.1 to 3%, and isopropyl alcohol 5 to 30 wt%. As a more preferable medium, water is 80 to 90 wt%, thiodiglycol 1 to 2.5 wt%, and isopropyl alcohol 10 to 20 wt%. The probe medium may be mixed with a water-soluble polymer material as necessary. Examples of the water-soluble polymer material include polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), paogen, carboxymethyl cellulose (CMC), hydroxyethyl cellulose (HEC), dextran, and pullulan. Specifically, a general-purpose polymer water-soluble material such as polyvinyl alcohol (PVA) or polyvinyl pyrrolidone (PVP) is preferable. As a method for mixing the water-soluble polymer material into the medium, a solution having a predetermined concentration in which the water-soluble polymer material is completely dissolved in advance is prepared. A method in which the prepared water-soluble polymer solution is weighed to a predetermined concentration and mixed is suitable. Specifically, as the water-soluble polymer material solution, polyvinyl alcohol powder is weighed and adjusted to be dissolved while heating to a concentration of 0.5 to 5 wt% in pure water. Is made. It is preferable to mix this water-soluble polymer aqueous solution in the probe medium so that the concentration in the probe medium is 0.01 to 1 wt%.
[0016]
There are several procedures for mixing the probe medium. As a first method, a probe and a substance having a functional group at a site capable of binding to the probe and a functional group at a site capable of binding to the substrate are mixed in advance, and then water, an organic solvent, or the like as a medium A certain amount of the solution is added and mixed. If necessary, the temperature may be adjusted and allowed to stand for a predetermined time, and the medium may be added and mixed repeatedly. Finally, a probe medium is prepared by adding a solution that has not been added or that has been added in a small amount until a predetermined ratio is reached. In mixing the probe and the substance, the reaction may proceed by mixing, and it is preferable to adjust the temperature, mixing time, pH, etc. of the mixed state as necessary. When the reaction proceeds by mixing the probe and the substance, it becomes easy to sufficiently advance the reaction between the probe and the substance by mixing in advance. Furthermore, it is easy to sufficiently bind the probe and the substance by adjusting the mixing, which is preferable. In addition, in adding and mixing a solution to a mixed medium of a probe and a substance, a method of sequentially dropping, mixing and adjusting water, an organic solvent, etc., which are part or all of the solution medium after mixing the probe and the substance Alternatively, a method of sequentially dropping, mixing, and adjusting a mixed medium in which a part or all of constituent media such as water and an organic solvent are mixed in advance may be used. When the probe and the substance react in the mixed medium, if the reaction is further promoted by the addition of the solution, it is preferable to adjust the order of the constituent media to be added, the interval, and the state at the time of addition. The second method can be applied only when the functional group at the site capable of binding to the probe and the functional group at the site capable of binding on the substrate are provided in different substances. A substance having a functional group at a site that can be bonded and a substance having a functional group at a site that can be bound on a probe or a substrate with a solution such as water or an organic solvent that is a part or all of the medium. After mixing, the remaining one is mixed. In this method, if it is difficult to mix and dissolve the probe or substance in the solution, the conditions for mixing and dissolving can be adjusted. It is suitable when an action occurs. Particularly when the substance is a silane coupling agent and the functional group capable of binding to the probe is stable when mixed or / and dissolved, sufficient silanol groups can be formed by mixing and dissolving the substance in the solution in advance. This is preferable because it can be formed. As a third method, a substance having a functional group at a site capable of binding to the probe and the probe and a functional group at a site capable of binding to the substrate and a part or all of the constituent medium of the solution are respectively weighed, It is the method of putting in one container and mixing at once. This method can be applied when the characteristics of the probe medium do not change depending on the mixing order of the probe, the substance, and the solution, and is a preferable method because the mixing process can be simplified.
[0017]
The probe medium thus obtained is suitable as a probe medium for detecting a target substance.
[0018]
A probe-fixing substrate can be prepared by spotting the obtained probe medium on the substrate. The substrate for fixing the probe is not particularly limited, but glass or quartz substrate material is preferable in consideration of detection of a target substance and versatility as a material. As a specific material, a slide glass substrate having a size of 1 inch × 3 inches is preferable, and a non-alkali material slide glass substrate in which an alkali component or the like is not included in the glass material if the fixing property of the probe is considered. Or it is preferable that it is a quartz glass material. Moreover, although the shape of the substrate is not limited depending on the detection method of the target substance, it is preferable that the substrate is in a state of versatility such as the detection method and apparatus. Furthermore, it is desired that the substrate material has a high surface smoothness.
[0019]
In addition, the substrate for fixing the probe is preferably a clean surface in order to fix the probe uniformly and reliably on the substrate. It is desirable to ensure a sufficiently clean surface by washing the substrate in advance before fixing the probe. As a method for cleaning the substrate, many methods such as water cleaning, chemical cleaning, plasma cleaning, UV ozone cleaning, etc. are known. A cleaning method using a chemical solution is appropriate. For example, there is a method in which the surface of the base material is sufficiently washed with a sodium hydroxide aqueous solution having a predetermined concentration to remove dirt adhering to the base material. Specifically, a 1 mol / l sodium hydroxide aqueous solution heated to about 50 ° C. is prepared, and the substrate surface is wiped in the aqueous solution or brushed while showering the aqueous solution. Make sure to remove the attached dirt. After removing the dirt, thoroughly wash away excess sodium hydroxide with water. Finally, moisture removal may be performed by N2 blow or the like. In this way, it is possible to obtain a substrate on which the probe medium can be fixed uniformly and reliably.
[0020]
Several methods are known for spotting a probe medium on a substrate. As specific methods, a pin method, an ink jet method, and a pin and ring method are known. Among these, the ink jet method is a preferred spotting method because it allows high-density and accurate spotting.
[0021]
In the spotting method based on the inkjet method, the components contained in the probe medium substantially affect the probe and the substance that fixes the probe onto the substrate when ejected from the inkjet head as the probe medium as described above. There is no particular limitation as long as it satisfies the medium composition that can be normally ejected onto a substrate using an inkjet head. For example, when the inkjet head is a bubble jet (registered trademark) head having a mechanism for applying thermal energy to the medium and discharging it, a liquid containing glycerin, thiodiglycol, or isopropyl alcohol is preferable as a component contained in the probe medium It is. More specifically, a liquid containing glycerin 5 to 10 wt% and thiodiglycol 5 to 10 wt% is suitably used as the probe medium. When the inkjet head is a piezo jet head that ejects a medium using a piezoelectric element, a liquid containing ethylene glycol and isopropyl alcohol is preferable as a component contained in the probe medium. More specifically, a liquid containing 5 to 10 wt% ethylene glycol and 0.5 to 2 wt% isopropyl alcohol is preferably used as the probe medium.
[0022]
When the probe medium thus obtained is ejected from the ink jet head and deposited on the substrate, the spot shape is circular and the ejected range does not widen. Even when the probe medium is spotted at a high density, the connection with adjacent spots can be effectively suppressed. The characteristics of the probe medium of the present invention are not limited to the above.
[0023]
It is effective to fix the probe contained in the probe medium applied on the substrate at a predetermined position and prevent contamination with the probe contained in the adjacent spot more reliably. As an effective means for firmly fixing, there is a method of using a material that is bonded to both a site that can be bonded to the substrate and the substrate by a chemical action. There are various types of bonds by chemical action, and examples thereof include covalent bonds and ionic bonds. A covalent bond is also preferable in order to firmly fix the probe on the substrate.
[0024]
Preferable examples of the functional group include a combination having a silanol (SiOH) group on the substance side and a hydroxyl (OH) group on the substrate. With this combination, the silanol group of the substance contained in the probe medium applied to the substrate by spotting reacts with the silanol group on the substrate to immobilize the substance on the substrate. If a specific base material is mentioned, the glass material and the quartz substrate material described above are preferable because they have a hydroxyl group on the base material.
[0025]
Furthermore, it is preferable that the probe and the site capable of binding to the probe are also bonded by chemical action. Examples of the bond as a chemical action include a covalent bond and an ionic bond. A covalent bond is also preferable in order to firmly fix the probe on the substrate. As a result, the probe is immobilized on the substrate, and the spot of the probe can be formed at a predetermined position. In particular, a probe medium comprising a probe having an amino group as a functional group and a substance having an epoxy group as a functional group at a site capable of binding to the probe and a silanol group as a functional group at a site capable of binding on the substrate is prepared. Then, a probe solution in which glycerin, thiodiglycol, isopropyl alcohol, or the like was mixed was adjusted to a predetermined ratio. When spotting is performed on a substrate having a hydroxyl group with this probe solution as a functional group using an inkjet head, the probe solution forms a spot having a stable size on the substrate. In this way, the probe can be fixed at a predetermined position on the substrate. Further, a probe medium comprising an amino group as a functional group, and a probe medium comprising a substance having an isocyanate group as a functional group capable of reacting with the probe and a silanol group as a functional group capable of reacting with a functional group on the substrate, A probe solution in which glycerin, thiodiglycol, isopropyl alcohol and the like were mixed so as to have a predetermined ratio was prepared. When spotting is performed on a substrate having a hydroxyl group with this probe solution as a functional group using an inkjet head, the probe solution forms a spot having a stable size on the substrate. In this way, the probe can be fixed at a predetermined position on the substrate.
[0026]
In addition, it is preferable to mix polyvinyl alcohol (PVA) which is a water-soluble polymer material in the probe medium. More preferably, it is desired to dissolve completely. The mixing of the water-soluble polymer material into the probe medium facilitates the observation of the spotting state on the substrate. By making the probe medium at the spot after spotting difficult to dry, the probe medium and the substrate are more reliably obtained. The top can be reacted and fixed. Furthermore, considering the storage condition of the probe-fixing substrate prepared by spotting, it is effective for detecting the target substance efficiently and stably even when the probe medium spot formed by spotting is dried. Because there is.
[0027]
For example, a probe medium containing a probe with a base length of 20 mer was adjusted so that the probe concentration was 8 μmol / l. The probe medium is a bubble jet (registered trademark) printer (trade name: BJF850; manufactured by Canon Inc., but modified so as to be able to print on a flat plate), the solid phase and the nozzle of the bubble jet (registered trademark) head. When the interval is set to about 1.2 to 1.5 mm and discharged from the nozzle (discharge amount is about 4 picoliters), a spot with a diameter of about 50 to 80 μm can be formed on the solid phase. In addition, no spot (hereinafter referred to as “satellite spot”) derived from splashes when the liquid landed on the solid phase surface was observed at all by visual observation using a loupe.
[0028]
Further, the probe fixing substrate (fixed with a probe medium not containing a water-soluble polymer material) was analyzed with a Fourier transform infrared spectrophotometer (microscopic FT-IR) for microscopic measurement. At sites other than the spots, Si—O bond peaks attributed to the substrate could be detected, but other bond peaks were hardly observed. On the other hand, in addition to being able to detect the broad peak of the substrate at the spot formation site, the peak of the —CH 2 — bond of the substance that fixes the probe contained in the probe medium on the substrate is Although it was very small, it was able to be detected.
[0029]
Further, the probe-fixing base material was analyzed by a temperature rising detriment method (TDS: Thermal Description Spectrometry). In performing the analysis, a 7 mm square substrate piece including a spot-formed portion and a 7 mm square substrate piece at a spot outer peripheral portion where no spot was formed were cut out and compared. The temperature was 25 to 500 ° C., and the heating rate was 5 ° C. per minute. As a result, when the desorption component masses of the two substrate pieces were compared, a large amount of CH4, CH3OH or other probe mass and the mass due to the substance fixing the probe on the substrate were detected at 150 ° C. or higher. From these facts, it was confirmed that the probe medium was spotted only at the spot portion, and the probe medium and the substance contained in the medium were not applied to the portion other than the spot portion.
[0030]
The probe-fixing substrate was analyzed by a time-of-flight secondary ion mass spectrometer (TOF-SIMS: TimeOfFlight-SecondaryIonMass Spectrometer). In performing the analysis, a surface scan was performed in the vicinity of the spot formation site and compared. As a result, at the spot formation site, the PO caused by DNA ^2- , PO ^3- Ions were detected. On the other hand, at a spot where no spot is formed or at the spot gap, PO ^2- , PO ^3- Not only ions were not detected, but secondary ions due to functional groups at sites that could bind to the probe were not detected.
[0031]
Here, for the purpose of improving detection accuracy (S / N ratio), for example, when detecting a target substance using this probe fixing substrate, for example, after fixing the probe to the solid phase surface, Blocking may be performed so that the non-probe binding portion on the substrate does not bind to a target substance or the like contained in the sample. The blocking is performed, for example, by immersing the substrate in a 2% bovine serum albumin aqueous solution, for example, for about 2 hours. However, in view of the effect of preventing the labeling substance from adsorbing to other than the probe fixing site on the substrate, an aqueous bovine serum albumin solution is preferable. The blocking step may be performed as necessary. For example, the sample is supplied to the probe-fixing base material in a limited manner for each spot, and the sample is not substantially attached to a site other than the spot. If not, you do not have to do it. Attachment of the sample to a site other than the spot varies depending on the material used as the base material. In particular, when the substrate is glass or quartz, the blocking process may not be performed.
[0032]
The probe fixing base material thus produced may be configured to have, for example, a plurality of spots including the same probe, or a plurality of spots each including a different type of probe, depending on the application. May be. The type, quantity, and arrangement of the probes can be changed as necessary. The probe-immobilized base material on which the probes are arranged at a high density by such a method is then used for detection of the target substance, identification of the target substance base sequence, and the like. For example, when it is used for detection of a single-stranded nucleic acid that may be contained in a sample and whose base sequence is a known target substance, it is complementary to the base sequence of the single-stranded nucleic acid of the target substance. A single-stranded nucleic acid having a simple base sequence is used as a probe, and a probe fixing substrate in which a plurality of spots including the probe are arranged on a solid phase is prepared. And is placed under conditions such that the single-stranded nucleic acid of the target substance and the probe hybridize, and then the presence or absence of a hybrid in each spot is detected by a known method such as fluorescence detection. Thereby, the presence or absence of the target substance in the sample can be detected.
[0033]
In addition, when used for specifying a base sequence in a single-stranded nucleic acid of a target substance contained in a sample, a plurality of candidates for base sequences in the single-stranded nucleic acid of the target substance are set, and the base sequence group A single-stranded nucleic acid having a complementary base sequence to each is spotted on the substrate as a probe. Next, after supplying a sample to each spot and placing it under conditions such that the single-stranded nucleic acid of the target substance and the probe hybridize, a known method such as fluorescence detection of the presence or absence of the hybrid in each spot Detect with. Thereby, the base sequence of the single-stranded nucleic acid of the target substance can be specified. As other uses of the probe-immobilizing substrate according to the present invention, for example, application to screening of a specific base sequence recognized by a DNA-binding protein and screening of a chemical substance having a property of binding to DNA can be considered.
[0034]
As the probe medium, not only those comprising the probe and the substance for fixing the probe on the substrate as described above, but also each of the probe and the substance for fixing the probe on the substrate are accommodated in a container, It may be mixed when fixed on a substrate.
[0035]
Examples of the probe include a single-stranded nucleic acid probe, a single-stranded DNA probe, a single-stranded RNA probe, and a single-stranded PNA probe. These probes may have a functional group. For example, functional groups such as amino (NH 2) group, isocyanate (NCO) group, mercapto (SH) group, and hydroxyl group (OH) group may be used. Can be mentioned. In particular, the probe functional group is preferably an amino group in consideration of the ease of probe functional group synthesis, and a mercapto group is preferable in consideration of the reactivity of the probe functional group.
[0036]
When the probe is housed in the container, the container is preferably a container that can be sealed so that other impurities do not enter, and can be easily mixed when fixed. So that it can be opened. The opening as a container is not particularly limited. The state of the probe housed in the container is not particularly limited, but it may be in the form of a solution or powder. When the functional group is a mercapto group or the like, it is preferably stored as a powdered product by freeze drying or the like in consideration of the stability of the probe. As the storage state, it is preferable to store in a frozen state in order to ensure the stability of the probe, but it is possible to change the storage state according to the storage period, probe type, probe functional group, and the like. In this way, the probe may be housed in a container and mixed when being fixed on the substrate in a stored state.
[0037]
Examples of the substance having a functional group at a site capable of binding to the probe and a functional group at a site capable of binding to the substrate include a primer, a coupling agent, a sealant, a surface treatment agent, and a surface modifier. Examples of functional groups that can be bonded to these probes include epoxy (COO) groups, amino (NH2) groups, mercapto (SH) groups, maleimide groups, succinimide ester groups, isocyanate (NCO) groups, and chloropropyl (C3H6Cl). ) Group and the like. In particular, the functional group at the site capable of binding to the probe is preferably an epoxy group, a maleimide group, a succinimide ester group, an isocyanate group or a chloropropyl group in consideration of the binding with the probe functional group. Examples of the sites that can be bonded to these substrates include alkoxysilyl (SiOR) groups, silanol (SiOH) groups, and alkoxy (OR) groups. In particular, an alkoxysilyl group and a silanol group are preferable from the viewpoint of ease of fixation on a substrate and reactivity. The silanol group may be formed by hydrolysis of an alkoxysilyl group. The functional group at the site where the probe can be bonded to the probe may be bonded by reacting in the medium, and the reaction is preferably a covalent bond by a chemical reaction between the functional groups.
[0038]
When storing a substance having a functional group at a site capable of binding to the probe and a functional group at a site capable of binding on the base material in the container, the container is further controlled so that other impurities are not mixed therein. It can be sealed so that it will not lose weight due to volatilization of water, or is preferably a sealed container, and can be opened easily so that mixing is simple when fixing It is preferable that However, the opening as a container is not particularly limited. When the functional group of the site capable of binding to the probe and the functional group of the site capable of binding to the substrate are in different substances, each substance is stored in the same container, or in separate containers. Although it is not particularly limited, it is preferable to store the probe medium so as to be suitable for forming the probe medium. The state of the substance stored in the container is not particularly limited, but it may be in the form of a solution or powder. Further, it may be a solution hydrolyzed to form a silanol group, which is an example of a functional group for fixing on a substrate. The storage state is preferably stored at room temperature in order to ensure the stability of the substance, but it is possible to change the storage state according to the storage period and the like. In particular, in the case of a hydrolyzed solution, it is preferable that the temperature during storage does not increase. Moreover, it is preferable to obtain stability by adjusting the pH after hydrolysis according to the functional group of the substance that fixes the probe on the substrate.
[0039]
Necessary for mixing the probe contained in these containers and the substance having the functional group of the site capable of binding to the probe and the substance having the functional group of the site capable of binding on the substrate when fixing the probe on the substrate. Depending on the case, a solution such as water may be added. In addition, a substance to be sufficiently mixed to obtain a probe medium, for example, a solvent such as water may be stored in a separate container and mixed at the time of adjusting the probe medium.
[0040]
Hereinafter, the present invention will be described in more detail with reference to examples.
[0041]
Example 1
(1) Probe synthesis
A single-stranded DNA probe was used as a probe that can specifically bind to the target substance. A single-stranded DNA probe of SEQ ID NO: 1 was synthesized using an automatic DNA synthesizer. An 18-mer oligomer in which an amino group was bonded to the hydroxyl group at the 5 ′ end via a phosphate group and hexamethylene was prepared at the end of the single-stranded DNA of SEQ ID NO: 1 and used in the following experiments.
SEQ ID NO: 1
5'H2N- (CH2) 6-O-PO2-O-ACTGGCCGTCGTTTTACA3 '
[0042]
(2) Substance having a functional group at a site capable of binding to the probe and a functional group at a site capable of binding on the substrate (probe binding substance)
Silane coupling including a silane compound having an epoxy group (γ-glycidoxypropyltrimethoxysilane) as a preparation of a substance having a functional group at a site capable of binding to a probe and a functional group at a site capable of binding on a substrate A probe binding substance solution was prepared by stirring a 500 μmol / l aqueous solution of an agent (trade name: KBM-403; manufactured by Shin-Etsu Chemical Co., Ltd.) for 30 minutes at room temperature. The prepared probe binding substance solution was used in the following experiment.
[0043]
(3) Adjustment of probe medium
The single-stranded DNA probe of SEQ ID NO: 1 is dissolved in TE solution (10 mM Tris-HCl (pH 8) / 1 mM ethylenediaminetetraacetic acid (EDTA) aqueous solution) so as to have a final concentration of about 40 μmol / l. A probe solution was prepared (the exact concentration was calculated from the absorption intensity).
[0044]
Next, polyvinyl alcohol (PVA), which is a water-soluble polymer material, was dissolved in pure water to a concentration of 0.5 wt%. For complete dissolution, the mixture was stirred for 60 minutes while warming to 80 ° C. in a hot bath. After confirming that there was no insoluble matter, filtration was performed so as to prevent nozzle clogging during spotting, and an aqueous PVA solution was prepared.
[0045]
100 μl of the probe solution prepared as described above was prepared, and 100 μl of the probe binding substance solution prepared as described above was added dropwise thereto, and then mixed for 5 minutes. After the mixed solution was allowed to stand for 10 minutes, 700 μl of pure water and 100 μl of the PVA aqueous solution prepared as described above were dropped and mixed for 5 minutes. After mixing, the probe medium was prepared by leaving it for 30 minutes.
[0046]
(4) Substrate cleaning
A 1 inch × 3 inch glass substrate (thickness: 1.1 mm) was put in a cassette, and the glass substrate was immersed in a 1 mol / l sodium hydroxide aqueous solution preheated to 60 ° C. for 10 minutes. Subsequently, it was sufficiently rinsed in running pure water, and sodium hydroxide adhering to the glass substrate and the cassette was washed and removed. After sufficiently rinsing, the glass substrate together with the cassette was immersed in pure water and subjected to ultrasonic cleaning for 10 minutes. After ultrasonic cleaning, the sample was sufficiently rinsed in pure water, and the particles adhering to the glass substrate and the cassette were washed and removed. The glass substrates after washing with water were dried one by one by blowing nitrogen gas. In order to confirm the cleaning of the substrate, the pure water contact angle on the substrate was measured. As a result, it was about 5 ° at all parts of the substrate.
[0047]
(5) Spotting of probe medium
The probe medium adjusted in the above (3) was filled in an ink tank for a bubble jet (registered trademark) printer (trade name: BJF850; manufactured by Canon Inc.) and mounted on a bubble jet (registered trademark) head. In addition, the bubble jet (registered trademark) printer (trade name: BJF850; manufactured by Canon Inc.) used here is modified so that printing on a flat plate is possible. Next, the glass substrate prepared in (4) above was mounted on the printer, and the probe medium was spotted on the glass substrate. Here, the distance between the liquid ejection surface of the bubble jet (registered trademark) head and the liquid adhesion surface of the glass substrate was 1.2 to 1.5 mm. After the spotting, the glass substrate was observed with a microscope, and it was confirmed that a matrix spot array was formed on the glass substrate surface. In addition, the pure water contact angle was measured on the outer periphery of the spot formation site. As a result, it was about 5 ° at all parts of the substrate. From this result, it was confirmed that no contamination due to the probe medium or the like was generated other than the spot portion. The glass substrate after spotting was left in a desiccator. In this way, a probe fixing substrate was produced.
[0048]
(6) Blocking process
The probe-immobilized substrate prepared in the above (5) was sufficiently washed with a 1M NaCl / 50 mM phosphate buffer (pH 7.0) solution to wet the probe-immobilized substrate so that a uniform blocking treatment could be performed. Next, the glass substrate was immersed in a 2% bovine serum albumin aqueous solution and allowed to stand for 2 hours for blocking treatment.
[0049]
(7) Hybridization treatment
A single-stranded DNA probe having a base sequence complementary to the single-stranded DNA probe of SEQ ID NO: 1 described in (1) above was synthesized by an automatic DNA synthesizer and labeled by binding rhodamine to the 5 ′ end. A single-stranded DNA probe was obtained. The labeled single-stranded DNA was dissolved in 1M NaCl / 50 mM phosphate buffer (pH 7.0) to a final concentration of 1 μM, and the blocking-immobilized probe-immobilized substrate obtained in (6) above was dissolved in this solution. And was subjected to a hybridization treatment at room temperature (25 ° C.) for 3 hours. Thereafter, the probe array was washed with a 1M NaCl / 50 mM phosphate buffer (pH 7.0) solution to wash away single-stranded DNA probes that did not hybridize with the probe nucleic acid. Then, after removing excess salt with pure water, the probe fixing substrate was dried by nitrogen blowing. Next, the fluorescence intensity of the spot on the probe-fixing substrate was evaluated using a fluorescence scanner (trade name: GenePix4000B; manufactured by Axon Instruments, Inc.). In the evaluation, the laser power was set to 100% and the PMT was set to 400V.
[0050]
(8) Results
When the fluorescent scanner evaluation results in (7) above were analyzed, the fluorescence intensity at 532 nm was 14830 in the spot of the DNA probe of SEQ ID NO: 1 that was a perfect match with the labeled single-stranded DNA probe. In addition, the fluorescence intensity other than the spot portion of the DNA probe was observed to be 614. When the spots of each DNA probe were observed with fluorescence, each spot shape was almost circular, and there was almost no difference in fluorescence intensity between spots spotted with the same probe medium. Further, it was observed that the distances between adjacent spots were almost uniform, and the spots were arranged in a lattice pattern at an interval of about 200 μm.
[0051]
(Example 2)
(1) Probe synthesis
A single-stranded DNA probe was prepared in exactly the same manner as in Example 1, and then used in the following experiment.
[0052]
(2) Substance having a functional group at a site capable of binding to the probe and a functional group at a site capable of binding on the substrate (probe binding substance)
Silane coupling including a silane compound having an epoxy group (γ-glycidoxypropyltrimethoxysilane) as a preparation of a substance having a functional group at a site capable of binding to a probe and a functional group at a site capable of binding on a substrate An agent (trade name: KBM-403; manufactured by Shin-Etsu Chemical Co., Ltd.) was prepared. This silane coupling agent was used in the following experiment.
[0053]
(3) Adjustment of probe medium
A single-stranded DNA probe solution and a PVA solution were prepared in exactly the same manner as in Example 1.
[0054]
10 ml of the probe solution prepared as described above was prepared, and 11 μl of the silane coupling agent was dropped onto the probe solution, and then mixed for 20 minutes. The mixed solution was allowed to stand for 30 minutes, then 5 ml of pure water was added dropwise, and a total of 80 ml of pure water was added dropwise while repeating the mixing. Next, 10 ml of the PVA aqueous solution prepared as described above was dropped and mixed for 10 minutes. After mixing, the sample was left for 30 minutes to adjust the probe medium (the exact concentration was calculated from the absorption intensity).
[0055]
(4) Substrate cleaning
Substrate cleaning was performed in exactly the same manner as in Example 1 to prepare a glass substrate.
[0056]
(5) Spotting of probe medium
The probe medium prepared in the above (3) was spotted in the same manner as in Example 1 to prepare a probe fixing base material. After the spotting, the glass substrate was observed with a microscope, and it was confirmed that a matrix spot array was formed on the glass substrate surface. The glass substrate after spotting was left in a desiccator. In this way, a probe fixing substrate was produced.
[0057]
(6) Blocking process
The blocking treatment was performed in exactly the same manner as in Example 1 above.
[0058]
(7) Hybridization treatment
Hybridization treatment was performed in exactly the same manner as in Example 1 above. Thereafter, the probe array was washed with a 1M NaCl / 50 mM phosphate buffer (pH 7.0) solution to wash away single-stranded DNA probes that did not hybridize with the probe nucleic acid. Then, after removing excess salt with pure water, the probe fixing substrate was dried by nitrogen blowing. Next, the fluorescence intensity of the spot on the probe-fixing substrate was evaluated using a fluorescence scanner (trade name: GenePix4000B; manufactured by Axon Instruments, Inc.). In the evaluation, the laser power was set to 100% and the PMT was set to 400V.
[0059]
(8) Results
When the fluorescent scanner evaluation result in the above (7) was analyzed, the fluorescence intensity at 532 nm was 23400 in the spot of the DNA probe of SEQ ID NO: 1 that was a perfect match with the labeled single-stranded DNA probe. In addition, the fluorescence intensity other than the spot portion of the DNA probe was observed to be 672. When the spots of each DNA probe were observed with fluorescence, each spot shape was almost circular, and there was almost no difference in fluorescence intensity between spots spotted with the same probe medium. Further, it was observed that the distances between adjacent spots were almost uniform, and the spots were arranged in a lattice pattern at an interval of about 200 μm.
[0060]
(Example 3)
(1) Probe synthesis
A single-stranded DNA probe was used as a probe that can specifically bind to the target substance. Single-stranded nucleic acids of SEQ ID NOs: 1 to 4 were synthesized using an automatic DNA synthesizer. Note that the single-stranded DNA probes of SEQ ID NOs: 2 to 4 are based on SEQ ID NO: 1 used in Example 1, SEQ ID NO: 2, 3 bases changed from SEQ ID NO: 3, The one obtained by changing 6 bases was designated as SEQ ID NO: 4. In addition, 18-mer oligomers in which amino groups were bonded to the 5′-terminal hydroxyl group via a phosphate group and hexamethylene were prepared at the ends of the single-stranded DNAs of SEQ ID NOS: 1-4 and used in the following experiments. The sequences of SEQ ID NOs: 2 to 4 are shown below.
SEQ ID NO: 2
5'H2N- (CH2) 6-O-PO2-O-ACTGGCCGTTGTTTTACA3 '
SEQ ID NO: 3
5 'H2N-(CH2) 6-O-PO2-O-ACTGGCCGCTTTTTTACA3'
SEQ ID NO: 4
5 'H2N-(CH2) 6-O-PO2-O-ACTGGCATCTTGTTTACA3'
[0061]
(2) Substance having a functional group at a site capable of binding to the probe and a functional group at a site capable of binding on the substrate (probe binding substance)
Silane coupling including a silane compound having an epoxy group (γ-glycidoxypropyltrimethoxysilane) as a preparation of a substance having a functional group at a site capable of binding to a probe and a functional group at a site capable of binding on a substrate An agent (trade name: KBM-403; manufactured by Shin-Etsu Chemical Co., Ltd.) was prepared. This silane coupling agent was used in the following experiment.
[0062]
(3) Adjustment of probe medium
Using the single-stranded DNAs of SEQ ID NOs: 1 to 4, four types of probe media were prepared in the same manner as described in Example 2 (3) (the exact concentration was calculated from the absorption intensity) .
[0063]
(4) Substrate cleaning
Substrate cleaning was performed in exactly the same manner as in Example 1 to prepare a glass substrate.
[0064]
(5) Spotting of probe medium
The four types of probe media prepared in (3) above were filled in each of the four ink tanks for a bubble jet (registered trademark) printer (trade name: BJF850; manufactured by Canon Inc.). The ink tank was attached to a bubble jet (registered trademark) head. In addition, the bubble jet (registered trademark) printer (trade name: BJF850; manufactured by Canon Inc.) used here is modified so that printing on a flat plate is possible. Next, the glass substrate prepared in (4) above was mounted on the printer, and each of the four types of probe media was spotted on each of four areas of 3 × 3 mm of the glass substrate. The spotting pattern in each area was the same as in Example 1. Here, the distance between the liquid ejection surface of the bubble jet (registered trademark) head and the liquid adhesion surface of the glass substrate was 1.2 to 1.5 mm. After the spotting, the glass substrate was observed with a microscope, and it was confirmed that a matrix spot array was formed on the glass substrate surface. The glass substrate after spotting was left in a desiccator. In this way, a probe fixing substrate was produced.
[0065]
(6) Blocking process
The blocking treatment was performed in exactly the same manner as in Example 1 above.
[0066]
(7) Hybridization treatment
Hybridization treatment was performed in exactly the same manner as in Example 1 above. Thereafter, the probe array was washed with a 1M NaCl / 50 mM phosphate buffer (pH 7.0) solution to wash away single-stranded DNA probes that did not hybridize with the probe nucleic acid. Then, after removing excess salt with pure water, the probe fixing substrate was dried by nitrogen blowing. Next, the fluorescence intensity of the spot on the probe-fixing substrate was evaluated using a fluorescence scanner (trade name: GenePix4000B; manufactured by Axon Instruments, Inc.). In the evaluation, the laser power was set to 100% and the PMT was set to 400V.
[0067]
(8) Results
When the fluorescent scanner evaluation result in the above (7) was analyzed, the fluorescence intensity at 532 nm was 14380 in the spot of the DNA probe of SEQ ID NO: 1 that was a perfect match with the labeled single-stranded DNA probe. On the other hand, the fluorescence amount of 8960 was obtained in the spot of the DNA probe of SEQ ID NO: 2 having a mismatch sequence of 1 base. In addition, the spot of the DNA probe of SEQ ID NO: 3 having a 3-base mismatch can only obtain a fluorescence amount less than half of 2032 and the DNA of SEQ ID NO: 4 having a 6-base mismatch can hardly recognize the fluorescent spot. The fluorescence intensity at the relevant site was 876. Further, the fluorescence intensity other than the spot portion of the DNA probe was observed to be 637. When the spots of each DNA probe were observed with fluorescence, each spot shape was almost circular, and there was almost no difference in fluorescence intensity between spots spotted with the same probe medium. Further, it was observed that the distances between adjacent spots were almost uniform, and the spots were arranged in a lattice pattern at an interval of about 200 μm. From the above, complete complementary single-stranded DNA could be specifically detected on the DNA array substrate.
[0068]
(Example 4)
(1) Probe synthesis
A single-stranded DNA probe was prepared in exactly the same manner as in Example 1, and then used in the following experiment.
[0069]
(2) Substance having a functional group at a site capable of binding to the probe and a functional group at a site capable of binding on the substrate (probe binding substance)
Silane coupling including a silane compound having an epoxy group (γ-glycidoxypropyltrimethoxysilane) as a preparation of a substance having a functional group at a site capable of binding to a probe and a functional group at a site capable of binding on a substrate An agent (trade name: KBM-403; manufactured by Shin-Etsu Chemical Co., Ltd.) was prepared. This silane coupling agent was used in the following experiment.
[0070]
(3) Adjustment of probe medium
A single-stranded DNA probe solution was prepared in exactly the same manner as in Example 1 above.
[0071]
10 ml of the probe solution prepared as described above was prepared, and 11 μl of the silane coupling agent was dropped onto the probe solution, and then mixed for 20 minutes. After the mixed solution was allowed to stand for 30 minutes, 5 ml of an aqueous solution containing 7.5 wt% glycerin, 7.5 wt% urea, and 7.5 wt% thiodiglycol was dropped, and 90 ml of pure water was dropped while repeating mixing. . Then mixed for 10 minutes. After mixing, the probe medium was prepared by leaving it for 30 minutes.
[0072]
(4) Substrate cleaning
Substrate cleaning was performed in exactly the same manner as in Example 1 to prepare a glass substrate.
[0073]
(5) Spotting of probe medium
The probe medium prepared in the above (3) was spotted in the same manner as in Example 1 to prepare a probe fixing base material. After the spotting, the glass substrate was observed with a microscope, and it was confirmed that a matrix spot array was formed on the glass substrate surface. The glass substrate after spotting was immersed in a container filled with a 1M NaCl / 50 mM phosphate buffer (pH 7.0) solution, and the whole container was put in a refrigerator and stored in a refrigerator (4 ° C.). In this way, a probe fixing substrate was produced.
[0074]
(6) Blocking process
The probe-immobilized substrate prepared in the above (5) was taken out from the 1M NaCl / 50 mM phosphate buffer (pH 7.0) solution. Next, the glass substrate was immersed in a container filled with 2% bovine serum albumin aqueous solution and allowed to stand for 2 hours for blocking treatment.
[0075]
(7) Hybridization treatment
Hybridization treatment was performed in exactly the same manner as in Example 1 above. Thereafter, the probe array was washed with a 1M NaCl / 50 mM phosphate buffer (pH 7.0) solution to wash away single-stranded DNA probes that did not hybridize with the probe nucleic acid. Then, after removing excess salt with pure water, the probe fixing substrate was dried by nitrogen blowing. Next, the fluorescence intensity of the spot on the probe-fixing substrate was evaluated using a fluorescence scanner (trade name: GenePix4000B; manufactured by Axon Instruments, Inc.). In the evaluation, the laser power was set to 100% and the PMT was set to 400V.
[0076]
(8) Results
When the fluorescent scanner evaluation result in the above (7) was analyzed, the fluorescence intensity at 532 nm was 23400 in the spot of the DNA probe of SEQ ID NO: 1 that was a perfect match with the labeled single-stranded DNA probe. Further, the fluorescence intensity other than the spot portion of the DNA probe was observed to be 628. When the spots of each DNA probe were observed with fluorescence, each spot shape was almost circular, and there was almost no difference in fluorescence intensity between spots spotted with the same probe medium. Further, it was observed that the distances between adjacent spots were almost uniform, and the spots were arranged in a lattice pattern at an interval of about 200 μm.
[0077]
(Example 5)
(1) Probe synthesis
A single-stranded DNA probe was prepared in exactly the same manner as in Example 1, and then used in the following experiment.
[0078]
(2) Substance having a functional group at a site capable of binding to the probe and a functional group at a site capable of binding on the substrate (probe binding substance)
Silane coupling agent comprising a silane compound having a chloropropyl group (γ-chloropropyltrimethoxysilane) as a preparation of a substance having a functional group at a site capable of binding to a probe and a functional group at a site capable of binding on a substrate After adjusting the pH of a 500 μmol / l aqueous solution (trade name: KBM-703; manufactured by Shin-Etsu Chemical Co., Ltd.) to pH 4.0, the mixture was stirred at room temperature for 30 minutes to prepare a probe fixing substance solution. did. The prepared probe fixing substance solution was used in the following experiment.
[0079]
(3) Adjustment of probe medium
A single-stranded DNA probe solution was prepared in exactly the same manner as in Example 1 above.
[0080]
100 μl of the probe solution prepared as described above was prepared, and 100 μl of the probe fixing substance solution prepared as described above was added dropwise thereto, and then mixed for 5 minutes. After the mixed solution was allowed to stand for 10 minutes, 800 μl of pure water was dropped and mixed for 5 minutes. After mixing, the probe medium was prepared by leaving it for 30 minutes.
[0081]
(4) Substrate cleaning
Substrate cleaning was performed in exactly the same manner as in Example 1 to prepare a glass substrate.
[0082]
(5) Spotting of probe medium
The probe medium prepared in the above (3) was spotted in the same manner as in Example 1 to prepare a probe fixing base material. After the spotting, the glass substrate was observed with a microscope, and it was confirmed that a matrix spot array was formed on the glass substrate surface. The glass substrate after spotting was immersed in a container filled with a 1M NaCl / 50 mM phosphate buffer (pH 7.0) solution, and the whole container was put in a refrigerator and stored in a refrigerator (4 ° C.). In this way, a probe fixing substrate was produced.
[0083]
(6) Blocking process
The blocking treatment was performed in exactly the same manner as in Example 4 above.
[0084]
(7) Hybridization treatment
Hybridization treatment was performed in exactly the same manner as in Example 1 above. Thereafter, the probe array was washed with a 1M NaCl / 50 mM phosphate buffer (pH 7.0) solution to wash away single-stranded DNA probes that did not hybridize with the probe nucleic acid. Then, after removing excess salt with pure water, the probe fixing substrate was dried by nitrogen blowing. Next, the fluorescence intensity of the spot on the probe-fixing substrate was evaluated using a fluorescence scanner (trade name: GenePix4000B; manufactured by Axon Instruments, Inc.). In the evaluation, the laser power was set to 100% and the PMT was set to 400V.
[0085]
(8) Results
When the fluorescent scanner evaluation result in the above (7) was analyzed, the fluorescence intensity at 532 nm was 11320 in the spot of the DNA probe of SEQ ID NO: 1 that was a perfect match with the labeled single-stranded DNA probe. In addition, the intensity of fluorescence other than the spot portion of the DNA probe was observed to be 641. When the spots of each DNA probe were observed with fluorescence, each spot shape was almost circular, and there was almost no difference in fluorescence intensity between spots spotted with the same probe medium. Further, it was observed that the distances between adjacent spots were almost uniform, and the spots were arranged in a lattice pattern at an interval of about 200 μm.
[0086]
(Example 6)
(1) Probe synthesis
A single-stranded DNA probe was prepared in exactly the same manner as in Example 1, and then used in the following experiment.
[0087]
(2) Substance having a functional group at a site capable of binding to the probe and a functional group at a site capable of binding on the substrate (probe binding substance)
Silane coupling agent comprising a silane compound having a chloropropyl group (γ-chloropropyltrimethoxysilane) as a preparation of a substance having a functional group at a site capable of binding to a probe and a functional group at a site capable of binding on a substrate (Trade name: KBM-703; manufactured by Shin-Etsu Chemical Co., Ltd.) was prepared. This silane coupling agent was used in the following experiment.
[0088]
(3) Adjustment of probe medium
A single-stranded DNA probe solution and a PVA solution were prepared in exactly the same manner as in Example 1.
[0089]
10 ml of the probe solution prepared as described above was prepared, and 10 μl of the above silane coupling agent was added dropwise thereto, and then mixed for 20 minutes. The mixed solution was allowed to stand for 30 minutes, then 5 ml of pure water was added dropwise, and a total of 80 ml of pure water was added dropwise while repeating the mixing. Next, 10 ml of the PVA aqueous solution prepared as described above was dropped and mixed for 10 minutes. After mixing, the probe medium was prepared by leaving it for 30 minutes.
[0090]
(4) Substrate cleaning
Substrate cleaning was performed in exactly the same manner as in Example 1 to prepare a glass substrate.
[0091]
(5) Spotting of probe medium
The probe medium prepared in the above (3) was spotted in the same manner as in Example 1 to prepare a probe fixing base material. After the spotting, the glass substrate was observed with a microscope, and it was confirmed that a matrix spot array was formed on the glass substrate surface. The glass substrate after spotting was left in a desiccator. In this way, a probe fixing substrate was produced.
[0092]
(6) Blocking process
The blocking treatment was performed in exactly the same manner as in Example 1 above.
[0093]
(7) Hybridization treatment
Hybridization treatment was performed in exactly the same manner as in Example 1 above. Thereafter, the probe array was washed with a 1M NaCl / 50 mM phosphate buffer (pH 7.0) solution to wash away single-stranded DNA probes that did not hybridize with the probe nucleic acid. Then, after removing excess salt with pure water, the probe fixing substrate was dried by nitrogen blowing. Next, the fluorescence intensity of the spot on the probe-fixing substrate was evaluated using a fluorescence scanner (trade name: GenePix4000B; manufactured by Axon Instruments, Inc.). In the evaluation, the laser power was set to 100% and the PMT was set to 400V.
[0094]
(8) Results
When the fluorescent scanner evaluation results in (7) above were analyzed, the fluorescence intensity at 532 nm was 15790 at the spot of the DNA probe of SEQ ID NO: 1 that was a perfect match with the labeled single-stranded DNA probe. Further, when the fluorescence intensity other than the spot portion of the DNA probe was observed, it was 608. When the spots of each DNA probe were observed with fluorescence, each spot shape was almost circular, and there was almost no difference in fluorescence intensity between spots spotted with the same probe medium. Further, it was observed that the distances between adjacent spots were almost uniform, and the spots were arranged in a lattice pattern at an interval of about 200 μm.
[0095]
(Example 7)
(1) Probe synthesis
A single-stranded DNA probe of SEQ ID NO: 1 was prepared in the same manner as in Example 1, and then used for the following experiment.
[0096]
(2) Substance having a functional group at a site capable of binding to the probe and a functional group at a site capable of binding on the substrate (probe binding substance)
In the same manner as in Example 1, a substance having a functional group at a site capable of binding to the probe and a functional group at a site capable of binding on the substrate was prepared and used in the following experiment.
[0097]
(3) Adjustment of probe medium
The probe medium was prepared in exactly the same manner as in Example 1 above.
[0098]
(4) Substrate cleaning
Substrate cleaning was performed in exactly the same manner as in Example 1 to prepare a glass substrate.
[0099]
(5) Spotting of probe medium
A probe-fixing substrate was produced in exactly the same manner as in Example 1 above.
[0100]
(6) Blocking process
Without performing the blocking treatment performed in Example 1 above, it was washed with a 1M NaCl / 50 mM phosphate buffer (pH 7.0) solution and wetted on the probe-immobilized substrate so that a uniform hybridization treatment could be performed. .
[0101]
(7) Hybridization treatment
Hybridization treatment was performed in exactly the same manner as in Example 1 above. Thereafter, the probe array was washed with a 1M NaCl / 50 mM phosphate buffer (pH 7.0) solution to wash away single-stranded DNA probes that did not hybridize with the probe nucleic acid. Then, after removing excess salt with pure water, the probe fixing substrate was dried by nitrogen blowing. Next, the fluorescence intensity of the spot on the probe-fixing substrate was evaluated using a fluorescence scanner (trade name: GenePix4000B; manufactured by Axon Instruments, Inc.). In the evaluation, the laser power was set to 100% and the PMT was set to 400V.
[0102]
(8) Results
When the fluorescent scanner evaluation result in the above (7) was analyzed, the fluorescence intensity at 532 nm was 23400 in the spot of the DNA probe of SEQ ID NO: 1 that was a perfect match with the labeled single-stranded DNA probe. In addition, the fluorescence intensity other than the spot portion of the DNA probe was observed to be 658. When the spots of each DNA probe were observed with fluorescence, each spot shape was almost circular, and there was almost no difference in fluorescence intensity between spots spotted with the same probe medium. Almost no increase in fluorescence intensity other than the spot portion due to the absence of the blocking treatment was observed. Further, it was observed that the distances between adjacent spots were almost uniform, and the spots were arranged in a lattice pattern at an interval of about 200 μm.
[0103]
(Example 8)
(1) Probe synthesis
A single-stranded DNA probe was used as a probe that can specifically bind to the target substance. A single-stranded nucleic acid of SEQ ID NO: 5 was synthesized using an automatic DNA synthesizer. A mercapto (SH) group was introduced into the single-stranded DNA end of SEQ ID NO: 5 by using a thiol modifier (manufactured by Glen Research) during synthesis with an automatic DNA synthesizer. Subsequently, normal deprotection was performed, the DNA was recovered, purified by high performance liquid chromatography, and used in the following experiments.
SEQ ID NO: 5
5'HS- (CH2) 6-O-PO2-O-ACTGGCCGTCGTTTTACA3 '
[0104]
(2) Substance having a functional group at a site capable of binding to the probe (probe binding substance) Bivalent reagent N- having a maleimide group and a hydroxysuccinimide ester group as a substance having a functional group at the site capable of binding to the probe A probe binding substance solution was prepared by stirring a 500 μmol / l aqueous solution of (6-Maleimidocaproyloxy) sulfocuccinimide, sodium salt (trade name: sulfo-EMCS; manufactured by Dojindo Laboratories) for 5 minutes at room temperature. The prepared probe binding substance solution was used in the following experiment.
[0105]
(3) Substance having a functional group at a site capable of binding on the substrate (substrate binding substance)
Silane coupling agent (trade name: KBM) containing a silane compound having an amino group (N-β (aminoethyl) γ-aminopropyltrimethoxysilane) as a preparation of a substance having a functional group at a site capable of binding on a substrate -603; manufactured by Shin-Etsu Chemical Co., Ltd.) was stirred at room temperature for 30 minutes to prepare a substrate-binding substance solution. The prepared substrate binder material solution was used for the following experiments.
[0106]
(4) Adjustment of probe medium
The single-stranded DNA probe of SEQ ID NO: 5 is dissolved in a TE solution (10 mM Tris-HCl (pH 8) / 1 mM ethylenediaminetetraacetic acid (EDTA) aqueous solution) so as to have a final concentration of about 40 μmol / l. A probe solution was prepared (the exact concentration was calculated from the absorption intensity).
[0107]
100 μl of the probe solution prepared as described above was prepared, and 100 μl of the probe binding substance solution prepared in (2) above was added dropwise thereto, followed by mixing for 5 minutes. Next, 100 μl of the substrate binding substance solution prepared in the above (3) was dropped and mixed for 5 minutes. After the mixed solution was allowed to stand for 10 minutes, 700 μl of an aqueous solution containing 7.5 wt% glycerin, 7.5 wt% urea, and 7.5 wt% thiodiglycol was dropped and mixed for 5 minutes. After mixing, the probe medium was prepared by leaving it for 30 minutes.
[0108]
(5) Substrate cleaning
A 1 inch × 3 inch glass substrate (thickness: 1.1 mm) was put in a cassette, and the glass substrate was immersed in a 1 mol / l sodium hydroxide aqueous solution preheated to 60 ° C. for 10 minutes. Subsequently, it was sufficiently rinsed in running pure water, and sodium hydroxide adhering to the glass substrate and the cassette was washed and removed. After sufficiently rinsing, the glass substrate together with the cassette was immersed in pure water and subjected to ultrasonic cleaning for 10 minutes. After ultrasonic cleaning, the sample was sufficiently rinsed in pure water, and the particles adhering to the glass substrate and the cassette were washed and removed. The glass substrates after washing with water were dried one by one by blowing nitrogen gas. In order to confirm the cleaning of the substrate, the pure water contact angle on the substrate was measured. As a result, it was about 5 ° at all parts of the substrate.
[0109]
(6) Spotting of probe medium
The probe medium adjusted in the above (4) was filled in an ink tank for a bubble jet (registered trademark) printer (trade name: BJF850; manufactured by Canon Inc.) and mounted on a bubble jet (registered trademark) head. In addition, the bubble jet (registered trademark) printer (trade name: BJF850; manufactured by Canon Inc.) used here is modified so that printing on a flat plate is possible. Next, the glass substrate prepared in (5) above was mounted on the printer, and the probe medium was spotted on the glass substrate. Here, the distance between the liquid ejection surface of the bubble jet (registered trademark) head and the liquid adhesion surface of the glass substrate was 1.2 to 1.5 mm. After the spotting, the glass substrate was observed with a microscope, and it was confirmed that a matrix spot array was formed on the glass substrate surface. In addition, the pure water contact angle was measured on the outer periphery of the spot formation site. As a result, it was about 5 ° at all parts of the substrate. From this result, it was confirmed that no contamination due to the probe medium or the like was generated other than the spot portion. The glass substrate after spotting was immersed in a container filled with a 1M NaCl / 50 mM phosphate buffer (pH 7.0) solution, and the whole container was put in a refrigerator and stored in a refrigerator (4 ° C.). In this way, a probe fixing substrate was produced.
[0110]
(7) Blocking process
The probe-immobilized substrate prepared in the above (6) was taken out from a 1M NaCl / 50 mM phosphate buffer (pH 7.0) solution. Next, the glass substrate was immersed in a container filled with 2% bovine serum albumin aqueous solution and allowed to stand for 2 hours for blocking treatment.
[0111]
(8) Hybridization treatment
A single-stranded DNA probe having a base sequence complementary to the single-stranded DNA probe of SEQ ID NO: 5 described in (1) above was synthesized by an automatic DNA synthesizer and labeled by binding rhodamine to the 5 ′ end. A single-stranded DNA probe was obtained. This labeled single-stranded DNA was dissolved in 1M NaCl / 50 mM phosphate buffer (pH 7.0) to a final concentration of 1 μM, and the blocking-immobilized probe-immobilized substrate obtained in (7) above was dissolved in this solution. And was subjected to a hybridization treatment at room temperature (55 ° C.) for 2 hours. Thereafter, the probe array was washed with a 1M NaCl / 50 mM phosphate buffer (pH 7.0) solution to wash away single-stranded DNA probes that did not hybridize with the probe nucleic acid. Then, after removing excess salt with pure water, the probe fixing substrate was dried by nitrogen blowing. Next, the fluorescence intensity of the spot on the probe-fixing substrate was evaluated using a fluorescence scanner (trade name: GenePix4000B; manufactured by Axon Instruments, Inc.). In the evaluation, the laser power was set to 100% and the PMT was set to 400V.
[0112]
(9) Results
When the fluorescent scanner evaluation result in the above (8) was analyzed, the fluorescence intensity at 532 nm was 25730 in the spot of the DNA probe of SEQ ID NO: 5 that was a perfect match with the labeled single-stranded DNA probe. Further, the fluorescence intensity other than the spot portion of the DNA probe was observed to be 639. When the spots of each DNA probe were observed with fluorescence, each spot shape was almost circular, and there was almost no difference in fluorescence intensity between spots spotted with the same probe medium. Further, it was observed that the distances between adjacent spots were almost uniform, and the spots were arranged in a lattice pattern at an interval of about 200 μm.
[0113]
Example 9
(1) Probe synthesis
A single-stranded DNA probe of SEQ ID NO: 5 was prepared in the same manner as in Example 8, and used for the following experiment.
[0114]
(2) Substance having a functional group at a site capable of binding to a probe (probe binding substance) Bivalent reagent N- (6-Maleimidocaproxy) having a maleimide group as an adjustment of a substance having a functional group at a site capable of binding to a probe sulfosuccinimide, sodium salt (trade name: sulfo-EMCS; manufactured by Dojindo Laboratories Co., Ltd.) was prepared. This probe binding substance was used in the following experiment.
[0115]
(3) Substance having a functional group at a site capable of binding on the substrate (substrate binding substance)
A substrate binding substance solution was prepared in exactly the same manner as in Example 8 above. The prepared substrate binder material solution was used for the following experiments.
[0116]
(4) Adjustment of probe medium
A single-stranded DNA probe solution was prepared in exactly the same manner as in Example 8.
[0117]
10 ml of the probe solution prepared as described above was prepared, and about 2.1 mg of the probe binding substance weighed above was added thereto, followed by mixing for 5 minutes. The mixed solution was allowed to stand for 30 minutes, and then 10 ml of pure water was dropped into the probe / probe binding substance mixed medium every 10 ml, and a total of 80 ml of pure water was dropped while repeating the mixing. Next, 10 ml of the substrate binding substance solution prepared in (3) above was dropped and mixed for 5 minutes. After mixing, the probe medium was prepared by leaving it for 30 minutes.
[0118]
(5) Substrate cleaning
Substrate cleaning was performed in exactly the same manner as in Example 8 to prepare a glass substrate.
[0119]
(6) Spotting of probe medium
The probe medium prepared in the above (4) was spotted in the same manner as in Example 8 to produce a probe fixing base material. After the spotting, the glass substrate was observed with a microscope, and it was confirmed that a matrix spot array was formed on the glass substrate surface. The glass substrate after spotting was immersed in a container filled with a 1M NaCl / 50 mM phosphate buffer (pH 7.0) solution, and the whole container was put in a refrigerator and stored in a refrigerator (4 ° C.). In this way, a probe fixing substrate was produced.
[0120]
(7) Blocking process
The blocking treatment was performed in exactly the same manner as in Example 8 above.
[0121]
(8) Hybridization treatment
Hybridization treatment was performed in exactly the same manner as in Example 8 above. Thereafter, the probe array was washed with a 1M NaCl / 50 mM phosphate buffer (pH 7.0) solution to wash away single-stranded DNA probes that did not hybridize with the probe nucleic acid. Then, after removing excess salt with pure water, the probe fixing substrate was dried by nitrogen blowing. Next, the fluorescence intensity of the spot on the probe-fixing substrate was evaluated using a fluorescence scanner (trade name: GenePix4000B; manufactured by Axon Instruments, Inc.). In the evaluation, the laser power was set to 100% and the PMT was set to 400V.
[0122]
(9) Results
When the fluorescent scanner evaluation result in the above (8) was analyzed, the fluorescence intensity at 532 nm was 25100 in the spot of the DNA probe of SEQ ID NO: 5 that was a perfect match with the labeled single-stranded DNA probe. In addition, the intensity of fluorescence other than the spot portion of the DNA probe was observed to be 769. When the spots of each DNA probe were observed with fluorescence, each spot shape was almost circular, and there was almost no difference in fluorescence intensity between spots spotted with the same probe medium. Further, it was observed that the distances between adjacent spots were almost uniform, and the spots were arranged in a lattice pattern at an interval of about 200 μm.
[0123]
(Example 10)
(1) Probe synthesis
A single-stranded DNA probe was used as a probe that can specifically bind to the target substance. Single-stranded nucleic acids of SEQ ID NOs: 5 to 8 were synthesized using an automatic DNA synthesizer. The single-stranded DNA probes of SEQ ID NOs: 6 to 8 are based on SEQ ID NO: 5 used in Example 8, SEQ ID NO: 6 is changed by 1 base, SEQ ID NO: 7 is changed by 3 bases, and The one obtained by changing 6 bases was designated as SEQ ID NO: 8. In addition, a thiol (SH) group was introduced into the single-stranded DNA ends of SEQ ID NOs: 5 to 8 by using Thiol-Modifier (manufactured by Glen Research) during synthesis with an automatic DNA synthesizer. Subsequently, normal deprotection was performed, and the DNA was collected and purified by high performance liquid chromatography, and used in the following experiments. The sequences of SEQ ID NOs: 6-8 are shown below.
SEQ ID NO: 6
5'HS- (CH2) 6-O-PO2-O-ACTGGCCGTTGTTTTACA3 '
SEQ ID NO: 7
5'HS- (CH2) 6-O-PO2-O-ACTGGCCGCTTTTTTACA3 '
SEQ ID NO: 8
5'HS- (CH2) 6-O-PO2-O-ACTGGCATCTTGTTTACA3 '
[0124]
(2) Substance having a functional group at a site capable of binding to the probe (probe binding substance) A probe binding substance solution was prepared in exactly the same manner as in Example 8. The prepared probe binding substance solution was used in the following experiment.
[0125]
(3) Substance having a functional group at a site capable of binding on the substrate (substrate binding substance)
A substrate binding substance solution was prepared in exactly the same manner as in Example 8 above. The prepared substrate binder material solution was used for the following experiments.
[0126]
(4) Adjustment of probe medium
A single-stranded DNA probe solution was prepared in exactly the same manner as in Example 8.
[0127]
Using the single-stranded DNAs of SEQ ID NOs: 5 to 8, four types of probe media were prepared by the same method as that described in Example 8 (4) (the exact concentration was calculated from the absorption intensity). .
[0128]
(5) Substrate cleaning
Substrate cleaning was performed in exactly the same manner as in Example 8 to prepare a glass substrate.
[0129]
(6) Spotting of probe medium
The four types of probe media prepared in the above (4) are filled in each of the four ink tanks for a bubble jet (registered trademark) printer (trade name: BJF850; manufactured by Canon Inc.). The ink tank was attached to a bubble jet (registered trademark) head. In addition, the bubble jet (registered trademark) printer (trade name: BJF850; manufactured by Canon Inc.) used here is modified so that printing on a flat plate is possible. Next, the glass substrate prepared in (5) above was mounted on this printer, and each of the four types of probe media was spotted on each of four areas of 3 × 3 mm of the glass substrate. The spotting pattern in each area was the same as in Example 8. Here, the distance between the liquid ejection surface of the bubble jet (registered trademark) head and the liquid adhesion surface of the glass substrate was 1.2 to 1.5 mm. After the spotting, the glass substrate was observed with a microscope, and it was confirmed that a matrix spot array was formed on the glass substrate surface. The glass substrate after spotting was immersed in a container filled with a 1M NaCl / 50 mM phosphate buffer (pH 7.0) solution, and the whole container was put in a refrigerator and stored in a refrigerator (4 ° C.). In this manner, a probe fixing substrate on which four types of probes were fixed was produced.
[0130]
(7) Blocking process
The blocking treatment was performed in exactly the same manner as in Example 8 above.
[0131]
(8) Hybridization treatment
A single-stranded DNA probe having a base sequence complementary to the single-stranded DNA probe of SEQ ID NO: 5 described in Example 8 above was synthesized by an automatic DNA synthesizer, and labeled by binding rhodamine to the 5 ′ end. A single-stranded DNA probe was obtained. This labeled single-stranded DNA was dissolved in 1M NaCl / 50 mM phosphate buffer (pH 7.0) to a final concentration of 1 μM, and the blocking-immobilized probe-immobilized substrate obtained in (7) above was dissolved in this solution. And was subjected to a hybridization treatment at room temperature (55 ° C.) for 2 hours. Thereafter, the probe array was washed with a 1M NaCl / 50 mM phosphate buffer (pH 7.0) solution to wash away single-stranded DNA probes that did not hybridize with the probe nucleic acid. Then, after removing excess salt with pure water, the probe fixing substrate was dried by nitrogen blowing. Next, the fluorescence intensity of the spot on the probe-fixing substrate was evaluated using a fluorescence scanner (trade name: GenePix4000B; manufactured by Axon Instruments, Inc.). In the evaluation, the laser power was set to 100% and the PMT was set to 400V.
[0132]
(9) Results
When the fluorescent scanner evaluation result in the above (8) was analyzed, the fluorescence intensity at 532 nm was 26120 in the spot of the DNA probe of SEQ ID NO: 5 that was a perfect match with the labeled single-stranded DNA probe. On the other hand, the fluorescence amount of 12870 was obtained in the spot of the DNA probe of SEQ ID NO: 6 having a mismatch sequence of 1 base. In addition, the spot of the DNA probe of SEQ ID NO: 7 having a 3 base mismatch can only obtain a fluorescence amount less than half of the perfect match of 4220, and the DNA of SEQ ID NO: 8 having a 6 base mismatch hardly recognizes the fluorescent spot. The fluorescence intensity at the corresponding site was 791. Further, the intensity of fluorescence other than the spot portion of the DNA probe was observed and found to be 675. When the spots of each DNA probe were observed with fluorescence, each spot shape was almost circular, and there was almost no difference in fluorescence intensity between spots spotted with the same probe medium. Further, it was observed that the distances between adjacent spots were almost uniform, and the spots were arranged in a lattice pattern at an interval of about 200 μm. From the above, complete complementary single-stranded DNA could be specifically detected on the DNA array substrate.
[0133]
(Example 11)
(1) Probe synthesis
A single-stranded DNA probe of SEQ ID NO: 5 was prepared in the same manner as in Example 8, and used for the following experiment.
[0134]
(2) Substance having a functional group at a site capable of binding to the probe (probe binding substance) A probe binding substance solution was prepared in exactly the same manner as in Example 8. The prepared probe binding substance solution was used in the following experiment.
[0135]
(3) Substance having a functional group at a site capable of binding on the substrate (substrate binding substance)
A substrate binding substance solution was prepared in exactly the same manner as in Example 8 above. The prepared substrate binder material solution was used for the following experiments.
[0136]
(4) Adjustment of probe medium
The probe medium was prepared in exactly the same manner as in Example 8 above.
[0137]
(5) Substrate cleaning
Substrate cleaning was performed in exactly the same manner as in Example 8 to prepare a glass substrate.
[0138]
(6) Spotting of probe medium
A probe-fixing substrate was produced in exactly the same manner as in Example 8 above.
[0139]
(7) Blocking process
Without performing the blocking treatment performed in Example 8 above, the substrate was washed with a 1M NaCl / 50 mM phosphate buffer (pH 7.0) solution so that a uniform hybridization treatment could be performed.
[0140]
(8) Hybridization treatment
Hybridization treatment was performed in exactly the same manner as in Example 8 above. Thereafter, the probe array was washed with a 1M NaCl / 50 mM phosphate buffer (pH 7.0) solution to wash away single-stranded DNA probes that did not hybridize with the probe nucleic acid. Then, after removing excess salt with pure water, the probe fixing substrate was dried by nitrogen blowing. Next, the fluorescence intensity of the spot on the probe-fixing substrate was evaluated using a fluorescence scanner (trade name: GenePix4000B; manufactured by Axon Instruments, Inc.). In the evaluation, the laser power was set to 100% and the PMT was set to 400V.
[0141]
(9) Results
When the fluorescent scanner evaluation result in the above (8) was analyzed, the fluorescence intensity at 532 nm was 24380 in the spot of the DNA probe of SEQ ID NO: 5 that was a perfect match with the labeled single-stranded DNA probe. In addition, the fluorescence intensity other than the spot portion of the DNA probe was observed to be 783. When the spots of each DNA probe were observed with fluorescence, each spot shape was almost circular, and there was almost no difference in fluorescence intensity between spots spotted with the same probe medium. Almost no increase in fluorescence intensity other than the spot portion due to the absence of the blocking treatment was observed. Further, it was observed that the distances between adjacent spots were almost uniform, and the spots were arranged in a lattice pattern at an interval of about 200 μm.
[0142]
Example 12
(1) Probe synthesis
A single-stranded DNA probe was used as a probe that can specifically bind to the target substance. A single-stranded DNA probe of SEQ ID NO: 5 was synthesized using an automatic DNA synthesizer. An 18-mer oligomer in which an amino group was bonded to the 5′-terminal hydroxyl group via a phosphate group and hexamethylene was prepared at the end of the single-stranded DNA of SEQ ID NO: 9 and used in the following experiments.
SEQ ID NO: 9
5'H2N- (CH2) 6-O-PO2-O-ACTGGCCGTCGTTTTACA3 '
[0143]
(2) Substance having a functional group at a site capable of binding to a probe (probe binding substance) Bivalent reagent N- (8-Maleimidocaproxy) having a maleimide group as an adjustment of a substance having a functional group at a site capable of binding to a probe A probe binding substance solution was prepared by stirring 500 μmol / l aqueous solution of sulfosuccinimide, sodium salt (trade name: sulfo-HMCS; manufactured by Dojindo Laboratories Co., Ltd.) at room temperature for 5 minutes. The prepared probe binding substance solution was used in the following experiment.
[0144]
(3) Substance having a functional group at a site capable of binding on the substrate (substrate binding substance)
Silane coupling agent (trade name: KBM-802; Shin-Etsu Chemical Co., Ltd.) containing a silane compound having a mercapto group (γ-mercaptopropylmethyldimethoxysilane) as a preparation of a substance having a functional group at a site capable of binding on a substrate 20 mmol / l aqueous solution manufactured by Kogyo Co., Ltd. was stirred at room temperature for 30 minutes to prepare a substrate-binding substance solution. The prepared substrate binder material solution was used for the following experiments.
[0145]
(4) Adjustment of probe medium
The single-stranded DNA probe of SEQ ID NO: 9 is dissolved in a TE solution (10 mM Tris-HCl (pH 8) / 1 mM ethylenediaminetetraacetic acid (EDTA) aqueous solution) so as to have a final concentration of about 40 μmol / l. A probe solution was prepared (the exact concentration was calculated from the absorption intensity).
[0146]
Next, polyvinyl alcohol (PVA), which is a water-soluble polymer material, was dissolved in pure water to a concentration of 0.5 wt%. For complete dissolution, the mixture was stirred for 60 minutes while warming to 80 ° C. in a hot bath. After confirming that there was no insoluble matter, filtration was performed so as to prevent nozzle clogging during spotting, and an aqueous PVA solution was prepared.
[0147]
100 μl of the probe solution prepared as described above was prepared, and 100 μl of the probe binding substance solution prepared in (2) above was added dropwise thereto, followed by mixing for 5 minutes. Next, 100 μl of the substrate binding substance solution prepared in the above (3) was dropped and mixed for 5 minutes. After the mixed solution was allowed to stand for 10 minutes, 600 μl of pure water and 100 μl of the PVA aqueous solution prepared as described above were dropped and mixed for 5 minutes. After mixing, the probe medium was prepared by leaving it for 30 minutes.
[0148]
(5) Substrate cleaning
Substrate cleaning was performed in exactly the same manner as in Example 8 to prepare a glass substrate.
[0149]
(6) Spotting of probe medium
The probe medium adjusted in the above (3) was filled in an ink tank for a bubble jet (registered trademark) printer (trade name: BJF850; manufactured by Canon Inc.) and mounted on a bubble jet (registered trademark) head. In addition, the bubble jet (registered trademark) printer (trade name: BJF850; manufactured by Canon Inc.) used here is modified so that printing on a flat plate is possible. Next, the glass substrate prepared in (4) above was mounted on the printer, and the probe medium was spotted on the glass substrate. Here, the distance between the liquid ejection surface of the bubble jet (registered trademark) head and the liquid adhesion surface of the glass substrate was 1.2 to 1.5 mm. After the spotting, the glass substrate was observed with a microscope, and it was confirmed that a matrix spot array was formed on the glass substrate surface. In addition, the pure water contact angle was measured on the outer periphery of the spot formation site. As a result, it was about 5 ° at all parts of the substrate. From this result, it was confirmed that no contamination due to the probe medium or the like was generated other than the spot portion. The glass substrate after spotting was left in a desiccator. In this way, a probe fixing substrate was produced.
[0150]
(7) Blocking process
The probe-immobilized substrate prepared in the above (5) was sufficiently washed with a 1M NaCl / 50 mM phosphate buffer (pH 7.0) solution to wet the probe-immobilized substrate so that a uniform blocking treatment could be performed. Next, the glass substrate was immersed in a 2% bovine serum albumin aqueous solution and allowed to stand for 2 hours for blocking treatment.
[0151]
(8) Hybridization treatment
A single-stranded DNA probe synthesized with a single-stranded DNA probe having a base sequence complementary to the single-stranded DNA probe of SEQ ID NO: 9 described above by an automatic DNA synthesizer and labeled with rhodamine bound to the 5 ′ end A DNA probe was obtained. This labeled single-stranded DNA was dissolved in 1M NaCl / 50 mM phosphate buffer (pH 7.0) to a final concentration of 1 μM, and the blocking-immobilized probe-immobilized substrate obtained in (7) above was dissolved in this solution. And was subjected to a hybridization treatment at room temperature (55 ° C.) for 2 hours. Thereafter, the probe array was washed with a 1M NaCl / 50 mM phosphate buffer (pH 7.0) solution to wash away single-stranded DNA probes that did not hybridize with the probe nucleic acid. Then, after removing excess salt with pure water, the probe fixing substrate was dried by nitrogen blowing. Next, the fluorescence intensity of the spot on the probe-fixing substrate was evaluated using a fluorescence scanner (trade name: GenePix4000B; manufactured by Axon Instruments, Inc.). In the evaluation, the laser power was set to 100% and the PMT was set to 400V.
[0152]
(9) Results
When the fluorescent scanner evaluation result in the above (8) was analyzed, the fluorescence intensity at 532 nm was 18710 in the spot of the DNA probe of SEQ ID NO: 5 that was a perfect match with the labeled single-stranded DNA probe. In addition, the fluorescence intensity other than the spot portion of the DNA probe was observed to be 664. When the spots of each DNA probe were observed with fluorescence, each spot shape was almost circular, and there was almost no difference in fluorescence intensity between spots spotted with the same probe medium. Further, it was observed that the distances between adjacent spots were almost uniform, and the spots were arranged in a lattice pattern at an interval of about 200 μm.
[0153]
(Example 13)
(1) Probe synthesis
A single-stranded DNA probe of SEQ ID NO: 5 was prepared in the same manner as in Example 12, and then used for the following experiment.
[0154]
(2) Substance having a functional group at a site capable of binding to the probe (probe binding substance) A probe binding substance solution was prepared in exactly the same manner as in Example 12 above. The prepared probe binding substance solution was used in the following experiment.
[0155]
(3) Substance having a functional group at a site capable of binding on the substrate (substrate binding substance)
A substrate binding substance solution was prepared in exactly the same manner as in Example 12 above. The prepared substrate binder material solution was used for the following experiments.
[0156]
(4) Adjustment of probe medium
A single-stranded DNA probe solution and a PVA solution were prepared in exactly the same manner as in Example 12 above.
[0157]
10 ml of the probe solution prepared as described above was prepared, and about 2.1 mg of the probe binding substance weighed above was added thereto, followed by mixing for 5 minutes. After the mixed solution was allowed to stand for 30 minutes, 10 ml of pure water was dropped into the probe / probe binding substance mixed medium in 10 ml portions, and a total of 70 ml of pure water was dropped while repeating the mixing. Next, 10 ml of the substrate binding substance solution prepared in (3) above was dropped and mixed for 5 minutes. After mixing, the mixture was allowed to stand for 30 minutes, and 10 ml of the PVA aqueous solution prepared as described above was added dropwise and mixed for 5 minutes. After the mixing was completed, the probe medium was adjusted by leaving it for 30 minutes.
[0158]
(5) Substrate cleaning
Substrate cleaning was performed in exactly the same manner as in Example 8 to prepare a glass substrate.
[0159]
(6) Spotting of probe medium
The probe medium prepared in the above (4) was spotted in the same manner as in Example 8 to produce a probe fixing base material. After the spotting, the glass substrate was observed with a microscope, and it was confirmed that a matrix spot array was formed on the glass substrate surface. The glass substrate after spotting was left in a desiccator. In this way, a probe fixing substrate was produced.
[0160]
(7) Blocking process
The blocking treatment was performed in exactly the same manner as in Example 12 above.
[0161]
(8) Hybridization treatment
Hybridization treatment was performed in exactly the same manner as in Example 12 above. Thereafter, the probe array was washed with a 1M NaCl / 50 mM phosphate buffer (pH 7.0) solution to wash away single-stranded DNA probes that did not hybridize with the probe nucleic acid. Then, after removing excess salt with pure water, the probe fixing substrate was dried by nitrogen blowing. Next, the fluorescence intensity of the spot on the probe-fixing substrate was evaluated using a fluorescence scanner (trade name: GenePix4000B; manufactured by Axon Instruments, Inc.). In the evaluation, the laser power was set to 100% and the PMT was set to 400V.
[0162]
(9) Results
When the fluorescent scanner evaluation result in (8) was analyzed, the fluorescence intensity at 532 nm was 17970 in the spot of the DNA probe of SEQ ID NO: 5 that was a perfect match with the labeled single-stranded DNA probe. In addition, the fluorescence intensity other than the spot portion of the DNA probe was observed to be 723. When the spots of each DNA probe were observed with fluorescence, each spot shape was almost circular, and there was almost no difference in fluorescence intensity between spots spotted with the same probe medium. Further, it was observed that the distances between adjacent spots were almost uniform, and the spots were arranged in a lattice pattern at an interval of about 200 μm.
[0163]
(Example 14)
(1) Probe synthesis
A single-stranded DNA probe of SEQ ID NO: 9 was prepared in the same manner as in Example 12 and used for the following experiment.
[0164]
(2) Substance having a functional group at a site capable of binding to the probe (probe binding substance) Bivalent reagent N- having a maleimide group and a hydroxysuccinimide ester group as a substance having a functional group at the site capable of binding to the probe A probe binding substance solution was prepared by stirring a 500 μmol / l aqueous solution of (6-Maleimidocaproyloxy) sulfocuccinimide, sodium salt (trade name: sulfo-EMCS; manufactured by Dojindo Laboratories) for 5 minutes at room temperature. The prepared probe binding substance solution was used in the following experiment.
[0165]
(3) Substance having a functional group at a site capable of binding on the substrate (substrate binding substance)
Silane coupling agent (trade name: KBM) containing a silane compound having an amino group (N-β (aminoethyl) γ-aminopropyltrimethoxysilane) as a preparation of a substance having a functional group at a site capable of binding on a substrate -603; manufactured by Shin-Etsu Chemical Co., Ltd.) was stirred at room temperature for 30 minutes to prepare a substrate-binding substance solution. The prepared substrate binder material solution was used for the following experiments.
[0166]
(4) Adjustment of probe medium
The single-stranded DNA probe of SEQ ID NO: 9 is dissolved in a TE solution (10 mM Tris-HCl (pH 8) / 1 mM ethylenediaminetetraacetic acid (EDTA) aqueous solution) so as to have a final concentration of about 40 μmol / l. A probe solution was prepared (the exact concentration was calculated from the absorption intensity).
[0167]
100 μl of the probe solution prepared as described above was prepared, and 100 μl of the probe binding substance solution prepared in (2) above was added dropwise thereto, followed by mixing for 5 minutes. Next, 100 μl of the substrate binding substance solution prepared in the above (3) was dropped and mixed for 5 minutes. After the mixed solution was allowed to stand for 10 minutes, 700 μl of an aqueous solution containing 7.5 wt% glycerin, 7.5 wt% urea, and 7.5 wt% thiodiglycol was dropped and mixed for 5 minutes. After mixing, the probe medium was prepared by leaving it for 30 minutes.
[0168]
(5) Substrate cleaning
A 1 inch × 3 inch glass substrate (thickness: 1.1 mm) was put in a cassette, and the glass substrate was immersed in a 1 mol / l sodium hydroxide aqueous solution preheated to 60 ° C. for 10 minutes. Subsequently, it was sufficiently rinsed in running pure water, and sodium hydroxide adhering to the glass substrate and the cassette was washed and removed. After sufficiently rinsing, the glass substrate together with the cassette was immersed in pure water and subjected to ultrasonic cleaning for 10 minutes. After ultrasonic cleaning, the sample was sufficiently rinsed in pure water, and the particles adhering to the glass substrate and the cassette were washed and removed. The glass substrates after washing with water were dried one by one by blowing nitrogen gas. In order to confirm the cleaning of the substrate, the pure water contact angle on the substrate was measured. As a result, it was about 5 ° at all parts of the substrate.
[0169]
(6) Spotting of probe medium
The probe medium adjusted in the above (4) was filled in an ink tank for a bubble jet (registered trademark) printer (trade name: BJF850; manufactured by Canon Inc.) and mounted on a bubble jet (registered trademark) head. In addition, the bubble jet (registered trademark) printer (trade name: BJF850; manufactured by Canon Inc.) used here is modified so that printing on a flat plate is possible. Next, the glass substrate prepared in (5) above was mounted on the printer, and the probe medium was spotted on the glass substrate. Here, the distance between the liquid ejection surface of the bubble jet (registered trademark) head and the liquid adhesion surface of the glass substrate was 1.2 to 1.5 mm. After the spotting, the glass substrate was observed with a microscope, and it was confirmed that a matrix spot array was formed on the glass substrate surface. In addition, the pure water contact angle was measured on the outer periphery of the spot formation site. As a result, it was about 5 ° at all parts of the substrate. From this result, it was confirmed that no contamination due to the probe medium or the like was generated other than the spot portion. The glass substrate after spotting was immersed in a container filled with a 1M NaCl / 50 mM phosphate buffer (pH 7.0) solution, and the whole container was put in a refrigerator and stored in a refrigerator (4 ° C.). In this way, a probe fixing substrate was produced.
[0170]
(7) Blocking process
The probe-immobilized substrate prepared in the above (6) was taken out from a 1M NaCl / 50 mM phosphate buffer (pH 7.0) solution. Next, the glass substrate was immersed in a container filled with 2% bovine serum albumin aqueous solution and allowed to stand for 2 hours for blocking treatment.
[0171]
(8) Hybridization treatment
A single-stranded DNA probe having a base sequence complementary to the single-stranded DNA probe of SEQ ID NO: 9 described in (1) above was synthesized by an automatic DNA synthesizer and labeled by binding rhodamine to the 5 ′ end. A single-stranded DNA probe was obtained. This labeled single-stranded DNA was dissolved in 1M NaCl / 50 mM phosphate buffer (pH 7.0) to a final concentration of 1 μM, and the blocking-immobilized probe-immobilized substrate obtained in (7) above was dissolved in this solution. And was subjected to a hybridization treatment at room temperature (55 ° C.) for 2 hours. Thereafter, the probe array was washed with a 1M NaCl / 50 mM phosphate buffer (pH 7.0) solution to wash away single-stranded DNA probes that did not hybridize with the probe nucleic acid. Then, after removing excess salt with pure water, the probe fixing substrate was dried by nitrogen blowing. Next, the fluorescence intensity of the spot on the probe-fixing substrate was evaluated using a fluorescence scanner (trade name: GenePix4000B; manufactured by Axon Instruments, Inc.). In the evaluation, the laser power was set to 100% and the PMT was set to 400V.
[0172]
(9) Results
When the fluorescent scanner evaluation result in the above (8) was analyzed, the fluorescence intensity at 532 nm was 25730 in the spot of the DNA probe of SEQ ID NO: 9 which was a perfect match with the labeled single-stranded DNA probe. Further, the fluorescence intensity other than the spot portion of the DNA probe was observed to be 639. When the spots of each DNA probe were observed with fluorescence, each spot shape was almost circular, and there was almost no difference in fluorescence intensity between spots spotted with the same probe medium. Further, it was observed that the distances between adjacent spots were almost uniform, and the spots were arranged in a lattice pattern at an interval of about 200 μm.
[0173]
(Example 15)
(1) Probe synthesis
A single-stranded DNA probe was prepared in exactly the same manner as in Example 1, and then used in the following experiment. When using a single-stranded DNA probe, it was dispensed into a microtube. After dispensing so that the amount of single-stranded DNA probe per microtube was 17.53 nmol, it was dried up and dried to obtain a single-stranded DNA probe.
[0174]
(2) Substance having a functional group at a site capable of binding to the probe and a functional group at a site capable of binding on the substrate (probe binding substance)
Silane coupling including a silane compound having an epoxy group (γ-glycidoxypropyltrimethoxysilane) as a preparation of a substance having a functional group at a site capable of binding to a probe and a functional group at a site capable of binding on a substrate Agent (trade name: KBM-403; manufactured by Shin-Etsu Chemical Co., Ltd.) was used. The epoxy silane coupling agent was used as a probe binding substance solution in the following experiments.
[0175]
(3) Adjustment of probe medium
The single-stranded DNA probe of SEQ ID NO: 1 was dispensed, and 10 μl of an epoxy silane coupling agent as a probe binding substance was added dropwise and mixed in a microtube that had been dried up and dried. Subsequently, 50 μl of pure water was dropped and mixed. After thoroughly mixing for 5 minutes, the mixture was allowed to stand for 30 minutes.
[0176]
Next, 500 μl of ethylene glycol, which is a high boiling point solvent, and 500 μl of ethyl alcohol were dropped into the microtube, and then mixed for 1 minute.
[0177]
Next, 100 μl of a 0.5 wt% aqueous solution of polyvinyl alcohol (PVA), which is a water-soluble polymer material, was dropped and mixed for 1 minute. Finally, pure water was added dropwise so that the total amount of the probe medium was 2000 μl, and mixed for 5 minutes. After mixing, the probe medium was adjusted by allowing it to stand for 60 minutes.
[0178]
(4) Substrate cleaning
A 1 inch × 3 inch glass substrate (thickness: 1.1 mm) was put in a cassette, and the glass substrate was immersed in a 1 mol / l sodium hydroxide aqueous solution preheated to 60 ° C. for 10 minutes. Subsequently, it was sufficiently rinsed in running pure water, and sodium hydroxide adhering to the glass substrate and the cassette was washed and removed. After sufficiently rinsing, the glass substrate together with the cassette was immersed in pure water and subjected to ultrasonic cleaning for 10 minutes. After ultrasonic cleaning, the sample was sufficiently rinsed in pure water, and the particles adhering to the glass substrate and the cassette were washed and removed. The glass substrates after washing with water were dried one by one by blowing nitrogen gas. In order to confirm the cleaning of the substrate, the pure water contact angle on the substrate was measured. As a result, it was about 5 ° at all parts of the substrate.
[0179]
(5) Spotting of probe medium
The probe medium adjusted in the above (3) was filled in an ink tank for a bubble jet (registered trademark) printer (trade name: BJF850; manufactured by Canon Inc.) and mounted on a bubble jet (registered trademark) head. In addition, the bubble jet (registered trademark) printer (trade name: BJF850; manufactured by Canon Inc.) used here is modified so that printing on a flat plate is possible. Next, the glass substrate prepared in (4) above was mounted on the printer, and the probe medium was spotted on the glass substrate. Here, the distance between the liquid ejection surface of the bubble jet (registered trademark) head and the liquid adhesion surface of the glass substrate was 1.2 to 1.5 mm. After the spotting, the glass substrate was observed with a microscope, and it was confirmed that a matrix spot array was formed on the glass substrate surface. In addition, the pure water contact angle was measured on the outer periphery of the spot formation site. As a result, it was about 5 ° at all parts of the substrate. From this result, it was confirmed that no contamination due to the probe medium or the like was generated other than the spot portion. The glass substrate after spotting was left in a desiccator. In this way, a probe fixing substrate was produced.
[0180]
(6) Blocking process
The probe-immobilized substrate prepared in the above (5) was sufficiently washed with a 1M NaCl / 50 mM phosphate buffer (pH 7.0) solution to wet the probe-immobilized substrate so that a uniform blocking treatment could be performed. Next, the glass substrate was immersed in a 2% bovine serum albumin aqueous solution and allowed to stand for 2 hours for blocking treatment.
[0181]
(7) Hybridization treatment
A single-stranded DNA probe having a base sequence complementary to the single-stranded DNA probe of SEQ ID NO: 1 described in (1) above was synthesized by an automatic DNA synthesizer and labeled by binding rhodamine to the 5 ′ end. A single-stranded DNA probe was obtained. The labeled single-stranded DNA was dissolved in 1M NaCl / 50 mM phosphate buffer (pH 7.0) to a final concentration of 1 μM, and the blocking-immobilized probe-immobilized substrate obtained in (6) above was dissolved in this solution. And was subjected to a hybridization treatment at room temperature (25 ° C.) for 3 hours. Thereafter, the probe array was washed with a 1M NaCl / 50 mM phosphate buffer (pH 7.0) solution to wash away single-stranded DNA probes that did not hybridize with the probe nucleic acid. Then, after removing excess salt with pure water, the probe fixing substrate was dried by nitrogen blowing. Next, the fluorescence intensity of the spot on the probe-fixing substrate was evaluated using a fluorescence scanner (trade name: GenePix4000B; manufactured by Axon Instruments, Inc.). In the evaluation, the laser power was set to 100% and the PMT was set to 400V.
[0182]
(8) Results
When the fluorescent scanner evaluation result in the above (7) was analyzed, the fluorescence intensity at 532 nm was 15750 in the spot of the DNA probe of SEQ ID NO: 1 that was a perfect match with the labeled single-stranded DNA probe. Further, when the fluorescence intensity other than the spot portion of the DNA probe was observed, it was 108. When the spots of each DNA probe were observed with fluorescence, each spot shape was almost circular, and there was almost no difference in fluorescence intensity between spots spotted with the same probe medium. Further, it was observed that the distances between adjacent spots were almost uniform, and the spots were arranged in a lattice pattern at an interval of about 200 μm.
[0183]
(Example 16)
(1) Probe synthesis
The single-stranded DNA probe was dispensed and dried up and dried in exactly the same manner as in Example 15.
[0184]
(2) Substance having a functional group at a site capable of binding to the probe and a functional group at a site capable of binding on the equipment (probe binding substance)
Silane coupling agent containing a silane compound having an isocyanate group (3-isocyanatepropyltriethoxysilane) as a preparation of a substance having a functional group at a site capable of binding to a probe and a functional group at a site capable of binding on a substrate Trade name: KBE-9007; manufactured by Shin-Etsu Chemical Co., Ltd.). The isocyanate silane coupling agent was used as a probe binding substance solution in the following experiments.
[0185]
(3) Adjustment of probe medium
20 μl of an isocyanate silane coupling agent, which is a probe binding substance, was dropped and mixed into a microtube in which the single-stranded DNA probe of SEQ ID NO: 1 was dispensed and dried up and dried. Subsequently, 50 μl of pure water was dropped and mixed. After thoroughly mixing for 5 minutes, the mixture was allowed to stand for 30 minutes.
[0186]
Next, 500 μl of ethylene glycol, which is a high boiling point solvent, and 500 μl of ethyl alcohol were dropped into the microtube, and then mixed for 1 minute.
[0187]
Next, 100 μl of a 0.5 wt% aqueous solution of polyvinyl alcohol (PVA), which is a water-soluble polymer material, was dropped and mixed for 1 minute. Finally, pure water was added dropwise so that the total amount of the probe medium was 2000 μl, and mixed for 5 minutes. After mixing, the probe medium was adjusted by allowing it to stand for 60 minutes.
[0188]
(4) Substrate cleaning
The substrate was cleaned in the same manner as in Example 15 to obtain a substrate.
[0189]
(5) Spotting of probe medium
The probe medium adjusted in the above (3) was filled in an ink tank for a bubble jet (registered trademark) printer (trade name: BJF850; manufactured by Canon Inc.) and mounted on a bubble jet (registered trademark) head. In addition, the bubble jet (registered trademark) printer (trade name: BJF850; manufactured by Canon Inc.) used here is modified so that printing on a flat plate is possible. Next, the glass substrate prepared in (4) above was mounted on the printer, and the probe medium was spotted on the glass substrate. Here, the distance between the liquid ejection surface of the bubble jet (registered trademark) head and the liquid adhesion surface of the glass substrate was 1.2 to 1.5 mm. After the spotting, the glass substrate was observed with a microscope, and it was confirmed that a matrix spot array was formed on the glass substrate surface. In addition, the pure water contact angle was measured on the outer periphery of the spot formation site. As a result, it was about 5 ° at all parts of the substrate. From this result, it was confirmed that no contamination due to the probe medium or the like was generated other than the spot portion. The glass substrate after spotting was left in a desiccator. In this way, a probe fixing substrate was produced.
[0190]
(6) Blocking process
The probe-immobilized substrate prepared in the above (5) was sufficiently washed with a 1M NaCl / 50 mM phosphate buffer (pH 7.0) solution to wet the probe-immobilized substrate so that a uniform blocking treatment could be performed. Next, the glass substrate was immersed in a 2% bovine serum albumin aqueous solution and allowed to stand for 2 hours for blocking treatment.
[0191]
(7) Hybridization treatment
A single-stranded DNA probe having a base sequence complementary to the single-stranded DNA probe of SEQ ID NO: 1 described in (1) above was synthesized by an automatic DNA synthesizer and labeled by binding rhodamine to the 5 ′ end. A single-stranded DNA probe was obtained. The labeled single-stranded DNA was dissolved in 1M NaCl / 50 mM phosphate buffer (pH 7.0) to a final concentration of 1 μM, and the blocking-immobilized probe-immobilized substrate obtained in (6) above was dissolved in this solution. And was subjected to a hybridization treatment at room temperature (25 ° C.) for 3 hours. Thereafter, the probe array was washed with a 1M NaCl / 50 mM phosphate buffer (pH 7.0) solution to wash away single-stranded DNA probes that did not hybridize with the probe nucleic acid. Then, after removing excess salt with pure water, the probe fixing substrate was dried by nitrogen blowing. Next, the fluorescence intensity of the spot on the probe-fixing substrate was evaluated using a fluorescence scanner (trade name: GenePix4000B; manufactured by Axon Instruments, Inc.). In the evaluation, the laser power was set to 100% and the PMT was set to 400V.
[0192]
(8) Results
When the fluorescent scanner evaluation result in the above (7) was analyzed, the fluorescence intensity at 532 nm was 15750 in the spot of the DNA probe of SEQ ID NO: 1 that was a perfect match with the labeled single-stranded DNA probe. Further, when the fluorescence intensity other than the spot portion of the DNA probe was observed, it was 108. When the spots of each DNA probe were observed with fluorescence, each spot shape was almost circular, and there was almost no difference in fluorescence intensity between spots spotted with the same probe medium. Further, it was observed that the distances between adjacent spots were almost uniform, and the spots were arranged in a lattice pattern at an interval of about 200 μm.
[0193]
(Example 17)
(1) Probe synthesis
A single-stranded nucleic acid of SEQ ID NO: 5 was synthesized using an automatic DNA synthesizer. A mercapto (SH) group was introduced into the single-stranded DNA terminal of SEQ ID NO: 1 by using a thiol modifier (manufactured by Glen Research) during synthesis with an automatic DNA synthesizer. Subsequently, normal deprotection was performed, the DNA was recovered, purified by high performance liquid chromatography, and used in the following experiments.
SEQ ID NO: 5
5'HS- (CH2) 6-O-PO2-O-ACTGGCCGTCGTTTTACA3 '
[0194]
(2) Probe immobilization material
A silane coupling agent containing a maleimide group-containing silane coupling agent (maleimidopropyltriethoxysilane: Compound I) as a material for fixing the probe on the substrate is prepared by the method described in Japanese Patent Application Publication No. 2001-72690. Synthesized. Confirmation of synthesis ¹ HNMR, ¹³ Performed by CNMR. This silane coupling agent was used in the following experiment.
[0195]
[Outside 1]

[0196]
[Outside 2]

Compound I
[0197]
(3) Adjustment of probe medium
The probe medium was prepared in exactly the same manner as in Example 1 above.
[0198]
(4) Substrate cleaning
Substrate cleaning was performed in exactly the same manner as in Example 1 to prepare a glass substrate.
[0199]
(5) Spotting of probe medium
A probe-fixing substrate was produced in exactly the same manner as in Example 1 above.
[0200]
(6) Blocking process
The blocking treatment was performed in exactly the same manner as in Example 1 above, washed with a 1M NaCl / 50 mM phosphate buffer (pH 7.0) solution, wetted on the probe-immobilized substrate, and allowed to perform a uniform hybridization treatment. did.
[0201]
(7) Hybridization treatment
Hybridization treatment was performed in exactly the same manner as in Example 1 above. Thereafter, the probe array was washed with a 1M NaCl / 50 mM phosphate buffer (pH 7.0) solution to wash away single-stranded DNA probes that did not hybridize with the probe nucleic acid. Then, after removing excess salt with pure water, the probe fixing substrate was dried by nitrogen blowing. Next, the fluorescence intensity of the spot on the probe-fixing substrate was evaluated using a fluorescence scanner (trade name: GenePix4000B; manufactured by Axon Instruments, Inc.). In the evaluation, the laser power was set to 100% and the PMT was set to 400V.
[0202]
(8) Results
When the fluorescent scanner evaluation result in the above (7) was analyzed, the fluorescence intensity at 532 nm was 23400 in the spot of the DNA probe of SEQ ID NO: 1 that was a perfect match with the labeled single-stranded DNA probe. Further, when the fluorescence intensity other than the spot portion of the DNA probe was observed, there were 658. When the spots of each DNA probe were observed with fluorescence, each spot shape was almost circular, and there was almost no difference in fluorescence intensity between spots spotted with the same probe medium. Almost no increase in fluorescence intensity other than the spot portion due to the absence of the blocking treatment was observed. Further, it was observed that the distances between adjacent spots were almost uniform, and the spots were arranged in a lattice pattern at an interval of about 200 μm.
[0203]
(Example 18)
(1) Probe synthesis
A single-stranded nucleic acid of SEQ ID NO: 5 was synthesized in exactly the same manner as in Example 17. Subsequently, normal deprotection was performed, the DNA was recovered, purified by high performance liquid chromatography, and used in the following experiments.
[0204]
(2) Probe immobilization material
A silane coupling agent including a silane coupling agent having a succinimide ester group (succinimidyl triethoxysilylpropionate: Compound II) as a preparation of a substance for immobilizing a probe on a substrate is shown below. It was synthesized by the method. Confirmation of synthesis ¹ HNMR, ¹³ Performed by CNMR. This silane coupling agent was used in the following experiment.
[0205]
[Outside 3]

[0206]
[Outside 4]

Compound II
[0207]
(3) Adjustment of probe medium
The probe medium was prepared in exactly the same manner as in Example 17 above.
[0208]
(4) Substrate cleaning
Substrate cleaning was performed in exactly the same manner as in Example 17 to prepare a glass substrate.
[0209]
(5) Spotting of probe medium
A probe-fixing substrate was produced in exactly the same manner as in Example 17 above.
[0210]
(6) Blocking process
The blocking treatment was performed in exactly the same manner as in Example 17, washed with a 1M NaCl / 50 mM phosphate buffer (pH 7.0) solution, wetted on the probe-immobilized substrate, and allowed to perform a uniform hybridization treatment. did.
[0211]
(7) Hybridization treatment
Hybridization treatment was performed in exactly the same manner as in Example 17 above. Thereafter, the probe array was washed with a 1M NaCl / 50 mM phosphate buffer (pH 7.0) solution to wash away single-stranded DNA probes that did not hybridize with the probe nucleic acid. Then, after removing excess salt with pure water, the probe fixing substrate was dried by nitrogen blowing. Next, the fluorescence intensity of the spot on the probe-fixing substrate was evaluated using a fluorescence scanner (trade name: GenePix4000B; manufactured by Axon Instruments, Inc.). In the evaluation, the laser power was set to 100% and the PMT was set to 400V.
[0212]
(8) Results
When the fluorescent scanner evaluation result in the above (7) was analyzed, the fluorescence intensity at 532 nm was 23400 in the spot of the DNA probe of SEQ ID NO: 1 that was a perfect match with the labeled single-stranded DNA probe. In addition, the fluorescence intensity other than the spot portion of the DNA probe was observed to be 658. When the spots of each DNA probe were observed with fluorescence, each spot shape was almost circular, and there was almost no difference in fluorescence intensity between spots spotted with the same probe medium. Almost no increase in fluorescence intensity other than the spot portion due to the absence of the blocking treatment was observed. Further, it was observed that the distances between adjacent spots were almost uniform, and the spots were arranged in a lattice pattern at an interval of about 200 μm.
[0213]
(Comparative example)
(1) Probe synthesis
A single-stranded DNA probe was used as a probe that can specifically bind to the target substance. A single-stranded nucleic acid of SEQ ID NO: 10 was synthesized using an automatic DNA synthesizer. A mercapto (SH) group was introduced into the single-stranded DNA terminal of SEQ ID NO: 1 by using a thiol modifier (manufactured by Glen Research) during synthesis with an automatic DNA synthesizer. Subsequently, normal deprotection was performed, the DNA was recovered, purified by high performance liquid chromatography, and used in the following experiments.
SEQ ID NO: 10
5'HS- (CH2) 6-O-PO2-O-ACTGGCCGTCGTTTTACA3 '
[0214]
(2) Adjustment of probe medium
A single-stranded DNA probe solution of SEQ ID NO: 10 was prepared in exactly the same manner as in Example 1 above (the exact concentration was calculated from the absorption intensity). The adjusted probe solution was used for the following experiment.
[0215]
100 μl of the probe solution prepared as described above was prepared, and 900 μl of an aqueous solution containing 7.5 wt% glycerol, 7.5 wt% urea, and 7.5 wt% thiodiglycol was added dropwise thereto. After mixing for 5 minutes, the probe medium was adjusted by leaving it for 30 minutes.
[0216]
(3) Substrate cleaning
Substrate cleaning was performed in the same manner as in Example 1 to prepare a glass substrate.
[0217]
(4) Probe immobilization process
A 1 wt% aqueous solution of a silane coupling agent (trade name: KBM-603; manufactured by Shin-Etsu Chemical Co., Ltd.) containing a silane compound having an amino group (N-β (aminoethyl) γ-aminopropyltrimethoxysilane) The mixture was stirred for 30 minutes at room temperature to prepare an aqueous silane coupling agent solution. Next, the glass substrate washed in (3) above was immersed in this aqueous solution at room temperature (25 ° C.) for 20 minutes, and then sufficiently rinsed with running water to remove the excess aqueous silane coupling agent solution. Nitrogen gas was sprayed onto both sides of the rinsed substrate and dried. Next, the substrate was baked in an oven heated to 120 ° C. for 1 hour to complete the silane coupling treatment. Then, 2.7 mg of N-maleimidocaproyloxysuccinimide N- (6-Maleimidocaproyloxy) sulfocuccinimide (trade name: EMCS; manufactured by Dojindo Laboratories) was weighed and 1: 1 of dimethylsulfoxide (DMSO) / ethanol. An EMCS solution was prepared by dissolving in the solution to a final concentration of 0.3 mg / ml. The substrate subjected to the silane coupling treatment was immersed in this EMCS solution for 30 minutes at room temperature. The substrate pulled up from the EMCS solution was sequentially washed with a mixed solvent of DMSO and ethanol and ethanol, and then dried in a nitrogen gas atmosphere. In this way, the probe immobilization process was completed. In order to confirm the effect of the probe immobilization treatment, the pure water contact angle was measured. As a result, the contact angle on the substrate was 25-30 °. From this result, it was confirmed that the probe immobilization treatment on the substrate was appropriately performed.
[0218]
(5) Spotting of probe medium
The probe medium adjusted in the above (2) was spotted in the same manner as in Example 1 to prepare a probe fixing base material. After the spotting, the glass substrate was observed with a microscope, and it was confirmed that a matrix spot array was formed on the glass substrate surface. In addition, in order to confirm the effect of the probe immobilization treatment, the pure water contact angle was measured. As a result, the contact angle on the substrate was 25-30 °. From this result, it was confirmed that no contamination due to the probe medium or the like was generated other than the spot portion. The glass substrate after spotting was immersed in a container filled with a 1M NaCl / 50 mM phosphate buffer (pH 7.0) solution, and the whole container was put in a refrigerator and stored in a refrigerator (4 ° C.). In this way, a probe fixing substrate was produced.
[0219]
(6) Blocking process
The blocking treatment was performed in exactly the same manner as in Example 1 above.
[0220]
(7) Hybridization treatment
A single-stranded DNA probe having a base sequence complementary to the single-stranded DNA probe of SEQ ID NO: 10 described in (1) above was synthesized with an automatic DNA synthesizer and labeled by binding rhodamine to the 5 ′ end. A single-stranded DNA probe was obtained. The labeled single-stranded DNA was dissolved in 1M NaCl / 50 mM phosphate buffer (pH 7.0) to a final concentration of 1 μM, and the blocking-immobilized probe-immobilized substrate obtained in (6) above was dissolved in this solution. And was subjected to a hybridization treatment at room temperature (25 ° C.) for 3 hours. Thereafter, the probe array was washed with a 1M NaCl / 50 mM phosphate buffer (pH 7.0) solution to wash away single-stranded DNA probes that did not hybridize with the probe nucleic acid. Then, after removing excess salt with pure water, the probe fixing substrate was dried by nitrogen blowing. Next, the fluorescence intensity of the spot on the probe-fixing substrate was evaluated using a fluorescence scanner (trade name: GenePix4000B; manufactured by Axon Instruments, Inc.). In the evaluation, the laser power was set to 100% and the PMT was set to 400V.
[0221]
(8) Results
When the fluorescent scanner evaluation result in the above (7) was analyzed, the fluorescence intensity at 532 nm was 12790 at the spot of the DNA probe of SEQ ID NO: 10 that was a perfect match with the labeled single-stranded DNA probe. In addition, the fluorescence intensity other than the spot portion of the DNA probe was observed to be 792. When the spots of each DNA probe were observed with fluorescence, each spot shape was almost circular, and there was almost no difference in fluorescence intensity between spots spotted with the same probe medium. Further, it was observed that the distances between adjacent spots were almost uniform, and the spots were arranged in a lattice pattern at an interval of about 200 μm.
[0222]
【The invention's effect】
As described above, according to the present invention, by including a probe and a substance that can bind to the probe and a substance that can bind to the substrate in the probe medium, the probe medium is simply applied on the substrate. The probe can be efficiently and stably fixed on the substrate. In addition, by using this method, it is possible not only to manufacture the probe fixing substrate with a small number of processes, but also to manufacture the probe fixing substrate with high quality and in a simplified manner. it can.
[0223]
Further, according to the present invention, when various types of probes are immobilized on a single substrate, it is possible to select the binding substance type and concentration corresponding to each probe type. Further, the binding reaction conditions between the probe, the substance that can bind to the probe, and the substance that can bind to the substrate can be adjusted according to each probe type. For this reason, it is possible to bind various probes on a single substrate under optimum conditions for each probe type.
[0224]
Furthermore, according to the present invention, it is possible to obtain a probe array capable of examining more information on a target substance more accurately even from a small amount of specimen, and whether or not the target substance is present in a sample by using it. Can be determined more accurately and quickly. Similarly, by using this probe array, the structure of the target substance in the sample can be identified more accurately and quickly.

Claims (9)

A single-stranded nucleic acid probe capable of specifically binding to a target substance and having a functional group modified at the terminal, and a functional group and a silanol group capable of binding to the terminal of the single-stranded nucleic acid probe A probe medium obtained by dissolving a silane coupling agent in an aqueous solution . A single-stranded nucleic acid probe capable of specifically binding to a target substance and having a functional group modified at the end, and a functional group capable of binding to the end of the single-stranded nucleic acid probe and a substrate. ur and the silane coupling agent having a site, a comprise in the presence of water, said single-stranded nucleic acid probe, the probe medium further containing a bifunctional reagent for coupling the silane coupling agent .   The probe medium according to claim 1 or 2, wherein the binding method described above is based on a covalent bond.   The bifunctional reagent is, N- (4-Maleimidocaproyloxy) sulfosuccinimide, sodium salt, N- (6-Maleimidocaproyloxy) sulfosuccinimide, sodiumsalt, N- (8-Maleimidocaproyloxy) sulfosuccinimide, sodium salt, N- (11-Maleimidocaproyloxy) sulfosuccinimide The probe medium according to claim 2, wherein the probe medium is at least one selected from the group consisting of:   A probe fixing method for fixing the probe medium according to any one of claims 1 to 4 by applying the probe medium onto a substrate by a spotting method.   A DNA array, wherein the probe on the probe-fixing substrate produced by the probe-fixing method according to claim 5 is a DNA probe.   A detection method for detecting a target substance using the probe-fixed substrate obtained by fixing the probe medium according to any one of claims 1 to 4 on a substrate by a spotting method. A single-stranded nucleic acid probe capable of specifically binding to a target substance and having a functional group modified at the terminal, and a functional group and a silanol group capable of binding to the terminal of the single-stranded nucleic acid probe A probe medium obtained by mixing each or a part of a substance containing a silane coupling agent in an aqueous solution and storing it in a container and fixing it on a substrate. A single-stranded nucleic acid probe capable of specifically binding to a target substance and having a functional group modified at the terminal, and a functional group and a silanol group capable of binding to the terminal of the single-stranded nucleic acid probe A DNA array, characterized in that a probe medium comprising a silane coupling agent in an aqueous solution is disposed in a spot-like manner on the substrate.