JP4741738B2 - Method for producing probe carrier - Google Patents

Description

【００９５】
のアミノシランカップリング剤（ＫＢＭ−６０３：化合物Ｉ 信越化学工業株式会社製）を１％の濃度で含む水溶液を室温下、１時間攪拌し、メトキシ基部分を加水分解させた。次に、上記基板を洗浄後速やかに前記シランカップリング剤水溶液に浸し、室温下、１時間浸漬した。その後、流水（超純水）洗浄し、窒素ガスを吹きつけて乾燥させ、次いで、１２０℃のオーブン中で１時間加熱定着させた。
【００９６】
冷却後、下記式（II）：
【００９７】
【化２】

【００９８】
のＮ−（６−マレイミドカプロキシ）スクシイミド（ＥＭＣＳ；化合物II）の０．３％溶液（エタノール：ジメチルスルホキシド＝１：１）に基板を室温下、２時間浸漬し、 ＥＭＣＳをアミノシランカップリング剤のアミノ基に反応させた。反応終了後、エタノール：ジメチルスルホキシド＝１：１で１回、エタノールで３回洗浄し、窒素ガスを吹きつけて乾燥させた。
５’−ATGAACCGGAGGCCCATC−３’ ▲１▼（配列番号：１）
３’−TACTTGGCCTCCGGGTAG−５’ ▲２▼（配列番号：２）
上記する▲１▼の塩基配列に相補的な塩基配列である▲２▼の塩基配列を有し、かつ、５’末端にリンカーを介して上記基板表面に最終的に精製したマレイミド基と反応結合可能なメルカプト基（ＳＨ基：スルフィドリル基ともいう）を有するオリゴヌクレオチド（化合物III ベックス株式会社）を本実施例の検証に用いるプローブに利用した。式（III）：
【００９９】
【化３】

【０１００】
のメルカプト基を導入したオリゴヌクレオチド（化合物III）を、液体吐出ユニットで吐出するための溶媒、すなわち、グリセリン７．５質量％、尿素７．５質量％、チオジグリコール７．５質量％、一般式（IV）：
【０１０１】
【化４】

【０１０２】
で示されるアセチレンアルコール（例えば、商品名：アセチレノールＥＨ 川研ファインケミカル株式会社）１質量％を含む水溶液に、吸光度が１．０になるように溶解させた。このオリゴヌクレオチド溶液を、実施例１で作製した液体吐出ユニットの液体リザーバーにマイクロディスペンサーを用いて供給した。なお、最終的なオリゴヌクレオチド溶液の充填を行う前に、作製した液体吐出ユニットに対して、溶媒と馴染ませる目的で、各液体リザーバー、ノズルは予め上記組成の溶媒による洗浄、ならびに必要に応じてオリゴヌクレオチド溶液で洗浄を行い、真空吸引による液体の除去を適宜繰り返した。
【０１０３】
その後、上記のマレイミド基を導入する処理を施した基板上にオリゴヌクレオチド溶液を吐出した。実施例１において作製したヘッドの設計仕様は、吐出される液滴１滴当たりの液量は２４ｐｌであり、この吐出条件では、基板上に付与される液滴１滴の占めるドットの直径は、用いる溶液の粘度によって、70〜100μｍの範囲となる。
【０１０４】
また、上記組成の溶媒は保湿性が高く、液体リザーバー内における乾燥、濃縮、ならびに、基板上に付与したオリゴヌクレオチド溶液の液滴が、次ぎ工程において、基板表面との反応による固定をなす前に、乾燥・固化を起こすことを防ぐことができる。
【０１０５】
この式（III）のオリゴヌクレオチド（化合物III）溶液を二次元アレイ状に付与した基板を、湿度１００％の保湿チヤンバー内に室温下で１時間保持し、オリゴヌクレオチドのメルカプト基と基板上のマレイミド基との反応を行わせた。取り出した後、未反応のオリゴヌクレオチドを除去するため、基板を流水（超純水）中で約３０秒洗浄した。
【０１０６】
次いで、上記オリゴヌクレオチドを固定するドットを二次元アレイ状に形成した基板について、ドット以外の表面にブロッキング処理を施すため、５０ｍＭリン酸緩衝液（ｐＨ＝７．０、１ＭＮａＣｌを含む）にＢＳＡ（牛血清アルブミンシグマアルドリッチジャパン）を２％の濃度で溶解したブロッキング用溶液に１時間浸漬した後、前記５０ｍＭリン酸緩衝液で適宜洗浄し、この５０ｍＭリン酸緩衝液中で保存し、DNAアレイとした。
【０１０７】
（ハイブリダイゼーション反応によるＤＮＡアレイのドット形状評価）
モデル標的ＤＮＡとして、式（Ｖ）：
【０１０８】
【化５】

【０１０９】
のＤＮＡ分子、すなわち、実施例２に示す▲１▼の配列を有し、蛍光標識としてテトラメチルローダミンを５’末端に結合した化合物Ｖ（べックス株式会社より購入）を用いて調製された二次元アレイ上のプローブとのハイブリダイゼーション反応を行った。
【０１１０】
このハイブリダイゼーション反応は、調製したＤＮＡアレイと、化合物Ｖを５ｎＭの濃度で含むリン酸緩衝液（１０ｍＭリン酸緩衝液ｐＨ＝７．０、５０ｍＭのＮａＣｌを含む）２ｍｌとを用い、ハイブリパック中で行った。DNAアレイをモデル標的ＤＮＡ溶液とともにハイブリパック中に封じ、恒温槽内で７０℃まで加熱し、その後、５０℃まで冷却し、その状態で１０時間放置した。
【０１１１】
次に、DNAアレイをハイブリパックから取り出し、未反応の標的ＤＮＡを除去する目的で、ハイブリダイゼーション用の緩衝液で洗浄する。洗浄後、緩衝液で覆われた状態でスライドグラス上に基板を置き、カバーガラスで覆って、蛍光標識からの蛍光を観察した。この観察に使用した蛍光顕微鏡は、ＥＣＬＩＰＳＥ Ｅ８００（株式会社ニコン）に２０倍対物レンズ（プランアポクロマート）と蛍光フイルタ（Ｙ−２Ｅ／Ｃ）をセットしたものである。また、蛍光顕微鏡で観測される画像は、イメージインテンシファイヤー付きＣＣＤカメラ（Ｃ２４００−８７ 浜松ホトニクス株式会社）と画像処理装置（Ａｒｇｕｓ５０ 浜松ホトニクス株式会社）を用いて、採り込みを行った。
【０１１２】
収録された画像に基づき、基板上に二次元アレイ状に形成した、化合物Ｖを固定したドット全てについて、蛍光が観察された。なお、その蛍光強度の平均値は、上記の測定装置の光強度指標で１７５０であった。また、その蛍光を発する各領域から、ドットの直径の平均値を算出したところ、約１００μｍであった。
【０１１３】
（256種のＤＮＡプローブからなるＤＮＡアレイの調製）
５’−ATGAACCGGAGGCCCATC−３’ ▲３▼（配列番号：１）
３’−TACTTGGCCTCCGGGTAG−５’ ▲４▼（配列番号：２）
▲３▼は実施例２の配列番号：１の塩基配列と同一であるが、実際は、発癌関連遺伝子ｐ５３のヌクレオチドの変異頻度が高い、二つのアミノ酸をコードする計４個の塩基部分（アンダーラインで示した）を含む１８ヌクレオチドの領域である。▲３▼の塩基配列に完全に相補的な塩基配列が▲４▼であり（すなわち実施例２の配列番号：２の配列と同一）、▲３▼のアンダーラインで示した塩基に対応する部分をアンダーラインで示している。
５’−GATGGGN¹N²TCN³N⁴GTTCAT−３’ ▲５▼（配列番号：３）
▲５▼は、▲４▼のアンダーラインで示した位置の塩基を、Ｎ¹、Ｎ²、Ｎ³、Ｎ⁴で表したものである。これらＮ¹、Ｎ²、Ｎ³、Ｎ⁴が、それぞれＡＴＧＣの塩基の何れかに置換し、かつ、５’末端にメルカプト基を結合した計256種のオリゴヌクレオチド・プローブを合成した。これを、実施例１で作製したＺＢＪヘッドを用いて、ガラス基板上に吐出し、反応結合することにより、256種のＤＮＡプローブからなる二次元状のＤＮＡプローブ・アレイを調製した。
【０１１４】
（ハイブリダイゼーションによる標的ＤＮＡの選別特性の評価）
調製した256種のＤＮＡプローブ・アレイを用いて、ハイブリダイゼーション反応を行い、目的とする塩基配列を有する標的ＤＮＡの選別特性を検証した。
【０１１５】
モデル標的ＤＮＡとして、以下の塩基配列を有し、かつ、蛍光標識としてテトラメチルローダミンを５’末端に結合した４種のＤＮＡを合成した。
５’−ATGAACCGGAGGCCCATC−３’ ▲３▼（配列番号：１）
５’−ATGAACGGGAGGCCCATC−３’ ▲６▼
（配列番号：４） Ｃ→Ｇ
５’−ATGAACGCGAGGCCCATC−３’ ▲７▼
（配列番号：５） Ｃ→Ｇ、Ｇ→Ｃ
５’−ATGAACGCGAAGCCCATC−３’ ▲８▼
（配列番号：６） Ｃ→Ｇ、Ｇ→Ｃ、Ｇ→Ａ
上述のように▲３▼はｐ５３遺伝子の正常配列に対して完全に相補的であり、▲６▼、▲７▼、▲８▼はそれに対して、塩基配列中に下線を付した塩基への置き換えが起こっている変異体のモデルである。
【０１１６】
この▲３▼、▲６▼、▲７▼、▲８▼の４種の配列をそれぞれ有する標的ＤＮＡについて、それぞれ個別にハイブリダイゼーションを行ったところ、調製したＤＮＡアレイの各ドットのうち、それぞれの標的ＤＮＡに完全に相補的塩基配列を有するプローブのドットからのみ蛍光が観察され、他のドットからは蛍光は観察されなかった。それぞれ、蛍光が観察されたドットにおいて、観測される蛍光強度は、標的ＤＮＡ▲３▼においては１８３０、標的ＤＮＡ▲６▼においては１２７０、標的ＤＮＡ▲７▼においては１５２０、標的ＤＮＡ▲８▼においては１９４０であった。
【０１１７】
若干のバラツキはあるものの、標的ＤＮＡ▲３▼における1830は、上記、ＤＮＡアレイのドット形状評価における平均値1750と統計誤差内で一致しており、その他の３種についても、概ね、その蛍光強度の差異は、有意なものとは言えない範囲である。従って、本発明の製造方法に従って、アレイ状の液体吐出装置を用いて、並列的にプローブ溶液を基板上に付与して調製したＤＮＡアレイは、標的ＤＮＡの定量的な検出に十分に使用できる程度に均一性と再現性を有するプローブ量の固定がなされていることがわかる。
【０１１８】
【発明の効果】
以上説明したように、プローブ担体の製造において、ノズル群を二次元に配置した液体吐出装置を用い、さらに1種のプローブ溶液を複数のノズルから吐出して基板上に二つのスポットを形成することにより、各プローブに対して検査が可能になり、プローブ溶液の注入ミスや装置の誤作動による間違った配列のプローブ・アレイの出荷を未然に防ぐことができる。
【０１１９】
【配列表】

【図面の簡単な説明】
【図１】液体吐出装置を構成するチップの模式図である。
【図２】本発明にかかる液体吐出装置の第１の実施例におけるノズル近傍部の拡大図である。
【図３】図２のＡ―Ａ'線での断面図である。
【図４】図２のＢ―Ｂ' 線での断面図である。
【図５】液体吐出装置を構成するチップの裏面の模式図（図１に示されたチップの裏面に相当）である。
【図６】液体吐出装置を構成するチップの液体供給口（リザーバー）の形状を説明するための模式図である。
【図７】液体吐出装置での液体の吐出を説明するための模式図である。
【図８】液体吐出装置の模式図である。
【図９】液体吐出装置の模式図である。
【図１０】液体吐出装置を液体吐出ユニットとして有するプローブ・アレイ調製用の装置の模式図である。
【図１１】プローブ・アレイ調製方法の模式図である。
【図１２】液体吐出装置の第２の実施例の模式図である。
【図１３】液体吐出装置の第３の実施例の模式図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for producing a probe carrier on a carrier, and more particularly to a method for producing a probe carrier that enables an accurate check for the occurrence of defective products.
[0002]
[Prior art]
When analyzing the base sequence of gene DNA, or simultaneously performing genetic diagnosis with high reliability for multiple items, it is necessary to select a DNA having the target base sequence using a plurality of types of probes. As a means for providing a plurality of types of probes used for this sorting operation, DNA microchips are attracting attention. In high-throughput screening and combinatorial chemistry for drugs, etc., a large number of target proteins and drug solutions (for example, 96 types, 384 types, 1536 types) can be arranged to perform orderly screening. Necessary. Developed a method for arranging many kinds of drugs for that purpose, automated screening technology in that state, a dedicated device, software for controlling a series of screening operations and statistically processing the results Has been.
[0003]
These parallel screening operations basically use a so-called probe array in which a large number of known probes are arranged for the substance to be evaluated, and are used for the probes under the same conditions. It detects the presence or absence of action or reaction. In general, what kind of probe action and reaction is used is determined in advance, and therefore, the probe types mounted on one probe array are, for example, a group of DNA probes having different base sequences, etc. It is a kind of substance when roughly classified. That is, a substance used for a group of probes is, for example, DNA, protein, synthesized chemical substance (drug), and the like. In many cases, a probe array consisting of a plurality of probe groups is often used. However, depending on the nature of the screening work, DNA having the same base sequence, protein having the same amino acid sequence, and the same chemistry may be used as the probe. It is possible to use a form in which a large number of substances are arranged in an array. These are mainly used for drug screening and the like.
[0004]
In a probe array consisting of a plurality of types of probes forming a group, specifically, a group of DNAs having different base sequences, a group of proteins having different amino acid sequences, or a group of different chemical substances are included in the group. In many cases, the seeds are arranged on a substrate or the like in an array according to a predetermined arrangement order. Among these, the DNA probe array is used for analyzing the base sequence of gene DNA and simultaneously performing highly reliable genetic diagnosis for many items.
[0005]
One of the problems in a probe array composed of a plurality of types of probes forming a group is to mount as many kinds of probes as possible, for example, DNA probes having many kinds of base sequences, on one substrate. In other words, how densely the probes can be arranged in an array.
[0006]
One method for fixing multiple types of probes on a substrate in an array is the sequential extension of DNA on a carrier using a photodegradable protective group and photolithography described in US Pat. No. 5,424,186 A method of preparing DNA probes having different base sequences in an array by reaction can be mentioned. Using this technique, for example, 1 cm²It is also possible to manufacture a DNA probe array on which more than 10,000 kinds of DNAs with different sequences are mounted. In this method, when DNA is synthesized by sequential extension reaction, a photolithographic process is performed for each of the four types of bases (A, T, C, G) using a dedicated photomask. By selectively extending one of the bases at a position, a plurality of types of DNA having a desired base sequence are synthesized on the substrate with a predetermined sequence. Therefore, as the DNA chain length increases, the cost required for production increases, and the production takes a long time. In addition, since the efficiency of nucleotide synthesis at each extension step is not 100%, the proportion of DNA in which the designed base sequence is defective is not small. Furthermore, when synthesizing, when using a photodegradable protecting group, the synthesis efficiency is lower than when using a normal acid-decomposable protecting group. There is also a problem that the proportion of DNA is reduced.
[0007]
Further, since the product directly synthesized on the carrier is used as it is, it is of course impossible to remove DNA having a defective base sequence from the DNA according to the designed base sequence by purification fractionation. In addition, in the finally obtained array, there is a problem that the base sequence of the DNA synthesized on the substrate cannot be confirmed. This is because if there is almost no extension of a given base at a certain extension stage due to a mistake in the process, and it is a completely defective product, screening using this defective product probe array results in an erroneous result. This means that there is no way to prevent it. The inability to confirm this base sequence is the biggest and essential problem in this method.
[0008]
As a method different from the above method, probe DNA is synthesized and purified in advance. In some cases, after confirming the base length, each DNA is supplied onto a substrate by a device such as a microdispenser,・ Methods for manufacturing arrays have also been proposed. PCT Publication No. WO95 / 35505 describes a technique for supplying DNA onto a membrane using a capillary. Applying this method, in principle, 1 cm²About 1000 DNA arrays can be manufactured per unit. Basically, a probe array is manufactured by supplying a probe solution to a predetermined position on a substrate with one capillary-like dispensing device for each probe and repeating the operation. If you prepare a dedicated capillary for each probe, there is no problem, but if you try to perform the same work using a small number of capillaries, in order to prevent cross-contamination, the capillary should be sufficient when replacing the probe type. Need to be cleaned. Moreover, it is necessary to control the supply position each time. Therefore, it cannot be said that the method is suitable for manufacturing an array in which many kinds of probes are arranged at high density. In addition, since the probe solution is supplied to the substrate by tapping the tip of the capillary on the substrate, reproducibility and reliability are not perfect.
[0009]
In addition, a micro dispenser device for supplying different drug solutions to individual wells, particularly for 96-well or 384-well microplates used for high-throughput screening of drugs, for example, Robbins HYDRA from Scientific^TMIt is marketed with the brand name. This is basically a two-dimensional arrangement of microsyringes, with a minimum discharge amount of 100 nl. If this is applied to array formation, the density is limited by the minimum discharge amount, and there is a limit to increasing the density.
[0010]
As another technique, when performing solid-phase synthesis of DNA on a substrate, a method of supplying a solution of a substance necessary for synthesis onto the substrate by an ink jet method at each extension stage has been proposed. For example, in European Patent Publication EP 0 703 825B1, a nucleotide monomer and an activator used in solid phase synthesis of DNA are supplied from separate piezo jet nozzles, respectively. A method for solid-phase synthesis of multiple types of DNA having sequences is described. The supply (application) by the ink jet method is more reliable than the supply (application) of the solution using the capillary, such as the reproducibility of the supply amount, and the nozzle structure can be miniaturized. It has characteristics suitable for increasing the density of the probe array. However, this technique also basically applies a sequential elongation reaction of DNA on a substrate, and is therefore the biggest problem in the technique described in US Pat. No. 5,424,186 described above. There still remains a problem that the base sequence of DNA synthesized on the substrate cannot be confirmed. Although the complexity of performing a photolithography process using a dedicated mask at each extension stage is eliminated, a predetermined probe is fixed to each point, which is an essential requirement for the probe array. , Including some problems. Note that the above-mentioned EP0,703,825B1 only describes a method of using a plurality of independently formed piezo jet nozzles, and when using this small number of nozzles, the aforementioned capillary is used. Similar to the approach, it is not necessarily suitable for manufacturing high density probe arrays.
[0011]
Japanese Patent Laid-Open No. 11-187900 discloses that a spot containing a probe is formed on a solid phase by attaching a liquid containing a probe to the solid phase as a droplet by a thermal ink jet head that discharges the droplet by heat. A method is disclosed. In this method, an ink jet head for a general printer is used as a unit for discharging liquid. However, the structure of the ink jet head for the printer may not be optimal for manufacturing a probe array.
[0012]
The ink jet head for printing has been developed for printing characters and images. Therefore, the liquid used is one color (black) ink for monochrome (typically black) printing, and generally three primary colors, ie, yellow (Y), cyan (color) for color printing. C) and magenta (M) ink of three colors. In the case of color printing, black or Y, M, and C dark and light inks may be used as necessary, but at most ten or more types of inks are not used.
[0013]
In addition, since a large amount of ink is used for printing on a paper surface, a conventional inkjet printing head has a tank (reservoir) for filling ink having a sufficient capacity for printing, and the ink to the nozzle. A flow path for guiding and a nozzle for discharging ink are provided.
[0014]
On the other hand, as described above, it is desired that the liquid discharge apparatus used for discharging the liquid at the time of manufacturing the probe array discharges as many kinds of liquid as possible. Therefore, it is desirable to provide a liquid ejecting apparatus having a number of accommodating portions for accommodating probe solutions corresponding to many types of probes required for manufacturing the probe array, and arranging an ejection port corresponding to each accommodating portion.
[0015]
Also, in the production of a probe array using a liquid ejection device for producing a probe array, the liquid is not consumed as much as when printing on a paper surface by a conventional general inkjet head for printing. A relatively small volume is sufficient.
[0016]
Furthermore, in a general inkjet head for printing, it is necessary to eject a desired ink at a desired position on the paper surface for forming characters and images, so that each nozzle can be independently selected at any timing. It has a head configuration. As a result, the head has a complicated configuration.
[0017]
On the other hand, a liquid ejecting apparatus used for ejecting liquid at the time of manufacturing a probe array does not necessarily require a configuration in which each nozzle can be independently selected at an arbitrary timing.
[0018]
As described above, the conventional general inkjet head for printing employs a head configuration in which each nozzle can be independently selected at an arbitrary timing. At this time, it is necessary to eject ink from a desired nozzle. Such power transistors and logic circuits may be provided outside the head or inside the head.
[0019]
By the way, as a method for discharging liquid, there are a bubble jet jet method in which liquid is discharged by heat energy generated from a heater, and a piezo jet method in which liquid is discharged by deformation of an element generated by applying a voltage to a piezo element. is there. Among these, the bubble jet method has a simpler structure than the piezo jet method, and is suitable for miniaturization of the head and the increase in the number of nozzles. From this point, it is considered that the thermal jet method is suitable when a large number of nozzles is desired for the liquid ejection unit for manufacturing the probe array.
[0020]
[Problems to be solved by the invention]
As described above, the method of manufacturing a probe array using an ink jet head that ejects liquid as droplets is excellent in that minute probe droplet amounts are arranged on a substrate at high density.
[0021]
Immobilized probe chips such as DNA microchips are used for academic research and various screenings in the medical field and often require high measurement sensitivity in various tests. In such a case, if a probe misalignment or a probe spot defect occurs during manufacture of the probe array, it cannot be used as a defective product. There is a need for a technique that can suppress the occurrence of such defective products and accurately check the occurrence of such defective products.
[0022]
For example, when using more than 100 types of probe solutions, there is no possibility that the solutions will be mixed or the probe will be misplaced due to a program error. In some cases, mistakes may be caused and human life may be involved.
[0023]
The present invention solves such problems, and its purpose is to achieve high speed formation of a large number of spots and high density on a base solid phase plate in a system in which a probe solution is discharged as droplets. Providing a method for manufacturing a probe array that maintains the advantages and can prevent the shipment of defective probe arrays that could lead to medical errors, etc., through accurate inspection of sequence errors in the resulting probe array There is to do.
[0024]
[Means for Solving the Problems]
As a result of diligent research in order to solve the above problems, the inventors of the present invention found that spots of probe droplets of the same type were formed when the spots of probe droplets were formed by a liquid ejecting apparatus using an ink jet method. By forming at least two spots, classifying those spots for quality inspection and for products used by users, and providing a quality inspection process for inspecting the quality of the probe array using the quality inspection spots The present inventors have found that spot missing and misalignment can be accurately checked, and the manufacturing yield of the probe array can be significantly improved.
[0025]
  That is, the method for producing a probe carrier of the present invention is a method for producing a probe carrier in which a plurality of types of probes are arranged on a carrier by ejecting a probe solution containing the probe from the liquid ejection device onto the carrier,
  The liquid ejection device is provided with at least the number of liquid ejection units corresponding to the plurality of types of probe solutions, and the liquid ejection units are configured to eject and fix the solutions of the plurality of types of probes from each of the liquid ejection units. And forming two or more spots for at least one of the plurality of types of probes;
A quality inspection step in which two or more spots of the same probe are classified into a quality inspection product and a product inspection, and a quality inspection reagent is further added to the quality inspection spot to perform a quality inspection;
It is a manufacturing method of the probe carrier characterized by having.
[0026]
  In the method for producing the probe carrier of the present invention,A liquid ejection device used for manufacturing a probe carrier in which a plurality of types of probes are arranged on a carrier, wherein a liquid container for storing a probe solution containing the probe, and a probe solution supplied from the liquid container A liquid discharge portion having a discharge port for discharging, a liquid path that allows the liquid storage portion and the discharge port to communicate in common, and discharge energy generating means that enables discharge of the probe solution from these discharge ports And at least one of the plurality of types of probes is formed on the carrier so that two or more spots are formed.Can be used.
[0027]
A probe carrier manufacturing apparatus can be obtained by using this liquid ejection apparatus, a holding means for holding the carrier, and an alignment means for aligning the relative position of the liquid ejection apparatus with respect to the carrier.
[0028]
According to the present invention, two types of spots for quality inspection and product are formed for probe spots that are considered to require quality inspection. By using the spots for quality inspection, the obtained probe array, etc. The quality inspection of the probe carrier can be performed accurately.
[0029]
In addition, by dropping the reagent in the quality inspection process by the liquid discharge method, it is possible to drop the reagent aiming only at the quality inspection spot among a plurality of spots, and to enlarge the substrate area. Therefore, the quality inspection can be performed accurately.
[0030]
The liquid ejection device used in the method of the present invention is formed to have at least the number of liquid ejection portions corresponding to a plurality of types of probe solutions. The liquid discharge section has a liquid storage section for storing a liquid, a discharge port for discharging the liquid, and a discharge energy generating means for discharging the liquid supplied from the liquid storage section. Can be suitably used. In the liquid discharge apparatus having such a configuration, it is preferable that a plurality of discharge ports of each liquid discharge unit are arranged in a one-dimensional or two-dimensional array.
[0031]
Furthermore, as this liquid discharge part, what has a structure which is provided with the some discharge opening with respect to the same liquid storage part, and forms simultaneously the spot which consists of a several same probe solution on a support | carrier can be used. In this way, by using a configuration in which a plurality of spots are simultaneously formed by a plurality of ejection openings provided corresponding to one liquid storage unit, the inspection spot can be formed without relative movement between the liquid ejection device and the carrier. Both product spots can be formed at once, eliminating the possibility of malfunctions related to relative movement, and making the reliability of various tests and inspections using probe carriers such as probe arrays almost 100% Can do. It is also possible to set the spot diameter for quality inspection to be smaller than the diameter of the product spot, thereby minimizing the amount of probe solution used.
[0032]
Furthermore, it is preferable that the liquid ejection apparatus is configured as a chip in which a plurality of liquid ejection units are provided on the same substrate. In such a configuration, the heater element is provided at a predetermined position on the substrate, the probe solution flow path provided at the corresponding position, and the probe solution supplied from the flow path in communication with the flow path. It can be obtained by forming a discharge port for discharging by heat generation, and further forming a liquid storage portion communicating with the flow path on the back side of the substrate. In this configuration, the discharge port, the flow path, and the liquid storage portion constitute a path that penetrates the substrate. Each heater element has a configuration in which both ends are connected to first and second wirings that are insulated from each other, and is driven by an electrical signal applied through these first and second wirings. it can.
[0033]
Further, a plurality of such chips can be provided side by side to constitute one liquid ejection device.
[0034]
The liquid discharge unit is configured such that liquid is discharged from the discharge port by generating bubbles in the flow path by heating from the heater element. During the discharge operation, the bubbles are discharged from the outside air through the discharge port. The structure which communicates can be utilized suitably.
[0035]
When the liquid discharge device is formed in such a chip shape, the liquid discharge direction from the discharge port is a direction perpendicular to the plane formed by the discharge ports arranged in a one-dimensional or two-dimensional array. A liquid discharge apparatus in which a liquid storage portion is provided corresponding to each discharge port on the side opposite to the side where the outlet is formed can be suitably used.
[0036]
Further, in the configuration using the liquid storage unit, the liquid storage unit for increasing the amount connected to the liquid storage unit is a plate integrally formed in an array shape, and the plate is formed in a flat chip shape. It is possible to use a liquid ejection device in which a liquid storage unit and an increase storage unit corresponding to each other are connected to the back surface provided with the liquid storage unit.
[0037]
In addition, the configuration for driving the heater element in the liquid discharge apparatus is a simple configuration including only wiring such as aluminum for applying a voltage to the heater, and a pad for connecting the liquid discharge apparatus and the outside. Those are preferred.
[0038]
In the present invention, if at least one of a plurality of types of probes is formed from a plurality of the same probes, a spot is formed. A part of the spot of the same probe can be used for quality inspection. A quality inspection reagent can be applied to the spot classified for quality inspection to perform the quality inspection. A liquid discharge device for reagent discharge can also be suitably used for applying the reagent for quality inspection.
[0039]
DETAILED DESCRIPTION OF THE INVENTION
In the method for producing a probe carrier according to the present invention, when producing a probe carrier such as a two-dimensional probe array including a plurality of types of probe spots, a probe prepared separately in advance is used as a solution in an array with a predetermined arrangement. Each probe solution is ejected onto the carrier in a desired liquid amount using a liquid ejection device in which an ejection port is disposed, and a dot-shaped probe solution spot is formed by applying the probe solution thereto. Probes in the spots of each probe solution are fixed to a carrier, and a probe carrier such as a probe array is obtained. According to the method of the present invention, a high density of various probes in a probe carrier such as a probe array can be achieved.
[0040]
The probe carrier production method and production apparatus of the present invention will be described in more detail below. In particular, a method for manufacturing a probe array having a step of discharging and applying each probe solution on a carrier in a desired amount by a so-called thermal jet type liquid discharge device that discharges droplets using a heater element. Explained.
[0041]
The amount of the probe solution discharged from one discharge port at a time is formed in consideration of various factors such as the viscosity of the probe solution, the affinity between the probe solution and the carrier, and the reactivity between the probe and the carrier. It is appropriately selected according to the size and shape of each dot spot constituting the probe array. Generally, an aqueous solvent is used for the probe solution. In the method of the present invention, the probe solution discharged from each discharge port at a time is generally 0.1 to 100 picoliters. It is preferable to select the range and to design and adjust the discharge port diameter according to the liquid amount.
[0042]
The area occupied by the array unit (unit area where one spot is formed) to which this probe solution is applied is 0.01 (for example, 0.1 μm × 0.1 μ) μm²To 40000 (for example, 200 μm x 200 μm) μm²Is generally determined by the size of the array itself and the density of the array matrix.
[0043]
The size and volume when using the liquid storage portion are also appropriately selected depending on the amount of probe solution discharged from the discharge port at a time and the number of arrays to be prepared. Since the amount of probe solution to be discharged at one time is naturally within a certain range depending on the setting of the discharge port diameter, a plurality of liquid discharge portions of the liquid discharge device are processed by processing a substrate made of a semiconductor such as silicon or glass. When forming into an integrated chip shape, a size capable of integrally forming the liquid storage portion on the back surface of the substrate facing the discharge port as described later may be sufficient (see FIGS. 4 and 5).
[0044]
On the other hand, if the amount of probe solution discharged from the discharge port at a time is relatively large and the number of arrays required to be manufactured is large, the probe solution is added to the liquid storage unit integrally formed on the substrate each time. A method that covers the amount of solution required when manufacturing the total number of arrays can be used. Alternatively, the liquid storage unit disposed on the back side of the substrate may be connected to a liquid storage unit for increasing the volume. At that time, the probe solution is added through the liquid storage unit for increasing the amount, and the shape of the liquid storage unit on the substrate side itself should be such that the probe solution can be easily supplied as necessary. it can.
[0045]
For each probe used for manufacturing the probe array, a liquid discharge portion is formed by associating a pair of liquid storage portions and discharge ports, and the discharge ports arranged in the plurality of liquid discharge portions are preferably one-dimensional. Alternatively, the discharge port surface is formed in a two-dimensional array. Therefore, the number of liquid ejection units including a pair of liquid storage units and ejection ports is not particularly limited, and is selected according to the required probe type of the array. The diameter of the dot-like spot, the number of spots, the density thereof, or the arrangement pattern shape of the entire array, and the total number of liquid ejection units are basically determined by the number of probe types required.
[0046]
In the method of the present invention, in general, the probes arranged in a two-dimensional array on the substrate are of the same type in a large sense.
[0047]
The probe immobilized on the carrier can be specifically recognized by a specific target (target) and is often called a ligand. In addition, the probes include oligonucleotides, polynucleotides, or other polymers that can be recognized by a particular target. The term “probe” means a probe molecule having a probe function such as an individual polynucleotide molecule or a probe having the same probe function such as a polynucleotide having the same sequence immobilized on the carrier surface in a dispersed state. May refer to a population of molecules. A probe can also bind to or become able to bind to a target as part of a ligand-antiligand pair. Probes and targets in the present invention may include bases as found in nature, or analogs thereof.
[0048]
The probe employed in the probe carrier produced by the method of the present invention is appropriately selected according to the purpose of use. DNA, RNA, cDNA (complementary DNA), PNA, oligonucleotides, polynucleotides, other nucleic acids, oligopeptides, polypeptides, proteins, enzymes, substrates for enzymes, antibodies, epitopes for antibodies, antigens, hormones, hormone receptors , A ligand, a ligand receptor, an oligosaccharide, or a polysaccharide is preferable, and two or more of these can be used in combination as necessary.
[0049]
In the present invention, a plurality of these probes, each of which is fixed to the carrier surface as an independent region, for example, a dot-like spot, is referred to as a probe carrier, and many of the probe spots are arranged in a plane. Those arranged in a two-dimensional array are called probe arrays. The probe carrier includes a DNA macroarray, a DNA chip, and a test plate or chip generally called a probe array.
[0050]
On the other hand, in the method of the present invention, the probe has a structure that can be bound to the surface of the carrier, and it is desirable that the probe be fixed on the carrier via the bondable structure. At that time, the structure that can be bonded to the carrier surface of the probe is an amino group, sulfhydryl group, carboxyl group, hydroxyl group, acid halide (haloformyl group; -COX), halide (-X), aziridine, maleimide group, Succinimide, isothiocyanate, sulfonyl chloride (-SO₂Cl), aldehyde (formyl group; —CHO), hydrazine, iodinated acetamide and the like, are preferably formed by treatment.
[0051]
【Example】
The present invention will be described more specifically with reference to the following examples. In addition, although the Example shown here is an example of preferable embodiment in this invention, this invention is not limited by these Examples.
[0052]
First, a probe array manufacturing apparatus using a liquid discharge apparatus in which discharge ports used in a probe solution discharge process are arranged in a two-dimensional array and heat energy is used for liquid discharge will be described.
[0053]
(Structure of the probe solution discharge liquid discharge device)
(Example 1)
FIG. 1 is a schematic plan view illustrating a structure of a discharge port forming surface of an example of a liquid discharge apparatus. This liquid ejection apparatus has a structure composed of a chip in which a large number of liquid ejection sections are arranged on a substrate made of silicon. As will be described in detail later, in this embodiment, a plurality of chips are used to form one liquid discharge device, which is used as a liquid discharge means (liquid discharge unit) of the probe array manufacturing apparatus. Note that most of the functions of the liquid ejection device depend on the configuration of the chip.
[0054]
FIG. 1 is a diagram for explaining a basic configuration of a liquid ejection apparatus used in the present invention, and shows a configuration in which each liquid ejection section has one ejection port (nozzle) for convenience. In the liquid ejection device used in the present invention, with respect to one liquid storage portion (reservoir) as shown in the enlarged view of the vicinity of the nozzle in FIG. 2 (corresponding to the portion surrounded by a circle in FIG. 1), A configuration in which two sets of one heater element and one nozzle are arranged is preferably used.
[0055]
1 and 2, 1 is a substrate made of silicon, 2 is a heater element made of TaN, TaSiN, TaAl or the like, 3 is a first wiring made of aluminum or the like, 4 is a second wiring made of aluminum or the like, 5 is A pad for making electrical contact between the unit and the outside, 6 is a nozzle, and 8 is a supply port which is provided on the back surface of the substrate and constitutes a portion for storing a probe solution to be supplied to the nozzle on the substrate surface side. The supply port functions as a liquid reservoir and is created by anisotropic etching of silicon, which will be described later. At this time, the supply port 8 has a quadrangular pyramid-shaped opening whose edge extends to a portion indicated by 9 (dotted line portion) on the back surface of the substrate 1. In this configuration, the supply port, the flow path (see FIGS. 3 and 4 described later), the heater element, and the nozzle form an independent liquid discharge section.
[0056]
The direction indicated by the white arrow in FIG. 1, which will be described in detail later, is the direction in which the unit moves when the liquid ejection device is mounted on the probe array manufacturing apparatus as a liquid ejection unit. Is described.
[0057]
As shown in FIG. 2, in this embodiment, two nozzles and heater elements correspond to one supply port in the same liquid ejection unit. Two portions of a pair of adjacent nozzles and heater elements in the same liquid discharge section are separated by a partition wall and are arranged at an interval of 1.27 mm (20 dpi). Each heater element is coupled to a pair of first wiring and second wiring, and the two wirings are connected to the outside through two pads (omitted) at the substrate end.
[0058]
In the configuration shown in FIG. 1, the chip has five one-dimensional (straight) nozzle rows each having eight nozzles arranged with an interval between adjacent nozzles of 1.27 mm (equivalent to 20 dpi) in the horizontal direction. Are arranged with an offset of 100 dpi in the vertical direction (shift in the horizontal direction). The interval between nozzle groups adjacent in the vertical direction in FIG. 1 is also set to 100 dpi. Each heater element in one nozzle row is connected to both ends in common with a pair of first and second wires.
[0059]
The nozzle spacing and nozzle row spacing in the configuration shown in FIG. 2 are also set in accordance with the mode of FIG. In the case of adopting the configuration of FIG. 2, the interval between the nozzle rows adjacent in the vertical direction of FIG. 1 is set with reference to the center position between two nozzles provided close to each other in the same liquid ejection unit, for example. can do.
[0060]
Also in the configuration in FIG. 2, each of the 16 heater elements included in a set of nozzle rows is commonly connected to a pair of first wiring and second wiring. The length of the chip in the long side direction is about 12 mm, and the length in the short side direction is about 7 mm. The number of nozzles in one chip is 80.
[0061]
FIG. 3 is a sectional view taken along line A-A ′ of FIG. FIG. 4 is a sectional view taken along line B-B ′ of FIG. FIG. 5 shows a schematic diagram of the back surface of the chip (corresponding to the back surface of the semiconductor chip shown in FIG. 1). 3 and 4, the same reference numerals as those used in FIGS. 1 and 2 denote the same components. Further, 10 is an insulating film, 11 is a protective film, 12 is a nozzle material, and 13 is a cavitation resistant film made of Ta or the like. The insulating film 10 may be any film such as a thermal oxide film formed by thermally oxidizing a silicon substrate, an oxide film, a nitride film, etc. formed by CVD. The protective film 11 may be any film such as an oxide film or a nitride film formed by CVD. The nozzle 6 and the channel 7 may be formed on the substrate by attaching a nozzle material having nozzles and channels in advance to the substrate or by extending the semiconductor process using photolithography technology.
[0062]
In particular, when the chip needs to be manufactured with a larger area, the method of attaching the nozzle material having the nozzle and the flow path to the semiconductor substrate is a wrinkle, a warp, Since problems such as misalignment may occur, a manufacturing method is desired in which a nozzle material is stacked on a substrate using a photolithography technique. As a method of forming a nozzle using photolithography, for example, there is a method described in JP-A-62-264957.
[0063]
The supply port 8 is produced by, for example, anisotropic etching of silicon using a TMAH solution, and opens at an angle of 54.7 degrees with respect to the horizontal plane on the back surface of the substrate, as shown in FIG. The shape of the supply port 8 is a quadrangular frustum shape as shown in FIG. In this embodiment, the width of the supply port on the substrate surface is set to 100 μm as shown in FIG. 3, and the thickness of the silicon substrate 1 is 625 μm, so the width on the back surface of the supply port is about 1 mm. It becomes.
[0064]
In a conventional inkjet head for printing, the main purpose of the supply port is to guide ink from a connection portion with an ink tank connected to the back surface of the substrate to the heater portion. However, as described above, in an apparatus for manufacturing a probe array, Since the total discharge amount of the liquid is small, the supply port can be used as a liquid reservoir.
[0065]
In the supply port of the above dimensions, the volume of the portion that can be used as the liquid reservoir is about 0.23 μl. In this embodiment, the total discharge amount from the two nozzles is 24 pl, so this volume is about 9600 probes. -It corresponds to the quantity which can produce an array. When the chip is viewed from the back surface, the supply port 8 has the shape shown in FIG. As shown in FIG. 4, the liquid is guided from the supply port 8 to the substrate surface from the back surface of the substrate, and is guided to the nozzle 6 through the flow path 7. That is, a path that penetrates the substrate is formed by the supply port (liquid reservoir), the flow path, and the nozzle.
[0066]
When voltage is applied to both ends of the heater, the liquid in the vicinity of the heater is overheated to cause film foaming, and the liquid is discharged as shown in FIG. The amount of liquid ejected from the nozzle was 12 pl. In order to obtain this droplet amount, the heater was 28 μm × 28 μm, and the nozzle was 22 μmφ.
[0067]
In order to discharge liquid stably, it is essential to cause film foaming stably. In order to cause stable film foaming, it is desirable to apply a voltage pulse of 0.1 to 5 μs to the heater. Further, in order to precisely control the amount of each probe included in the probe array, the amount of droplets ejected from the nozzle is stable, Japanese Patent Laid-Open No. 4-10940 and Japanese Patent Laid-Open No. 4-10941, As described in JP-A-4-10942, a method in which bubbles generated by heating with a heater communicate with the outside air through a discharge port is preferable.
[0068]
Further, FIG. 8 shows a schematic diagram of a liquid ejection apparatus in which a plurality of chips are arranged in parallel. In FIG. 8, 21 is a liquid discharge apparatus having a portion in which 25 chips are arranged in parallel, 22 is the chip described in FIGS. 1 to 7, and 23 is a nozzle. In FIG. 8, the tip 22 shows only the nozzle 23 for the sake of clarity. In this liquid ejection apparatus, the chips 22 are arranged in 5 rows and 5 columns, and each chip has 80 nozzles, so one unit has a total of 2000 nozzles.
[0069]
FIG. 9 shows an example of a configuration when a plurality of chips are arranged in parallel. As shown in FIG. 9, a window frame-shaped fixing frame 24 made of alumina, resin, or the like and having openings 25 formed therein is used to insert and fix a chip 22 in each opening. It is possible to obtain a liquid ejection device in which the chips are arranged in parallel. The pads of each chip are electrically connected to the outside of the head by a flexible wiring board (not shown) disposed on the frame body 24. The solution is injected into the supply port 8.
[0070]
(Structure of probe array manufacturing equipment)
FIG. 10 shows a schematic diagram of the structure of the main part of an example of a probe array manufacturing apparatus using the liquid discharge device as a liquid discharge unit. In FIG. 10, 22 is a shaft that guides the movement of the liquid ejection unit 31 substantially in parallel, 32 is a stage to which a carrier for manufacturing the probe array is fixed, 33 is a carrier on which the probe array is formed (this example) Then, a glass substrate is used.
[0071]
The unit moves in the X direction in FIG. 10, the stage moves in the Y direction, and the unit can move relative to the stage in two dimensions by these operations. In FIG. 10, a structure in which a plurality of glass substrates serving as probe arrays are fixed and probes are applied is shown. However, a probe array is manufactured on a single glass substrate serving as a large probe array, The glass substrate may be cut to obtain a probe array.
[0072]
(Method of applying probe solution using liquid discharge unit)
Next, a probe array manufacturing method will be described. FIG. 11 is a schematic diagram for explaining a method of applying a probe solution using a probe array manufacturing apparatus. In FIG. 11, reference numerals 41 to 42 denote chips, and 33 denotes a probe array. In FIG. 11, since the surface of the probe array is drawn face up, the arrangement of the nozzles of the chip and the main scanning direction are opposite to those shown in FIG.
[0073]
11, 41 is the chip indicated by “1” in FIG. 8, and 42 is the chip indicated by “2” in FIG. As shown in FIG. 1, the chip configuration includes eight nozzle groups, in which nozzle groups are offset. Since each heater in one nozzle group is commonly connected to a pair of first and second aluminum wirings as described above, a voltage pulse is generated between a pair of pads connected to these wirings. By applying this, it is possible to discharge liquid from all nozzles in the same nozzle group. That is, discharge occurs simultaneously with 16 nozzles.
[0074]
In FIGS. 8 to 11, numerals 1, 2, 3, 4, 5 and A, B, C, D, E, F, G, and H displayed adjacent to the chip indicate nozzle groups. It is the thing displayed for. Each nozzle group includes two nozzles shown in FIG.
[0075]
First, by the head of the first nozzle group, eight sets and 16 spots indicated by 1A, 1B, 1C, 1D, 1E, 1F, 1G, and 1H in FIG. ) Is formed. Next, ejection is performed sequentially from the 16 nozzles of the second nozzle group at the timing when the head has moved 1.27 mm (equivalent to 20 dpi) in the main scanning direction, and 2A, 2B, 2C, 2D, and 2E in FIG. , 2F, 2G, 2H, 8 sets of 16 spots are arranged in two rows on the probe array. In the same manner, the nozzles are sequentially discharged from the third to fifth nozzle groups, and 80 probe groups in the first row of the probe array 33 (indicated by black circles and white circles in FIG. 11) are arranged. . At this time, the distance between the centers of adjacent probes is 254 μm (corresponding to 100 dpi). That is, the 40 types of liquid discharged from the first chip form a first row of probe groups in the probe array. This is schematically shown at 43 in FIG. (Only 5 spots of 1A to 5A are shown in 40 spots.)
Next, liquid was discharged from the second chip by the same operation, and the probes in the second row (indicated by white circles in FIG. 11) of the probe array were arranged. At this time, the center-center distance between the first row of probe groups produced from the first chip and the second row of probe groups produced from the second chip is 254 μm (corresponding to 100 dpi). The drive timing was adjusted so that
[0076]
Further, droplets were discharged from the third to 25th chips by the same operation, and a probe array was produced. (Spots surrounded by dotted lines in Fig. 11)
As described above, a probe array of 40 rows and 25 columns (1000 sets, 2000 spots) of 100 dpi can be produced from the head shown in FIG. At this time, since the two spots of each set are connected to the same reservoir, it is guaranteed that the same probe solution is discharged, and either spot (for example, the black circle in FIG. 11) is used as a spot for quality inspection. The other (for example, the white circle in FIG. 11) can be used as a product. Further, since the distance between the two spots is determined by the interval between the nozzles on the liquid ejecting apparatus, it does not include a substrate movement error or the like, and is very accurate. For this reason, it is possible to arrange the two spots as close as possible so that the two spots do not overlap, and the size of the probe array can be reduced.
[0077]
A quality inspection step can be provided after the probe array is manufactured using the quality inspection spots formed in this way. That is, it is possible to confirm that a desired probe is formed at a desired position by dropping a reagent on the quality inspection spot and observing the reaction. In this case, it is necessary to precisely control the position and the amount of the reagent so that the reagent is dropped only on the target spot and the reagent does not come into contact with other spots. Is preferably used. The configuration of the reagent dropping device in this case is preferably similar to that shown in FIG. However, it is not necessary to have the same number of nozzle groups and reservoir groups as the number of probes. For example, if a reagent that reacts with a plurality of probes is used, the liquid discharge unit can be simplified, and preferably one or two chips 22 are used. It is also possible to use a configured liquid discharge unit.
[0078]
In addition, the number of quality inspection spots is not limited to one, and by providing two or more inspection spots and applying different reagents to each, it is possible to reliably inspect with fewer types of reagents. . In this example, two spots are formed by two nozzles, but three or more nozzles may be installed for one liquid reservoir.
[0079]
It is also possible to form two spots with one nozzle for quality inspection and product, respectively. However, since the substrate is moved during the formation of the two spots, it is necessary to increase the distance between the spots in consideration of a movement error, and the substrate may become large. Therefore, in order to achieve spot arrangement with higher density, a plurality of nozzles are provided in advance for one liquid reservoir, thereby simplifying the control system when scanning the unit with respect to the carrier. Since it can be used, it is preferable.
[0080]
The case where silicon is used as the chip constituting the unit has been described so far. However, as described above, since the present invention has a simple structure including a heater and wiring connected to the heater, a silicon substrate is not necessarily used. However, the liquid discharge unit can be manufactured using a less expensive substrate such as a glass substrate.
[0081]
In the present embodiment, the case where a semiconductor chip having 80 nozzles is arranged in 5 rows and 5 columns to create a head has been described. However, the number of nozzles and the arrangement arrangement are limited to the configuration shown in the embodiment. You can choose freely. However, as described above, when a one-dimensional array of liquid discharge units such as one row and 25 columns is produced using a semiconductor chip, a probe array can be created only by main scanning. The structure shown in FIG. 10 can be made simpler.
[0082]
(Example 2)
FIG. 12 shows a schematic diagram of another example of a semiconductor chip constituting the liquid discharge unit according to the present invention. FIG. 12 is an enlarged view of the vicinity of the nozzle, which corresponds to FIG. 2, and the same numbers indicate the same parts.
[0083]
As shown in FIG. 12, in this embodiment, two nozzles and a heater are arranged on both sides of the supply port for one supply port. The sizes of the two sets of nozzles and heaters are different from each other, and are a combination of a heater 36 μm × 36 μm, a discharge port 26 μmφ, a heater 29 μm × 29 μm, and a discharge port 14 μmφ. The former is designed to eject 24 pl droplets and the latter is designed to eject 8 pl droplets. Since the appropriate driving conditions (voltage, pulse width) differ depending on the heater size, each heater is connected to a different set of first and second wires, for a total of two sets of wires. Are connected to the outside through two sets of pads (omitted) at the end of the substrate. As shown in FIG. 1, the chip is composed of 8 nozzle groups arranged in the horizontal direction in FIG. 1 at intervals of adjacent nozzle groups of 1.27 mm (equivalent to 20 dpi). The columns are arranged with an offset of 100dpi. The interval between adjacent nozzle groups is also set to 100 dpi.
[0084]
The shape of the supply port is a quadrangular frustum shape as shown in FIG. In this prototype, the width of the supply port on the substrate surface is set to 100 μm as shown in FIG. 3, and the thickness of the silicon substrate 1 is 625 μm, so the width on the back surface of the supply port is about 1 mm. It becomes. The length of the chip in the long side direction is about 12 mm, and the length in the short side direction is about 7 mm.
[0085]
(Method of applying probe solution using liquid discharge unit)
A probe array was manufactured in the same manner as in Example 1 using this liquid discharge unit. The difference is that the formed spots are 90 μmφ and 60 μmφ in different combinations of sizes. When the reagent was dropped onto the 60 μmφ spot and observed, the reaction could be confirmed. As a result, the amount of probe used was reduced from 48 pl (24 + 24) to 32 pl (24 + 8). In addition, since the sizes of the spot for quality inspection and the spot for product are different, it is expected that there will be no confusion in the determination at the medical site.
[0086]
Example 3
FIG. 13 shows a schematic diagram of another example of a semiconductor chip constituting the liquid discharge unit according to the present invention. FIG. 13 is an enlarged view of the vicinity of the nozzle, which corresponds to FIG. 2, and the same numbers indicate the same parts.
[0087]
As shown in FIG. 13, in this embodiment, 11 nozzles and heaters are arranged for one supply port, one on one side and 10 on the opposite side across the supply port. Adjacent nozzles and heaters are separated by a partition wall (not shown) and are arranged at an interval of 42.5 μm (600 dpi). Each heater is coupled to a different set of first wiring and second wiring, and a total of 11 sets of wirings are connected to the outside through 11 sets of pads (omitted) at the substrate end. As shown in FIG. 1, the chip is composed of 8 nozzle groups arranged in the horizontal direction in FIG. 1 at intervals of adjacent nozzle groups of 1.27 mm (equivalent to 20 dpi). The columns are arranged with an offset of 100 dpi. The interval between adjacent nozzle groups is also set to 100 dpi.
[0088]
The shape of the supply port is a quadrangular frustum shape as shown in FIG. In this prototype, the width of the supply port on the substrate surface is set to 100 μm as shown in FIG. 3, and the thickness of the silicon substrate 1 is 625 μm, so the width on the back surface of the supply port is about 1 mm. It becomes. Further, as can be seen from FIG. 13, the long side of the supply port is 42.5 × 10 = 425 μm. As a result, the length in the long side direction of the chip is about 12 mm, and the length in the short side direction is about 9 mm.
[0089]
(Method of applying probe solution using liquid discharge unit)
When this liquid discharge unit is used, first the first nozzle in two rows discharges, then the liquid ejecting apparatus is moved in the main scanning direction, and the remaining nine nozzles are discharged in synchronization with the moving speed. The spot formed by the solution ejected from one nozzle of the left nozzle row and the right 10 spots are ejected on the substrate so that the droplet completely overlaps the first droplet on the substrate. It is possible to obtain two spots having different sizes from spots formed by the solution discharged from the nozzle. In this example, spots of two sizes of 40 μm and 90 μm were obtained by discharging 2.4 pl droplets using a 24 × 24 μm heater and a discharge port of 11 μmφ. As a result, the amount of probes for quality inspection was further reduced, and the efficiency was improved.
[0090]
The advantage of this method is that it is possible not only to easily obtain different spot diameters by changing the number of nozzles, but also to form a spot for a product with a total of droplets ejected from a plurality of nozzles. Even if the discharge of one nozzle is irregular, the influence may be small. In this example, the spot for inspection is formed by using one nozzle, but it is of course possible to prepare for irregular discharge by using a plurality of nozzles. Moreover, it is also possible to form three or more spots having different sizes by simultaneously discharging from a plurality of nozzles.
[0091]
(Method of applying probe solution using liquid discharge unit)
When this liquid discharge unit is used, first, main scanning and sub-scanning are simultaneously performed, and the same circular movement as the nozzle arrangement is performed in the relative movement between the substrate and the head, thereby sequentially discharging from the nozzles on the circumference. The probe solution is driven to land at the same point on the substrate. Thereafter, the probe array is completed by repeating the circular motion by moving 1.27 mm in the main scanning direction.
[0092]
Reference example
(Manufacture of DNA array using liquid discharge unit)
25.4mm x 25.4mm x 0.5mm^tThe fused quartz substrate was ultrasonically washed in 1% ultrasonic detergent GP-III (Branson) for 20 minutes, and then ultrasonically washed with tap water and washed with running water as appropriate. Next, it was immersed in 1N NaCl at 80 ° C. for 20 minutes, washed with running water (tap water), ultrapure water ultrasonic washing, and running water (ultra pure water).
[0093]
Purified by distillation under reduced pressure, the following formula (I):
[0094]
[Chemical 1]

[0095]
An aminosilane coupling agent (KBM-603: Compound I manufactured by Shin-Etsu Chemical Co., Ltd.) at a concentration of 1% was stirred at room temperature for 1 hour to hydrolyze the methoxy group portion. Next, the substrate was immediately immersed in the silane coupling agent aqueous solution after cleaning, and immersed at room temperature for 1 hour. Thereafter, it was washed with running water (ultra pure water), dried by blowing nitrogen gas, and then heat-fixed in an oven at 120 ° C. for 1 hour.
[0096]
After cooling, the following formula (II):
[0097]
[Chemical 2]

[0098]
The substrate was immersed in a 0.3% solution (ethanol: dimethyl sulfoxide = 1: 1) of N- (6-maleimidocaproxy) succinimide (EMCS; Compound II) for 2 hours at room temperature, and EMCS was added as an aminosilane coupling agent. The amino group was reacted. After completion of the reaction, it was washed once with ethanol: dimethyl sulfoxide = 1: 1 and three times with ethanol, and dried by blowing nitrogen gas.
5'-ATGAACCGGAGGCCCATC-3 '(1) (SEQ ID NO: 1)
3'-TACTTGGCCTCCGGGTAG-5 '(2) (SEQ ID NO: 2)
It has a base sequence of (2) which is a base sequence complementary to the base sequence of (1) described above, and is finally bonded to the purified maleimide group on the substrate surface via a linker at the 5 ′ end. An oligonucleotide (Compound III Bex Co., Ltd.) having a possible mercapto group (SH group: also referred to as a sulfhydryl group) was used as a probe used for verification of this example. Formula (III):
[0099]
[Chemical Formula 3]

[0100]
Solvent for discharging the oligonucleotide (compound III) introduced with the mercapto group of the liquid discharge unit, that is, 7.5% by mass of glycerin, 7.5% by mass of urea, 7.5% by mass of thiodiglycol, Formula (IV):
[0101]
[Formula 4]

[0102]
In an aqueous solution containing 1% by mass of acetylene alcohol (for example, trade name: acetylenol EH Kawaken Fine Chemical Co., Ltd.), the absorbance was 1.0. This oligonucleotide solution was supplied to the liquid reservoir of the liquid discharge unit prepared in Example 1 using a microdispenser. Before filling the final oligonucleotide solution, each liquid reservoir and nozzle should be washed with a solvent having the above composition in advance and used as necessary for the purpose of making the prepared liquid discharge unit compatible with the solvent. Washing with the oligonucleotide solution was repeated as appropriate to remove the liquid by vacuum suction.
[0103]
Thereafter, the oligonucleotide solution was discharged onto the substrate subjected to the treatment for introducing the maleimide group. The design specification of the head manufactured in Example 1 is that the liquid amount per droplet to be ejected is 24 pl. Under this ejection condition, the diameter of the dot occupied by one droplet to be applied on the substrate is Depending on the viscosity of the solution used, it is in the range of 70-100 μm.
[0104]
In addition, the solvent having the above composition is highly moisturizing, so that the drying, concentration in the liquid reservoir, and droplets of the oligonucleotide solution applied on the substrate are not fixed by reaction with the substrate surface in the next step. , Can prevent drying and solidification.
[0105]
The substrate to which the oligonucleotide (compound III) solution of the formula (III) was applied in a two-dimensional array was held in a moisture retention chamber at 100% humidity for 1 hour at room temperature, and the mercapto group of the oligonucleotide and the maleimide on the substrate Reaction with the group was carried out. After removal, the substrate was washed in running water (ultra pure water) for about 30 seconds in order to remove the unreacted oligonucleotide.
[0106]
Next, with respect to the substrate on which the dots for immobilizing the oligonucleotides are formed in a two-dimensional array, in order to perform blocking treatment on the surface other than the dots, BSA (containing 1M NaCl, pH = 7.0) is applied to the surface other than the dots. Bovine serum albumin (Sigma Aldrich Japan) was soaked in a blocking solution dissolved at a concentration of 2% for 1 hour, washed with the 50 mM phosphate buffer, stored in the 50 mM phosphate buffer, did.
[0107]
(Evaluation of dot shape of DNA array by hybridization reaction)
As a model target DNA, the formula (V):
[0108]
[Chemical formula 5]

[0109]
2 prepared using Compound V (purchased from Vex Co., Ltd.) having the sequence of (1) shown in Example 2 and having tetramethylrhodamine bound to the 5 ′ end as a fluorescent label. Hybridization reactions with probes on a dimensional array were performed.
[0110]
This hybridization reaction uses a prepared DNA array and 2 ml of a phosphate buffer (compound V containing 10 mM phosphate buffer pH = 7.0, 50 mM NaCl) containing Compound V at a concentration of 5 nM. I went there. The DNA array was sealed in a hybrid pack together with the model target DNA solution, heated to 70 ° C. in a thermostatic bath, then cooled to 50 ° C., and left in that state for 10 hours.
[0111]
Next, the DNA array is taken out from the hybrid pack and washed with a hybridization buffer for the purpose of removing unreacted target DNA. After washing, the substrate was placed on a slide glass in a state covered with a buffer solution, covered with a cover glass, and the fluorescence from the fluorescent label was observed. The fluorescence microscope used for this observation is a set of ECLIPSE E800 (Nikon Corporation) with a 20 × objective lens (plan apochromat) and a fluorescence filter (Y-2E / C). The image observed with a fluorescence microscope was captured using a CCD camera with an image intensifier (C2400-87 Hamamatsu Photonics Co., Ltd.) and an image processing device (Argus 50 Hamamatsu Photonics Co., Ltd.).
[0112]
Based on the recorded images, fluorescence was observed for all the dots formed by fixing the compound V formed in a two-dimensional array on the substrate. The average value of the fluorescence intensity was 1750 as the light intensity index of the above measuring apparatus. Moreover, when the average value of the diameter of a dot was computed from each area | region which emits the fluorescence, it was about 100 micrometers.
[0113]
(Preparation of DNA array consisting of 256 kinds of DNA probes)
5'-ATGAACCGGAGGCCCATC-3 '(3) (SEQ ID NO: 1)
3'-TACTTGGCCTCCGGGTAG-5 '(4) (SEQ ID NO: 2)
(3) is the same as the base sequence of SEQ ID NO: 1 of Example 2, but actually, a total of four base parts (underlined) encoding two amino acids having a high nucleotide mutation frequency of the carcinogenesis-related gene p53. A region of 18 nucleotides). The base sequence completely complementary to the base sequence of (3) is (4) (that is, the same as the sequence of SEQ ID NO: 2 of Example 2), and the part corresponding to the base indicated by the underline of (3) Is underlined.
5'-GATGGGN¹N²TCN^ThreeN^FourGTTCAT-3 '(5) (SEQ ID NO: 3)
(5) indicates the base at the position indicated by the underline of (4), N¹, N², N^Three, N^FourIt is represented by. These N¹, N², N^Three, N^FourHowever, a total of 256 types of oligonucleotide probes were synthesized by substituting any of the bases of ATGC and having a mercapto group bonded to the 5 'end. This was discharged onto a glass substrate using the ZBJ head produced in Example 1, and reacted and bonded to prepare a two-dimensional DNA probe array composed of 256 types of DNA probes.
[0114]
(Evaluation of selection characteristics of target DNA by hybridization)
Using the prepared 256 kinds of DNA probe arrays, a hybridization reaction was performed, and the selection characteristics of the target DNA having the target base sequence were verified.
[0115]
As the model target DNA, four types of DNA having the following base sequences and having tetramethylrhodamine bound to the 5 'end as a fluorescent label were synthesized.
5'-ATGAACCGGAGGCCCATC-3 '(3) (SEQ ID NO: 1)
5'-ATGAACGGGAGGCCCATC-3 '(6)
(SEQ ID NO: 4) C → G
5'-ATGAACGCGAGGCCCATC-3 '(7)
(SEQ ID NO: 5) C → G, G → C
5'-ATGAACGCGAAGCCCATC-3 '(8)
(SEQ ID NO: 6) C → G, G → C, G → A
As described above, (3) is completely complementary to the normal sequence of the p53 gene, and (6), (7), and (8) are compared to the base sequence underlined in the base sequence. It is a model of a mutant that is undergoing replacement.
[0116]
When the target DNAs having the four types of sequences (3), (6), (7), and (8) were individually hybridized, each of the dots in the prepared DNA array Fluorescence was observed only from the dots of the probe having a completely complementary base sequence to the target DNA, and no fluorescence was observed from the other dots. In each of the dots where fluorescence was observed, the observed fluorescence intensity was 1830 for target DNA (3), 1270 for target DNA (6), 1520 for target DNA (7), and target DNA (8). Was 1940.
[0117]
Although there is some variation, 1830 in the target DNA (3) agrees with the above average value 1750 in the dot shape evaluation of the DNA array within the statistical error. The difference in the range is not significant. Therefore, according to the manufacturing method of the present invention, a DNA array prepared by applying a probe solution on a substrate in parallel using an array-like liquid ejection device can be sufficiently used for quantitative detection of target DNA. It can be seen that the amount of probe having uniformity and reproducibility is fixed.
[0118]
【The invention's effect】
As described above, in the production of the probe carrier, a liquid ejecting apparatus in which nozzle groups are two-dimensionally arranged is used, and further, one type of probe solution is ejected from a plurality of nozzles to form two spots on the substrate. Thus, it is possible to inspect each probe, and it is possible to prevent a probe array having an incorrect arrangement from being shipped due to an injection error of the probe solution or a malfunction of the apparatus.
[0119]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 is a schematic view of a chip constituting a liquid ejection apparatus.
FIG. 2 is an enlarged view of the vicinity of a nozzle in the first embodiment of the liquid ejection apparatus according to the present invention.
3 is a cross-sectional view taken along line AA ′ of FIG.
4 is a cross-sectional view taken along line BB ′ of FIG.
FIG. 5 is a schematic diagram of the back surface of a chip constituting the liquid ejection apparatus (corresponding to the back surface of the chip shown in FIG. 1).
FIG. 6 is a schematic diagram for explaining the shape of a liquid supply port (reservoir) of a chip constituting the liquid ejection apparatus.
FIG. 7 is a schematic diagram for explaining liquid ejection by the liquid ejection apparatus.
FIG. 8 is a schematic diagram of a liquid ejection device.
FIG. 9 is a schematic diagram of a liquid ejection apparatus.
FIG. 10 is a schematic diagram of an apparatus for preparing a probe array having a liquid ejection device as a liquid ejection unit.
FIG. 11 is a schematic diagram of a probe array preparation method.
FIG. 12 is a schematic diagram of a second embodiment of the liquid ejection apparatus.
FIG. 13 is a schematic diagram of a third embodiment of the liquid ejection apparatus.

Claims (13)

A method for producing a probe carrier in which a plurality of types of probes are arranged on a carrier by ejecting a probe solution containing the probe from the liquid ejection device onto the carrier,
The liquid ejection device is provided with at least the number of liquid ejection units corresponding to the plurality of types of probe solutions, and the liquid ejection units are configured to eject and fix the solutions of the plurality of types of probes from each of the liquid ejection units. And forming two or more spots for at least one of the plurality of types of probes;
A quality inspection process in which two or more spots of the same probe are classified into quality inspection and product, and a quality inspection reagent is further added to the quality inspection spot to perform quality inspection. A method for producing a probe carrier, comprising: Each of the liquid ejection units has a liquid storage unit for storing a liquid, a discharge port for discharging a droplet, and generation of liquid discharge energy for discharging the liquid supplied from the liquid storage unit from the discharge port. The method for producing a probe carrier according to claim 1, further comprising: means.   The method for producing a probe carrier according to claim 1 or 2, wherein two or more spots from the same probe solution are formed adjacent to each other. 2 or more spots of the same probe solution, method for producing a probe carrier according to claim 1, comprising two or more spots of different diameters.   The probe carrier according to claim 4, wherein two or more spots from the same probe solution having different diameters have different diameters depending on a difference in the number of droplets of the probe solution applied to the carrier from the discharge port. Manufacturing method.   The two or more spots from the same probe solution having different diameters have different diameters depending on the volume of the probe solution for forming one spot applied from the discharge port to the carrier. A method for producing the probe carrier as described. The probe according to the discharge port is any one of the claims 2-6 forming the outlet group consisting of one-dimensional or two-dimensional array to be arranged a discharge opening on the front face of the liquid ejection apparatus A method for producing a carrier.   The probe carrier manufacturing method according to claim 7, wherein the ejection port group includes a plurality of rows of ejection ports arranged in parallel. The liquid discharge energy generating means, a manufacturing method of a probe carrier according to any one of claims 2-8 which is a heater element for generating heat energy with each of the liquid ejecting portions. Each of the liquid discharge portions includes a substrate on which the heater element is provided, a probe solution channel provided at a position corresponding to the heater element, a probe communicating with the channel and supplied from the channel A discharge port for discharging the solution by heat generation of the heater element, and a liquid reservoir in communication with the flow path,
The heater element is provided on the front side surface of the substrate, and the liquid reservoir is provided on the back side of the substrate,
These discharge ports, flow paths, and liquid reservoirs are configured in a chip that forms a path through the substrate,
The probe according to claim 9, wherein both ends of the heater element are connected to first and second wirings insulated from each other and driven by an electrical signal applied through the first and second wirings. A method for producing a carrier.   The liquid discharge unit has a structure in which bubbles are generated in the probe solution in the flow path when the probe solution is discharged from the discharge port by driving the heater element, and the bubbles communicate with the outside air through the discharge port. The method for producing a probe carrier according to claim 10, comprising: The method for producing a probe carrier according to any one of claims 1 to 11, wherein the reagent for quality inspection is applied from a liquid discharge device for discharging the reagent. The process according to any one of claims 1 to 12 wherein the probe is specifically recognizes substance target substance.

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