JP2004532382A - Method and apparatus for producing a support carrying a plurality of different polynucleotide and / or peptide sequences - Google Patents

Abstract

The present invention relates to the manufacture of electronic micro-sensors (chips) for analyzing nucleotide and / or peptide sequences. The aim is to easily and economically immobilize a large number of different probe sequences on the surface of the chip. The method of the present application aims at producing an array of sequenced probes on a support by immobilization and / or local chemical synthesis in the presence of a volatile solvent. The method uses a support on which a grid of reaction wells made from a photocured (epoxy) resin is made; in a reaction chamber saturated with vapors of a synthetic solvent (acetonitrile). Finally, using a localized supply of liquid / reagent in each well corresponding to a particular probe-sequence to immobilize the probe sequence in that well and optionally Ensure that in situ localized chemical synthesis occurs, and collectively supply common liquids / reagents to the wells, and ensure removal of liquid contained in each well prior to each localized supply operation It is characterized by including doing. The invention also relates to an apparatus for performing the method.

Description

【０１６５】
（１．２．装置）
使用される装置は、図３を参照して上記に記載されるものである。
【０１６６】
この装置は、表１の試薬Ａ、ＣおよびＤを射出するために図３Ａに示される型の６つのマイクロバルブ装置から作製される射出システム６を包含する（試薬Ａについて１つのバルブ、試薬Ｄについて１つのバルブ、Ｃにおける４塩基についてのバルブ）。
【０１６７】
この装置はまた、表１の試薬Ａ、Ｂ、Ｅ、ＦおよびＧについて図３に示される型の６つの非局在化供給手段１０を備える。
【０１６８】
製品Ａ（アセトニトリル）は、局在化様式および非局在化様式の両方で送られ得る。この装置はまた、アルゴンガス（すなわち、表１の試薬Ｈ）で基体を乾燥するための手段を備える。
【０１６９】
支持体は、２５６のリソグラフィー樹脂ウェル（エポキシ）を備えるシリコンまたはガラスプレートによって構成される。
【０１７０】
各マイクロウェルは、４１５μｍ×４１５μｍの表面および約４１５μｍの深さを有した。
【０１７１】
ウェルの容量は、塩基の液滴および活性剤の液滴をあふれさせることなく射出することによって充填される必要がある。
【０１７２】
このサイズのシリコンに対するマイクロウェル作製プロトコルは、以下の通りである：
１．支持体を洗浄する（アセトン、アルコール．．．）
２．乾燥アルゴンで乾燥する
３．例えば、カチオン性重合エポキシ樹脂（例えば、γ−ブチロラクトンで異なる比で希釈して供給される、ＩＢＭによって開発された樹脂ＥＰＯＮ ＳＵ８−１００）を使用する、スピナーを使用して基体上にリソグラフィー樹脂の層を堆積させる；スピナーの速度は、１μｍ〜１０００μｍ（代表的に、１００μｍ）の樹脂の厚みを制御する；例として引用された樹脂は、マイクロシステムを作製する際に使用され、そして１／２０までの形状係数（ｆｏｒｍ ｆａｃｔｏｒ）で、厚い垂直なウェルを作製し得る；
４００μｍの深さのマイクロウェルを、以下：
・低い回転速度で単一の樹脂層をゆっくりと塗布するか；
・または２〜４の層を連続的に塗布し、そして各層について以下の工程４、５および６を繰り返すことによって、
のいずれかによって作製した；
４．樹脂および支持体の厚みに依存して、９０℃で５分〜３０分間硬化する；
５．ＵＶ供給源（１組のマスクを用いるＵＶランプまたはＵＶレーザーで直接）を用いて選択的に暴露する。基体によって吸収される１平方メートルあたりのマイクロジュール（ｍＪ／ｍ^２）のエネルギー線量は、異なる硬化工程および現像（ｄｅｖｅｌｏｐｉｎｇ）工程の間に優れた樹脂／基体接着を獲得するように調整されなければならない。マイクロウェルのサイズは、５μｍ×５μｍから１０００μｍ×１０００μｍ（代表的に、４１５μｍ×４１５μｍ）まで変化する；
６．樹脂および支持体の厚みに依存して、５０℃〜１００℃で５分〜３０分間二次硬化する；
７．任意の超音波活性化とともに溶媒（アセトン、γ−ブチロラクトン、ＰＧＭＥＡ．．．）を用いて非暴露樹脂を溶解する；
８．樹脂の架橋を増加させるために最終硬化する：９０℃〜１５０℃で５分〜３０分。この工程は、オリゴヌクレオチド合成試薬に対するマイクロウェルの挙動を決定する．
９．スルホクロム混合物で５分間、支持体を洗浄し、次いで二回蒸留した水でリンスする；
１０．ＧＰＴＳ／水混合物（１％ｖ／ｖ）中に４５分、次いで１２５℃で４５分間オーブン中で浸漬することによって、水性シラン化処理する。
【０１７３】
該方法のパラメーターが調整され、その結果：
・オリゴヌクレオチド合成において使用される溶媒（アセトニトリル、ジクロロメタン、ＴＨＦ、ピリジンなど）および試薬（ヨウ素など）は、樹脂上部構造（ｓｕｐｅｒｓｔｒｕｃｔｕｒｅ）の分解を引き起こさないか、またはその分離を引き起こさない；
・上部構造がオリゴヌクレオチド合成に対して不活性であるように、樹脂の硬化を十分に促進する。
【０１７４】
【表２】

【０１７５】

【０１７６】
移動手段の制御は、ノズルの位置間の距離が、局在化された液体の射出の間に、ウェル４の開口部から約１ｍｍであるように調整される。局在化された試薬を含むボトル中の圧力は、以下の範囲で存在する：
・試薬が完全には射出されないが、塩基に接着する最小値から；
・ウェルの容量に対応する最大値まで。
【０１７７】
ウェルからウェルの汚染の危険を減少させるために、１つおきのウェルのみを使用し、その結果、基体中の２５６ウェルのうち６４のみが、ＯＧＮアレイについての交点（ｎｏｄｅ）を構成する。
【０１７８】
メモリー１５および中央ユニット１６を、以下のＯＧＮの以下のバリエーションの全てを含むアレイが作製されるように搭載する（３’から５’方向で）：
３’−ＧＡＧ ＧＡＴ ＧＧＴ Ｘ_１Ｘ_２Ｘ_３ ＣＣＴ ＧＣＴ ＡＧＧ ＴＡＴ−５’
Ｘ_１、Ｘ_２およびＸ_３は、Ａ、Ｃ、ＧおよびＴの６４のありうる組み合わせを形成する。特定の場合のＸ_１Ｘ_２Ｘ_３＝ＡＣＧが生物学的関心（ｂｉｏｌｏｇｉｃａｌ ｉｎｔｅｒｅｓｔ）のＬ２２６ ＯＧＮであるので、この実施例を選択した。
【０１７９】
実際上、ハイブリダイゼーションの間の立体障害に関して、ＯＧＮは基体から空間をあけておくことが有用である。この目的に対して、配列を１０Ｃヘッダーで伸長し、例えば、以下を生じる：
３’−ＣＣＣ ＣＣＣ ＣＣＣ ＣＧＡ ＧＧＡ ＴＧＧ ＴＸ_１Ｘ_２Ｘ_３ＣＣ ＴＧＣ ＴＡＧ ＧＴＡ ＴＣ−５’
アレイの第一および第三の部分は、以下の配列である：
ＣＣＣ ＣＣＣ ＣＣＣ ＣＧＡ ＧＧＡ ＴＧＧＴおよびＣＣ ＴＧＣ ＴＡＧ ＧＴＡ ＴＣを、基体を使用し得るように改変されたＰｅｒｋｉｎ Ｅｌｍｅｒ Ｅｘｐｅｒｔｉｓｅ ＯＧＮ合成器を用いて作製した。
【０１８０】
中心部分Ｘ_１Ｘ_２Ｘ_３を、図１０の関数として、図１〜４の装置で作製した。図１０は、ＯＧＮ Ｌ２２６が中心位置にあり、そして非常に異なる組成を有するプローブによって囲まれ、その結果、ＡＣＧピン（ｐｉｎ）のハイブリダイゼーションは、検出するのが容易であることを示す。このようなアレイを用いて、Ｌ２２６に相補的なＵ２２６ ＯＧＮを検出することが可能であった。
【０１８１】
（産業上適用）
本発明の好ましい適用は、複数のポリヌクレオチド配列（好ましくは、オリゴヌクレオチド配列および／またはペプチドを保有する支持体を作製することであり、これらの配列は、互いに異なり、そしてその面の少なくとも一つの上でこれらの相補体と対合し得る。
【図面の簡単な説明】
この詳細な説明は、添付の図面を参照してなされる。
【図１】
図１は、その上に本発明のＯＧＮ配列アレイが製造される支持体の斜視図である。
【図２】
図２は、図１のラインＩＩ−ＩＩに沿う垂直断面図である。
【図３】
図３は、本発明の装置の第１実施形態の概略図である。
【図３Ａ】
図３Ａは、局在化試薬の液滴を射出するためのシステムの改良型の概略図である。
【図４Ａ】
図４Ａは、反応チャンバ、支持体および局在化供給手段を含む図３の詳細であり、そしてＸ軸に沿って局在化供給手段に対して該支持体および反応チャンバの一部の移動を示す。
【図４Ｂ】
図４Ｂは、図４ＡのラインＩＶ−ＩＶに沿う断面図であり、そしてＹ軸に沿って局在化供給手段に対して該支持体および反応チャンバの一部の移動を示す。
【図４Ｃ】
図４Ｃは、図４Ａと同一のエレメントを繰り返すが、Ｚ軸に沿って局在化供給手段に対して該反応チャンバの下部および該支持体の移動を示す。
【図５】
図５および図６は、化学固定および／またはＯＧＮ合成反応のためのシートとして機能するように意図された数十のマイクロウェルを規定するフォトレジンのグリッド３を含む支持体１の拡大写真である。図５と図６との差異は、図６の支持体において円で囲まれたマイクロウェル中の局在化された試薬の液滴の存在にある。
【図６】
図５および図６は、化学固定および／またはＯＧＮ合成反応のためのシートとして機能するのうに意図された数十のマイクロウェルを規定するフォトレジンのグリッド３を含む支持体１の拡大写真である。図５と図６との差異は、図６の支持体において円で囲まれたマイクロウェル中の局在化された試薬の液滴の存在にある。
【図７】
図７は、本装置の第２実施形態の一般的な概略斜視図である
【図８】
図８は、支持体１およびその移動手段７および局在化供給手段およびそれらの移動手段３０を示す図７の詳細な斜視図である。
【図９Ａ】
図９Ａ、９Ｂ、９Ｃおよび９Ｄは、非局在化供給手段を示す。図９Ａは、該非局在化供給手段８のピストン８０の正面図である。
【図９Ｂ】
図９Ａ、９Ｂ、９Ｃおよび９Ｄは、非局在化供給手段を示す。図９Ｂは、図９Ａのピストンを通る直径の断面図（ｄｉａｍｅｔｒｉｃａｌ ｃｒｏｓｓ ｓｅｃｔｉｏｎ）である。
【図９Ｃ】
図９Ａ、９Ｂ、９Ｃおよび９Ｄは、非局在化供給手段を示す。図９Ｃは、図９Ａおよび図９Ｂのピストン８０の上面図である。
【図９Ｄ】
図９Ａ、９Ｂ、９Ｃおよび９Ｄは、非局在化供給手段を示す。図９Ｄは、支持体１へ適用されたピストン８０の部分直径断面図（ｐａｒｔｉａｌ ｄｉａｍｅｔｒｉｃａｌ ｃｒｏｓｓ ｓｅｃｔｉｏｎ）である。
【図１０】
図１０は、実施例において各々製造された３ｍｅｒ長オリゴヌクレオチドのアレイ６４を示す。
図１および図２は、本発明の装置の一体化部品を形成する支持体１を示す。[0001]
(Field of the Invention)
The field of the invention is that of making microarrays for use in analyzing nucleotide and / or peptide sequences. This type of analysis opens the door to detecting and identifying some or all of the genes or peptides that may be markers for higher biological entities (cells, tissues, bacteria, viruses, fungi, yeasts, etc.) I do.
[0002]
Productions of interest here are the localized immobilization and / or chemical synthesis of polymer probes (oligonucleotides and / or peptides) on a support intended to form an array of probes, The probe can pair with a target sequence contained in the medium to be analyzed. Said pairing can be revealed using fluorescent, colored or radioactive markers or by electronic transconductance.
[0003]
More precisely, the present invention relates to poly-, preferably oligo-nucleotide and / or peptide sequences (OGN) which advantageously differ from each other and can be paired with their complement (target sequence) by base pairing. Array) on at least one of its faces, wherein the OGN array is obtained by localized chemical synthesis in the presence of a volatile solvent.
[0004]
The invention also relates to an apparatus for performing the method, and a support included in the apparatus and used in the method.
[0005]
The present invention also provides a method for detecting and / or identifying a target OGN sequence using a bioarray produced by the above method.
[0006]
In the context of the present description, an OGN sequence corresponds to a poly-, preferably an oligo-nucleotide and / or peptide sequence. In other words, these can be:
Oligonucleotides each carrying a nitrogen-containing ATCG base;
An oligopeptide whose repeating unit is an amino acid;
Or peptide nucleic acids (PNAs), which are polymers having a peptide backbone comprising, for example, N- (2-aminoethyl) glycine repeat units linked together via amide bonds, each unit being a pyrimidine or purine Possesses a nitrogen-containing base);
-Or chimeras with mixed nucleic acids and peptide-nucleic acid structures.
[0007]
The explosion of genetics, genomics, and biotechnology throughout the medical field and industry has created a need for tools for analyzing nucleic acid or amino acid sequences. This tool must be a high resolution tool, and it must be extremely quick and reliable. Using nucleic acid sequences, this need for effective analytical tools is felt in a number of applications, such as studying gene mutations, detecting pathogens, or genomic sequencing.
[0008]
The principle of analyzing nucleic acid and / or peptide sequences is relatively simple. Because, in the case of polynucleotides and PNAs, this involves reading the sequences of the four nucleotide co-monomers A, C, T and G, and the sequence of about 20 amino acids for the polypeptide. is there. This simple principle is complicated by the considerable amount of data processed; the genome for the bacterium Escherichia Coli (one of the shortest bacteria) contains 4.7 million nucleotides and 4000 genes, while the human genome is It is composed of about 100,000 genes and represents 3 billion nucleotides. In addition, there is the fact that decoding genes or proteins is incomparable to understanding the complex action of these nucleic acid or peptide polymers in the normal or abnormal mechanisms of living organisms. .
[0009]
This further emphasizes the fact that it is particularly useful to have available tools or arrays that can rapidly and reliably analyze large numbers of nucleic acid or amino acid sequences.
[0010]
(Prior art)
To meet this need, different analysis tools and techniques for making said tools have been proposed in the prior art.
[0011]
PCT International Patent Application WO-A-89 / 10977 (Inventor: Southern) describes a method and apparatus for analyzing polynucleotide sequences. In accordance with the invention, there is provided a support (eg, a glass plate) comprising an array or complete set of oligonucleotide sequences (OGN probes) capable of hybridizing to complementary target OGN sequences contained in the medium to be analyzed. . Hybridization that can occur on the OGN array is revealed using markers that can be carried by the probe and / or target. The concept of a bioarray with an array of OGN probes is that the digital imprint of the genetic content of the medium for analysis involves the partial or complete removal of said genetic content in a single analytical step. Allowing it to be acquired with sequencing. In theory, this concept is increasingly fascinating. Because, using an array of OGN probes that are different and large (hundreds to millions) (which can also be referred to as "pixels" by analogy with photography), multiple polynucleotide sequences without the need for cloning It should be possible to perform the analysis in a medium containing a population of However, apart from obvious theoretical interest, a difficult practical problem arises: the creation of a support that supports an array of probe OGN sequences, each of 10-mer, 20-mer, or 30-mer length. Macromolecular OGN probes must be immobilized on units with extremely small surface area with known accuracy for each probe.
[0012]
A specific solution to the practical problem employed in the embodiment of PCT WO-A-89 / 10977 is as follows. Microscope slides were used as supports. These plates undergo silanization (ie, they are treated with 3-glycidoxypropyltriethoxysilane, which adheres to the surface of the glass, forming anchoring points for the OGN sequence). The silane moiety is then subjected to an acid treatment to produce hydroxyl groups that can react with the OGN monomer head monomer (Lung 1). OGN oligonucleotides were synthesized using phosphoramidite chemistry. The 5 'end of the nucleotide was protected by a dimethoxytrityl (DMT) group and the 3' end was protected by β-cyanoethyl phosphoramidite. Coupling of nucleotides in Lang 1 and on silane, and Lang 2 n Was performed using tetrazole. The solvent was acetonitrile. The reaction was performed under anhydrous conditions in a sealed vessel. The vessel contained a microscope slide coated with a strip of silicone rubber hollowed out leaving only the edges to define a single reaction site. A Teflon plate of the same size and thickness as the glass support plate covered the reaction zone and silicone interface. A short plastic tube allowed the reaction volume to communicate with the outside and allowed injection and removal of the reaction solution, and also allowed the reaction volume to be filled with argon, and the operation was performed using a syringe. After each nucleotide coupling step, the assembly is separated and the reaction is continued by immersing the support in a reaction solution or reagent for reassembly to perform the next coupling step.
[0013]
Obviously, techniques for making arrays of OGN probes are not industrially available, especially because of their cumbersomeness and complexity. In addition, the examples in the PCT document only disclose arrays containing a very small number of oligonucleotides (ie, 24 and 72 units). These arrays are shown in Tables 1a and 1b of the PCT application. The composition of the OGN sequence varies very slightly and a significant number of bases have been deleted. Thus, techniques for making OGN arrays according to PCT WO-A-89 / 10977 appear to be ideally suited for improvement.
[0014]
The authors of PCT WO-A-89 / 10977 produce OGN matrices by synthesizing oligonucleotides in cells of an array using a computer controlled printing device and using an automated device including an inkjet printhead. Suppose that it should be possible. However, this is just an idea that has not been put into practical use. In reality, putting it into practical use has proven to be very difficult.
[0015]
Because the reactions performed are so complex and numerous, the generation of OGN arrays cannot be merely the employment of known techniques for immobilizing and synthesizing oligonucleotides on a support.
[0016]
The technique described in PCT WO-A-89 / 10977 derives from a mode of fabrication that can be referred to as "in-situ", which increases the set of pixels on a support simultaneously, one base at a time, on a substrate. To synthesize a probe.
[0017]
There is a further group of fabrication techniques that can be referred to as "ex situ", which are based on processing of the support and pre-production of a set of OGN probes independent of the support and subsequent to said production, The probe is fixed.
[0018]
These techniques are difficult to implement when the set of OGN probes exceeds a few tens of units, and become impractical above a few hundred units.
[0019]
International PCT application WO-A-92 / 10092 (inventor: FODOR et al.), Corresponding to the article in Science, Vol. 251, 767-773, 1991, discloses an in situ synthesis located on a support by the photochemical route. . For this purpose, the nucleotide base is protected at its 5 'end by a photolabile group such as nitroverertryloxycarbonyl (NVOC), which is a protecting group used in phosphoramidite synthesis ( DMT). The support is a glass plate with amine or hydroxyl groups that are also protected with NVOC. The effect of localized light irradiation on the substrate results in photochemical cleavage of the photolabile groups and deprotection of the irradiated surface. It is then possible to proceed locally with the addition of an oligonucleotide that is also protected by the NVOC group. FODOR et al. Synthesized an array of OGN probes by irradiating different areas of the support and then by a combinatorial synthesis strategy. A major disadvantage of the FODOR et al. Technique lies in the deleterious effect of the NVOC group on the synthesis yield. This results in sequencing errors and a reduction in the useful surface of the array. In addition, the FODOR method is expensive and inflexible in its implementation (masks, reagents are not commercially available).
[0020]
A further disadvantage of this fabrication method is that the combinatorial strategy used results in an array of compositions that varies little from one pixel to the next; typically, the composition of the probe is from one pixel to the next. Only one base changes. Therefore, distinguishing hybridization in neighboring pixels can prove difficult.
[0021]
OGN synthesis techniques involving photolabile NVOC-type protecting groups are also described in International PCT Application WO-A-93 / 09668, which describes a combinatorial strategy for synthesizing an array of nucleotide or peptide polymers on a substrate. Describe. This PCT application refers to the localized in situ synthesis of different OGN monomers in a given region of a substrate. This PCT application recommends two types of techniques for chemical synthesis.
[0022]
1-circulating a flow of the reaction solution inside the channels formed in the block, wherein one of the arrays is formed by the surface of the substrate in a defined area or in a predetermined area;
2- depositing or spraying a droplet of the reaction solution on a predetermined area of the substrate;
3- Combination of techniques 1 and 2.
[0023]
In technique 1, a channel block is used, comprising a set of parallel grooves in contact with the surface of the substrate, so as to define a channel through which the reaction liquid circulates, where the reaction liquid circulates. As a result, the method comprises fixing one or more different monomers of rung 1, removing the substrate-channel block assembly for the washing and deprotection steps, the monomer of rung 2 and the rung n Up to reassemble the assembly to fix up the monomers. Combinatorial strategies consist of moving a substrate relative to a channel block, either by rotation (90 °) or translation.
[0024]
In a variation of Technique 1, as shown in FIG. 12, a support is provided that includes a plurality of reaction wells and a reagent distributor that includes an assembly of tubes for dispensing reagents or reaction solutions to the support. In this variant, it is possible to insert a mask between the distributor tube and the support well to control the supply of the reaction solution to a given area of the support.
[0025]
With respect to Technology 2, the PCT application filed in a general manner one square centimeter (cm ² A) comprising a support comprising at least 10,000 reaction zones per). There is no specific example for embodying such a support. This PCT application also describes means for distributing the reaction solution in the reaction zone of the support. An inkjet printhead is cited as an example of a means for distributing a reaction liquid to a reaction area of a support.
[0026]
One of the proposed techniques for creating a reaction zone on a support is to create a hydrophobic zone around the hydrophilic zone (e.g., of a hydrophilic resin bearing protected hydrophobic groups). By applying a layer and deprotecting these hydrophobic groups in the area intended to form the contour of the surface tension well) to obtain a surface tension well on the surface of the support . Alternatively, the reaction area may be formed by a three-dimensional well defined by walls as shown in FIG.
[0027]
In this PCT application, hydrophobic groups are advantageously deprotected by photolysis.
[0028]
PCT application WO-A-93 / 09668 also suggests a problem with solvent evaporation that can affect the synthesis of OGN sequences. To overcome that difficulty, the use of a sealed reaction chamber is proposed, together with reducing the vapor pressure (low temperature) and / or saturating the reaction chamber with volatile solvents.
[0029]
It should be noted that the gap between the speculative generality and practical working practical embodiments cannot be ignored. This is confirmed by the fact that the examples given in that PCT application only relate to the description of technique 1 using a reaction fluid circulation channel. As described in the examples, this technique allows arrays to be created that include 2500 reaction regions. OGN oligonucleotide sequences are synthesized in all of the reaction regions. To adhere the seventh and eighth monomers, the reaction solution circulation channel block used is immobilized against a substrate surface that already contains each 6 mer long OGN sequence. The seventh nucleotide is distributed by the channel assembly that is parallel to the predetermined direction, and the eighth nucleotide circulates in the channel in a direction that is perpendicular to the direction of the channel intended to receive the seventh nucleotide. Distributed by In that example, an 8-mer OGN sequence is created only at the intersection between the channels.
[0030]
The method of application PCT WO-A-93 / 09668 does not seem to be able to quickly, reliably and easily produce arrays of oligonucleotides of different composition. With each rotation of the substrate with respect to the channel block, the device needs to be removed, which makes the method very cumbersome for in situ OGN synthesis and localization.
[0031]
U.S. Pat. No. 5,658,802 (DJ HAYES et al.) Relates to ex situ and in situ synthesis methods based on the local ejection of reagent droplets onto a substrate using a piezoelectric system. In fact, the most frequently used substrate for diluting nucleotide bases is acetonitrile, and considering that this solvent has a low wetting contact angle, more than a few hundred microns of solvent on the substrate. Problems with spreading the droplets arise, making it difficult to localize the reaction to an appropriate scale. Further, the acetonitrile used in this manner is volatile and is therefore subject to large-scale evaporation.
[0032]
The device described in U.S. Pat. No. 5,658,802 comprises a substrate of the type used to make integrated circuits with patterns as required. In that patent, there is no problem defining very low surface area reaction sites. Each piezoelectric injection means is associated with a liquid reservoir. The piezoelectric injection means and these reservoirs together form a distribution assembly. The device also provides a means for positioning the liquid ejection means and / or the substrate.
[0033]
The above shows that the method and apparatus described in US-A-5 658 802 is not operable when producing an array of OGN sequences by localized chemical synthesis using volatile solvents. .
[0034]
With regard to the problem of controlling the spread of the reaction liquid droplets on the substrate and confining them to separate reaction sites separated from each other, the aforementioned International PCT Patent Application WO-A-93 / 09668 and Reference should be made to the description of surface tension well technology, which surrounds a hydrophilic region having a diameter of about 100 micrometers (μm) to define multiple reaction sites by surface tension differences. It consists in creating a hydrophobic region. A. P. A document by BLANCHARD et al. (Biosensors-Bioelectronics 11, 687-690, 1966) also deals with this technique. However, it should be recognized that the technique solves the problem in a satisfactory manner only for water-soluble solvents. Unfortunately, aqueous solvents are not suitable for oligonucleotide or oligopeptide synthesis reactions.
[0035]
In fact, the surface tension well technique is not appropriate when the reaction is constituted by a volatile organic solvent such as acetonitrile. Due to the surface tension characteristics of such volatile solvents, it is not very sufficient to separate the reaction parts by the difference in surface tension. In addition, the problem of evaporation of solvents distributed in very small quantities remains a major problem in propositions involving surface tension wells.
[0036]
International PCT application WO-A-94 / 27719 relates to a method and apparatus for making a chemically reactive array on the surface of a support. In this PCT application, surface tension wells are made on the surface of a support using photoresins of the phenol / formol type or polymethacrylate type. This PCT application recommends a piezoelectric inkjet printhead type means for dispensing the reaction liquid.
[0037]
Therefore, it must be appreciated that the prior art also does not propose an efficient and sophisticated way of producing arrays containing large amounts of OGN sequences of different and varying properties on the surface of the support. Many methods and devices described in the literature do not have any actual application and do not appear to have been made. Furthermore, exceptions to the general rules result in an OGN array containing a small number of pixels, or have a large number of incorrect OGN arrays. This last point constitutes a weakness of these methods with respect to the synthesis yield. An average yield of 92% is known to reduce the useful surface of each pixel by up to 51% for the 8-mer probe and up to 36% for the 12-mer probe. The presence of incorrect sequences throughout most of the array complicates the detection of hybridization or base pairing and requires overlapping array designs or probe length limitations. Such deficiencies are particularly true for photochemical methods involving NVOC protecting groups. In addition, known fabrication methods are not very flexible in that it is difficult to change the composition of the OGN sequence that easily constitutes the array.
[0038]
Finally, known methods are generally complex, unreliable, not very quick, expensive and, most importantly, can be considered not industrially available.
[0039]
Under these circumstances, an essential object of the present invention is a method and apparatus for making a support bearing a plurality of poly-preferably oligo-nucleotide and / or peptide sequences) on at least one side thereof. Wherein the OGN sequences differ from each other and can pair with their complement by base pairing, and are obtained by chemical synthesis in the presence of a volatile solvent:
The method is required to be flexible, reliable, fast and simple, while at the same time actually functioning correctly; and
-The method also produces arrays containing tens to hundreds of millions of OGN sequences that have good chemical yields and then have a low percentage of errors in the sequences.
[0040]
In a further essential aspect, it is an object of the present invention to provide a simple, inexpensive and industrial device for performing the method outlined above.
[0041]
In a still further essential aspect, the present invention aims to provide a support for use in a method and apparatus having the above-mentioned object, which is easy to make and which comprises an OGN sequence array Optimize the production of
[0042]
In a still further essential aspect, the present invention aims at providing a method for detecting and / or identifying a target OGN sequence, said method comprising a support for supporting an array of OGN probes obtained by a method having said purpose. Including bioarrays comprising the body, such detection and / or identification methods are also more reliable and more specific.
[0043]
Disclosure of the invention
Said subject is achieved, inter alia, in a first aspect, by the present invention, which provides a method of producing a support bearing a plurality of poly-, preferably oligo-nucleotide and / or peptide sequences on one of its faces, which is advantageous. The sequences that differ from each other (OGN sequences) can pair with their complement by base pairing, and these are obtained by chemical synthesis in the presence of volatile solvents;
The method essentially comprises:
1) using a support [where the surface intended to carry the OGN has an array of reaction wells, each well being localized and / or fixed and / or of a given OGN sequence; Acts as a sheet for chemical synthesis; the wells are coated with a photocurable resin, the resin is cured by exposure to actinic radiation in areas intended to define the wells, and the cured By removing the resin and obtaining a grid of resin that forms the walls defining the wells];
2) placing the support in a reaction chamber and using the vapor of a volatile solvent used in the localized immobilization and chemical synthesis of the OGN, preferably through the reaction chamber (5) Saturating the chamber by circulating a gas stream containing a suitable saturated vapor;
3) performing (by increasing) localized immobilization and / or chemical synthesis of the OGN sequence in at least a portion of the well;
And this operation is accomplished by:
i. Locally and separately supplying reagents and liquid consumables for fixing and / or synthesizing the corresponding OGN sequences to each associated well;
ii. Collectively (non-locally) supplying reagents and / or liquid consumables common to reactions intended to be performed in all the wells to the associated wells;
iii. Prior to each operation for localized supply to said well (4), at least a portion of any liquid contained in each well intended to be supplied in a localized manner is removed. Make sure that ,
The method of the present invention corresponds to producing an array of oligonucleotides by in situ (or ex situ) chemical synthesis that better meets practical needs than prior art methods.
[0044]
The present invention has many advantages. Examples mentioned include the fact that arrays of OGN sequences can be manufactured in a simple and flexible manner in a wide variety of compositions, sizes and densities. In particular, it meets the requirements of genetics research. Furthermore, the method is performed on a scale where the consumption of very expensive reagents (especially those used for OGN synthesis) is particularly low. This makes it possible to mass produce arrays at low cost. Further, it should be noted that the method of the present invention uses only commercial reaction liquids and equipment.
[0045]
Furthermore, the method of the present invention makes OGN array technology, and especially that of oligonucleotides, widely available for all genetic analyzes applicable to the pharmaceutical and bio industries.
[0046]
The method of the present invention can be used from several square millimeters to 300 cm ² [The surface area of one pixel is in the range of 1000 micrometers (μm) × 1000 μm to 10 μm × 10 μm) on a support (eg, a silicon wafer) of a wide variety of compositions in a flexible manner. (Which may or may not be combinatorial). Thus, the number of pixels can be from a few to hundreds of millions of leaves.
[0047]
OGN sequences immobilized in situ on a support and optionally produced can be considered as probes capable of reacting with a complementary target by base pairing. These OGN sequences are advantageously constituted by several comonomers, which are repeating units and / or amino acid units of nucleotides and / or peptide nucleic acids (PNA). The number of nucleotide comonomers and / or PNAs and / or amino acids of these polymers or more precisely oligomers can range from a few units to tens of units, preferably 15 units to 25 units.
[0048]
Preferably, according to the invention, the OGN immobilization as well as the localized in situ synthesis is performed in the wells. However, it can also be expected to synthesize OGNs ex situ and immobilize them individually to the wells of the support in a localized manner.
[0049]
Ideally, the constituent materials of the support are selected from the group consisting of:
Glass, quartz, silicon optionally coated with at least one layer of oxide or nitride.
[0050]
Silicon or other semiconductor materials are preferred, especially if the support is intended to be integrated into a bioarray that functions by exposing a pair of electronic transconductance types.
[0051]
In a further feature, the surface of the support is preferably from the group constituted by positive or negative resins, preferably from a sub-group comprising negative resins, and more preferably from radicals and / or It has an array of reaction wells formed by edging of a photocurable resin selected from the class comprising (meth) acrylate, epoxy, polyester and / or polystyrene resins that can be photocured by a cationic route.
[0052]
The term "positive resin" as used in the present invention means a resin that becomes highly soluble upon irradiation.
[0053]
The term "negative resin" as used in the present invention means a resin that becomes insoluble upon irradiation.
[0054]
Negative resins are preferably used and have the advantage of creating a thick chemical resistant layer that adheres to the substrate.
[0055]
Advantageously, the uncured resin in the unmasked areas corresponding to the wells is removed by dissolving the resin in an organic solvent. It can be, for example, acetone, gamma-butyrolactone, propylene-glycol methyl ethyl acetate (PGMEA), acetonitrile, or other solvents known in the art. Such formation of microwells in lithographic resin follows known experimental protocols of washing, depositing, curing, exposing to actinic radiation (UV), and dissolving the uncured resin. .
[0056]
The examples and figures provided below and particularly in FIGS. 1 and 2 and the illustrations provide other useful details of the support and its non-limiting description of its manufacture.
[0057]
In an advantageous variant of the invention, the support is surface-treated to provide an anchoring site thereon, on which a non-labile bond can be formed with the head comonomer of the OGN sequence, said treatment preferably comprising the microwell Silanization using alkoxysilanes or epoxyalkoxysilanes with ester functionality before and / or after making the array.
[0058]
Conventionally, the silanization agent is glycidoxypropyltrimethoxysilane. Other silanizing agents that may be cited are glycidoxypropyldimethoxysilane, aminopropyltriethoxysilane and carboxymethyltriethoxysilane.
[0059]
Silanization is used for arrays produced in situ. n It provides a solid anchor point for the set of rung 1 monomers (eg, nucleotides) that make up the OGN sequence. The silane is advantageously crosslinked with the monomer (nucleotide) of Lang 1 by, for example, a hydroxyl group.
[0060]
The non-volatile solvent evaporation required for the immobilization reaction and OGN sequence synthesis is fully considered in the method of the invention. Thus, in this method, a saturation step is provided, preferably using a gas stream comprising suitable saturated steam and neutral gas. This controls the rate of evaporation of the solvent used, and controls all micro-reactions required to make the array.
[0061]
If the evaporation is controlled by saturation, it can be expected to cause evaporation of the solvent to stop the localization reaction on the support.
[0062]
Of course, it is also possible to limit the evaporation, for example by reducing the temperature (preferably of the support), thereby reducing the vapor pressure of all volatile solvents used.
[0063]
Advantageously, the vector gas used for saturation is argon or helium.
[0064]
Preferably, the gas flow is such that it determines an over-pressure in the reaction chamber compared to the ambient atmosphere.
[0065]
In a non-limiting example, the gas flow determines a pressure of 0.01 bar to 3 bar with respect to atmospheric pressure.
[0066]
With regard to the localized immobilization and / or chemical synthesis of OGN on the support, according to the invention, means for localized supply of the support to the reaction wells and the non-localized supply ( Means for non-localized supply) are used.
[0067]
Advantageously, the localized supply means and the support are movable relative to each other in three dimensions X, Y and Z by means of the moving means.
[0068]
According to the present invention, n In order to fix and / or synthesize OGN sequences on the support, x Including comonomer:
・ Created n The composition of the array of OGN sequences is determined by comparing the composition of each of the n Respond to a series of actions of removing, dispensing and depositing reagent liquid localized in wells x By storing the composition in a continuous rung configuration, it is stored in memory. n Lang Ri = 1 to well x Determine the properties of the comonomer of
And firstly, instructing the localized supply means and the mobile means to perform various series of actions as defined above, and secondly, instructing the delocalized supply means with the fixed And at various times during the OGN sequence synthesis procedure, n Instruct the wells to remove, transport and supply the delocalized liquid [drain the liquid present in the well and more generally in the reaction chamber following each localized supply Process is performed];
[The instructions and the actions they induce are R _i At 1 until i = x i Is repeated by increasing.
[0069]
During the various steps to produce an array of OGN sequences (eg, oligonucleotides), the reaction chamber may be supplied with fluids (gases, solvents, etc.) and reagents (bases, deprotecting agents, coupling agents, etc.). is necessary. The fluids (liquid consumables) and reagents can be divided into two categories:
1) Fluids and reagents intended to be sent pixel by pixel to obtain an array (hereinafter referred to as “localized liquid”);
2) Fluids and reagents intended to be delivered simultaneously to all or a portion of the support in a manner that is common to all the wells, and more particularly to all or a portion of the reaction wells of the support. . The fluids and reagents are hereinafter referred to as “non-localized liquids”.
[0070]
According to a preferred feature of the invention, the liquid is removed in step 3 (iii) of the method, so that the associated wells have at least 90% of their volume, preferably at least 95%, and more. Preferably, there is no liquid to a depth of at least 98%.
[0071]
Advantageously, the liquid is dried, by injecting a gas (preferably a neutral gas) into the reaction chamber, and / or by increasing the temperature of the reaction chamber and / or the support, and / or Elimination is achieved by reducing the vapor pressure of the liquid removed from the reaction chamber.
[0072]
These treatments allow for accurate projection or deposition of the localization liquid and splashes, especially in the wells adjacent to the well in which the given localization liquid was provided. [This constitutes a danger if the well already contains liquid before starting the considered supply], so that it does not create smears.
[0073]
Said removal of the liquid by drying using a neutral gas may also remove traces of water, which may compromise OGN synthesis and / or fixation. It is well known that bases and solvents that can be used as reaction liquids must be stored in anhydrous media.
[0074]
The reaction chamber can also be left under partial and controlled vacuum.
[0075]
Obviously, the method of the invention can achieve the following:
Providing a support with a superstructure defining a microreactor corresponding to the pixels of the array and controlling the spreading of the localized liquid;
Controlling the evaporation rate of the localized liquid solvent in the microreactor to control the chemical microreactions required to obtain the OGN array;
-At the end of the microreaction, drain the used chemical product to continue the production of the array.
[0076]
In a further aspect, the present invention provides an apparatus for performing the method as defined above. The device comprises:
At least one support, wherein the surface intended to carry the OGN has an array of reaction wells, each well for the localization and chemical synthesis of a given OGN sequence Acting as a sheet of the well, applying a photocurable resin, curing the resin by exposure to actinic radiation in the area intended to define the well, and the uncured resin. To obtain a resin grid (3) that forms the walls defining the wells];
At least one reaction chamber intended to contain the support;
Means for the localized supply of specific localization liquids (reagents / consumables) to the reaction wells, allowing the fixation and synthesis of a given OGN sequence in each of the wells of the support;
Means for moving the localized supply means and / or vice versa with respect to the support;
Means for delocalized supply of delocalized liquid (product / consumable) to the reaction well, common to reactions intended to be performed in all or part of the well;
• any means for moving the delocalized supply means;
Means for discharging a delocalized liquid; said discharge means being associated with said delocalized supply means;
An apparatus for saturating the reaction chamber using the volatile solvent vapor used in the immobilization and synthesis of localized OGNs;
A container for the localized liquid;
A container for the delocalized liquid;
A container for emptying liquid waste;
-Any device for supplying gas to the chamber;
Means for moving the support with respect to the localized supply means and / or vice versa;
• optionally, at least one memory for various OGN sequences immobilized and synthesized on the support; and
At least one central unit for reading said memory and generating signals, if necessary, to control the supply of fluid and the movement of said localized supply means and said support relative to each other;
It is characterized by including.
[0077]
The invention will be better understood from the following description of a preferred, non-limiting example of implementation of the method and of an embodiment of the apparatus.
[0078]
Best mode for implementing the present invention
Preferably, the support 1 comprises:
At least one substrate plate 2 made of a material selected from the group consisting of glass, quartz and optionally silicon coated with at least one layer of oxide or nitride;
At least one array 3 (preferably a grid) of photo-cured resin integral with one of the faces of the substrate [the array defining a matrix in a reaction well 4 and the photoresin , Epoxy resins and (meth) acrylate resins that are photocurable by cationic and / or radical routes, with epoxy resins being more particularly preferred].
[0079]
Advantageously, the resin grid 3 is compatible with the processing performed on the substrate to fix the rung 1 monomer (nucleotide). The grid 3 can be manufactured before or after the treatment. The grid 3 of resin adheres to the support plate 2 during OGN array array production. The grid does not interfere with the operation to read the array on the bioarray, but it can be removed before it is used on the bioarray, if desired.
[0080]
It is important that the grids formed in the resin not completely cured do not contaminate the synthesis reagents, and, reciprocally, that the reagents do not degrade the grid or cause grid / substrate separation.
[0081]
The reaction wells 4 defined by the grid 3 define as many reaction sites as pixels in the array. The wells limit the spreading of highly wetting solvents such as acetonitrile. The array or grid 3 of reaction wells 4 is formed by depositing the resin on a substrate using conventional lithographic techniques with a positive or negative resin, and, for example, by removing the wall in the case of a negative resin. By exposing the areas corresponding to actininc radiation to cure them and then selectively dissolving the uncured areas to form wells. Without limitation, the invention recognizes that it produces microwells having a depth in the range 0.1 μm to 1000 μm, preferably in the range 50 μm to 500 μm, and a surface in the range 10 μm × 10 μm to 1000 μm × 1000 μm. I can do it.
[0082]
In an advantageous arrangement of the method of the invention, the array or grid 3 of reaction microwells 4 localizes the chemical reaction independently of the nature and / or preparation of the support, so that it is heterogeneous It is possible to use supports of various properties which have undergone a physicochemical surface treatment (oxidation, silanization, nitridation, etc.).
[0083]
A first embodiment of the device of the invention shown in FIG. 3 comprises a support 1 having a reaction well 4 defined on its surface, defined by a resin grid 3, wherein the support 1 is generally Included in the reaction chamber indicated by the general reference number 5. The device of FIG. 3 also comprises a localized supply means 6, a means 7 for moving the support 1 relative to the localized supply means 6.
[0084]
Means 8 are provided to ensure a delocalized supply of the delocalized reaction liquid. Said means 8 is associated with means 9 for discharging the delocalized liquid.
[0085]
The apparatus also includes an apparatus 10 for saturating the reaction chamber 5 using a gas stream filled with a neutral gas and a solvent. Reference numbers 11, 12, and 13 in FIG. 3 denote a container for the localized liquid, a container for the delocalized liquid, and a container for emptying the waste liquid, respectively. Apparatus 14 is provided for supplying gas to reaction chamber 5.
[0086]
Finally, in a preferred feature of the invention, the automated implementation of the method is ensured by a memory 15 and a central unit 16 for reading the memory 15 and issuing control signals (dotted arrows). To be.
[0087]
The various components of the device are supported by a suitable chassis and / or frame and / or base.
[0088]
In a first implementation of this first embodiment, the apparatus comprises a reaction chamber receiving the support in opposition to the supply means, one end carrying the localized supply means. With sealed bellows having an opposite end with a reaction site intended for, and having delocalized supply means, the end (5) carrying the localized supply means. ₂ ) Is preferably fixed and the other end (5) carrying the support ₁ ) Is preferably three-dimensional using the moving means (7) x , y ,and z Is movable.
[0089]
In a second implementation of the first embodiment, the apparatus comprises: Three-dimensional under the action of transportation x , y ,and z It can be moved in.
[0090]
FIG. 3 corresponds to an example of an apparatus according to a first implementation of the first embodiment, wherein the localized supply means is a moving part of the reaction chamber intended to receive the support. Fixed against. This moving part is x , y ,and z Provided by means 7 for movement.
[0091]
As shown in FIG. 3, the reaction chamber 5 comprises a lower part 5 used as a receptacle for the support 1. ₁ Upper part 5 with localization supply means 6 ₂ And the lower part 5 ₁ And upper part 5 ₂ Part 5 constituted by sealed bellows connected to ₃ It is composed of Upper part 5 of reaction chamber ₂ Is bellow 5 ₃ It may or may not be integrated with. Sealed bellows 5 ₃ Is advantageously made from a chemically inert material such as polytetrafluoroethylene (TEFLON®).
[0092]
In a preferred feature of the invention, the localized supply means 6 comprises a projection system. The injection system is advantageously selected from the following systems:
1) Mechanical injection using the following types of equipment:
a) drop-on-demand using the piezoelectric effect;
b) continuous jet interrupted by electrostatic effects;
2) Injection under pressure using a microvalve device.
[0093]
In the embodiment shown in FIG. 3, the injection system is of type 1a) and at least one nozzle 6 for injecting or depositing a localized liquid. ₁ Is provided. This piezoelectric nozzle 6 ₁ Is line 6 ₂ To one or more localized liquid reservoirs 11.
[0094]
A system 1a) for drop-on-demand ejection using the piezoelectric effect is described in J. The paper "Techniques d'impression d'images numbers" by ELTGEN [Digital Image Printing Techniques] (described in TECHNIQUES D'INGENIEUR-elements Electronics 56).
[0095]
One of the advantages of the piezoelectric system 1a is that they have a very small volume of localized liquid (reagent) over a very small surface area microwell 4 (eg 70 μm × 70 μm to 300 μm × 300 μm). For example, 50 picoliters) can be deposited on the microwells 4 of the support 1.
[0096]
A further advantage of the piezoelectric system 1a is that the component 6 with 64 to 128 nozzles independently controlled at a frequency of 1 to 10 kHz ₁ [Eg, P64 and P128 systems from MODULAR INK TECHNOLOGY (Sweden)]. Such components can then be used to simultaneously feed multiple wells by injecting reagents diluted in acetonitrile.
[0097]
As a variant, the injection system is of the type 1b), as described in J.C. J. It can be a continuous jet interrupted by electrostatic effects as described in the article by ELTGEN. This system can provide high resolution (typically 30 μm × 30 μm).
[0098]
Finally, a variant with a type 2) injection system as shown in FIG. ₁ ) Instead of using mechanical parts 20 made of a material that is inert to the delocalized liquid reagent. It can be made from stainless steel, polytetrafluoroethylene (PTFE) or polyamide (NYLON®). Each component 20 includes a fin that passes through a channel 21. The inlet opening 22 to the channel 21 is connected via a line 23 to an electrically-controlled valve 24 which manages the admission of the localized liquid to the channel 21 of the component 20. I have. The localized reagent liquid 25 is supplied to the valve 24 via the reservoir 26. Line 27 connects reservoir 26 to valve 24. Neutral gas 28 (eg, argon) drives liquid 25 to valve 24 at overpressure (eg, 0.5 bar). The lines 23, 27 having a diameter of, for example, 1 millimeter (mm) are advantageously made of the same material as the part 20, for example PTFE.
[0099]
The outlet opening 29, ie the nozzle of the part 20, is arranged opposite the substrate 1 containing the microwells 4 defined by the photoresin grid 3.
[0100]
The diameter of the nozzle 29 is, for example, 100 μm.
[0101]
The valve 24 is controlled by a central electronic control unit 16 (not shown in FIG. 3A), as shown in FIG. 3. This unit 16 has a short control period (eg, 0. Opening for 1 millisecond (ms) to 10 ms), then controlling the closing of valve 24 to release a small amount 30 of localized reagent liquid 25, and into microwell 4 of support 1 Can supply.
[0102]
Using 1 mm diameter lines 23, 27, an overpressure of 0.5 bar of argon, an electric control valve 24 with an opening period of 0.1 ms to 10 ms, a nozzle 29 of 100 μm diameter, the delivered quantity 3 is 400 μm × 400 μm And a microwell 4 having a surface area of 400 μm and a depth of 400 μm.
[0103]
The elements 20, 21, 29 can be, for example, components of a microsyringe having a diameter of less than 100 μm.
[0104]
In fact, valve 24 may be comprised of an INKX 0510100 valve from LEE COMPANY (USA), which also creates a system with 7 nozzles 29 with reference number INZX 0510100. To increase the compatibility of this valve with acetonitrile, it would be advantageous to replace the elastomer flap with a fluorinated elastomer flap (KALREZ® or CHEMRAZ®).
[0105]
The variation of the injection system shown in FIG. 3A is advantageous because of its reduced cost, simplicity of function, and reliability.
[0106]
In the embodiment shown by way of example in FIG. 3, the localized supply means 6 also comprises a line 6 for supplying gas under pressure. ₃ Which is adapted to transfer the localized liquid contained in the reservoir 11 to the line 6 ₂ Through the nozzle 6 ₁ Can be transported to This line is an electric control valve 6 for controlling injection. ₄ It has. Element 6 forming localized supply means 6 ₁ ~ 6 ₄ Is preferably a valve 6 ₄ Is controlled by a central control unit 16 that manages the opening and closing of the. The central unit 16 also has line 6 ₃ To control the supply of high-pressure gas. In the most usual case where there are multiple reservoirs 11 for the localized liquid, the central unit 16 also manages the selection of the liquid to be ejected using a suitable device.
[0107]
In the example of FIG. 3, only one injection nozzle 6 ₁ It should be noted that it is possible to recognize in the variant that there are as many injection nozzles as there are localized liquids to be injected.
[0108]
The delocalized supply means 8 comprises a lower part 5 of the reaction chamber containing the support. ₁ 8 for supplying delocalized liquid to the ₁ It has. This line 8 ₁ Is the lower part 5 of the reaction chamber 5 ₁ Opening 8 provided in ₂ Is connected to the localized liquid reservoir 12. This line 8 ₁ Has an electrical control valve 8 for controlling the delocalized liquid flow intended to supply the support. ₃ Is provided. The delocalized supply means 8 also transfers the delocalized liquid stream to the line 8. ₁ 8 for supplying high pressure gas (propellant gas) generated in ₄ including.
[0109]
By providing on the support 1 a means for selecting a delocalized liquid to be introduced into the lower part of the reaction chamber 5, another delocalized liquid is provided in said delocalized supply means 8. It can be readily appreciated that a reservoir 12 can be included. In a similar manner to the localized supply means 6, the delocalized supply means 8 is controlled by a central unit 16 (dashed arrow), which ₄ And valve 8 ₃ Controls the transport of the delocalized liquid by the high pressure gas supplied via the
[0110]
Said means 8 is associated with means 9 for discharging the delocalized liquid, supplied by means 8 and temporarily staying in the lower part 51 of the reaction chamber 5 and immersing the support 1. The evacuation means 9 comprises an outlet orifice 9 provided in the lower part 51 of the chamber 5. ₂ 9 connecting to a waste reservoir (effluent reservoir) 13 ₁ including. Line 9 ₁ Has an electric control valve 9 controlled by a central unit 16, which controls the drainage of wastewater at a suitable time. ₃ Is provided. The driving force for the evacuation can be, for example, the action of a suction pump (not shown in FIG. 3) which allows the overpressure of the gas and / or the liquid to be removed.
[0111]
The device 10 enabling the circulation of a saturated volatile solvent stream comprises a lower part 5 of the reaction chamber. ₁ To and lower part 5 ₁ Solvent reservoir 10 for the saturated gas stream from ₁ , Entrance 10 ₂ And exit 10 ₃ including. The flow is in line 10 ₄ Into the solvent reservoir 10 and with it the solvent vapor ₅ Generated by a neutral gas (eg, argon) that carries the gas to the inlet 10 ₂ Into the chamber via exit and exit 10 ₃ (This is discharge line 10 ₆ Can be exhausted therefrom. Line 10 ₅ And 10 ₆ Is controlled by a central unit 16 (which therefore controls the saturation gas flow). ₇ And 10 ₈ including.
[0112]
Preferably, the reaction chamber saturator 10 is designed to allow circulation of a saturated gas stream containing the volatile solvent vapor used and at least one neutral gas.
[0113]
An optional gas supply 14 communicates with the reaction chamber via an inlet provided in the lower part. This device 14 is equipped with an electronic control valve (not shown) and is also controlled by a central unit.
[0114]
With regard to the means 7 for moving the support 1 relative to the means 6, it can be seen that in a first implementation of the first embodiment of the device of the invention, the means 7 is symbolically shown in FIG. , FIG. 3 also shows the control of the moving means 7 by the central unit 16 by means of dotted arrows. Generally, the transfer means have sufficient chemical resistance to the reagents used for OGN synthesis, especially to the solvent used for saturation. Solvents are flammable, so the design of the vehicle must avoid the danger of fire (hence the importance of neutral gases). These conditions are fulfilled for the apparatus of FIG. 3 since the transfer means is located outside the chemical reactor for the bellows.
[0115]
The moving means 7 comprises a lower part 5 of the reaction chamber 5. ₁ May be configured by any known means suitable for moving Bellow 5 ₃ For this lower part 5 ₁ Is the injection nozzle 6 ₁ Upper part 5 of chamber 5 containing ₂ Has a certain degree of mobility. Examples that may be mentioned are: pin / rack systems in three dimensions, which can be driven mechanically, slide systems, or cross-motion carriages with crossing movement and / or translation and / or translation and / or Examples include systems with rotation-powered micrometric displacement plates. Commercially available products corresponding to the above system are provided by MICROCONTROL, NEWPORT and OWIS.
[0116]
4A, 4B and 4C show the injection nozzle 6 ₁ Lower part 5 ₁ Of the support 1 carried by x , y ,and z Indicates movement along an axis.
[0117]
Such a transfer means may supply a localized liquid to the reaction well 4 of the support 1.
[0118]
This example of the first embodiment of the device of the invention shown in FIG. 3 allows the localization liquid to be distributed in very small volumes of droplets to the microwells 4 of the support 1.
[0119]
By way of example, FIGS. 5 and 6 show enlarged pictures of the support 1 comprising dozens of microwells 4, each having a surface of 150 μm × 150 μm and a depth of about 100 μm.
[0120]
The microwell is defined by a grid 3 of photocured epoxy resin. The photograph in FIG. 5 shows an array of microwells 4, in particular, the encircled microwells 4 ′ (which also do not contain traces of localization liquid). The photograph of FIG. 6 shows the array of FIG. 5, where the encircled microwells 4 ′ are the drop-on-demand type piezoelectric nozzles 6 of the injection system. ₁ It contains about 50 picoliters of acetonitrile droplet G sent by (1a). The support 1 containing the droplet G is absent under the conditions provided by the apparatus of FIG. 3 (saturated), the droplet evaporates in another fraction and it is possible to take a picture Did not. In contrast, using the apparatus of FIG. 3, the droplets are seen in a volume saturated with acetonitrile in the reaction chamber. It stays there for 5 minutes (min) to 30 minutes, which is enough time to cause a chemical fixation and / or OGN synthesis reaction.
[0121]
The implementation of the method of the invention described in FIGS. 3, 3A and 4A, 4B and 4C has the advantage that it is easy to perform with flexible PTFE bellows. The saturated volume is low, which facilitates control (chemical composition, solvent concentration, pressure, temperature, etc.). The transfer means is completely outside the saturation volume and is therefore protected from corrosion. In contrast, the width of movement of the microdeposition / bellows means is limited to a sweep of the bellows, ie a few centimeters (cm).
[0122]
To make arrays on large supports or substrates, for example, 4-8 inch silicon wafers (10.16 cm-20.32 cm), the second embodiment of FIGS. The device is preferably used as described in more detail in FIGS. 9A, 9B, 9C and 9D.
[0123]
FIGS. 7, 8 and 9 show a second embodiment of the device of the invention. As can be seen in the figures, the essential difference between this second embodiment and the first embodiment is that the support using the means 7 for moving the support 1 along the axis X In addition to moving the body, the localized supply means 6 is movable along the Y axis, and the delocalized supply means 8 is at least X, Y along the Z axis with large movement. Along the axis is the fact that it is movable and allows large substrates to be worked on.
[0124]
The means 7 for moving the support 1 and the moving means 30 and 40 for the localized supply means 6 and the delocalized supply means 8 are at least partially contained in the reaction chamber 5. This chamber is advantageously larger than the device of the first embodiment (eg 1.5 m (m) x 1.5 m x 1 m). The chamber 5 is designed such that it can be saturated, for example, with a gas stream filled with a neutral gas (eg, argon) and a volatile solvent vapor. To this end, an apparatus 10 for saturating the chamber 5 is provided. It is the same as that described for the first embodiment of the device of the present invention. Identical elements have identical reference numbers.
[0125]
The device of the second embodiment is more suitable for industrial operation. The important point here is that the saturation of the reaction chamber can be kept constant. It is clear that an interruption in saturation will require a significant amount of time to resume saturation due to the large volume of the chamber.
[0126]
A further feature of this second embodiment of the device is that a part of the moving means 7, 30, 40 arranged outside the chamber 5 comprises a motor 31, 41 and 71, and a power supply for the motor (FIG. Are not shown), while the mechanical part inside the moving means 7, 30, 40 5 (described in more detail below) is made of TEFLON® (PTFE), stainless steel It is made from an inert material such as steel, perfluorinated elastomers (KALREZ®, CHEMRAZ®), or polyamide (NYLON®).
[0127]
When loading and unloading the support 1 carried by the moving means 7 so as not to interrupt the saturation of the chamber 5, the chamber 5 is provided with a side airlock 50.
[0128]
FIG. 8 shows in detail the localized supply means 6 lifted from their means 30 for movement along the Y axis, and the support 1 and its means 7 for movement along the X axis. The walls of the reaction chamber 5 are marked in FIG. 8 by a dot-dash line.
[0129]
The means 7 for moving the support or substrate 1 comprising the resin grid 3 defining an array of reaction microwells 4 generates a rotational movement which can be transformed into a translation by a transmission worm screw 74. It includes a translation carriage that can slide on a guide rail 73 under the action of a motor 71 that can be driven. The guide rail 73 and the transmission worm screw 74, which are, for example, metal rods of polygonal or circular cross-section, are parallel to each other. At each end of the slide 73, a buffer block 75 is provided. The opposite end of the transmission worm screw 74 connected to the motor 71 is also provided with a buffer block 76. The end portions of the elements 73, 74, the buffer blocks 75, 76, and the motor 71 are arranged outside the chamber 5.
[0130]
Advantageously, the path for the guide rail 73 and the transmission worm screw 74 through the walls of the chamber is sealed by a formed O-ring, for example made of KALREZ. In an advantageous variant of the invention, the buffer block 75 for the slide 73 can form an integral part of the wall of the chamber 5 and can save an opening that has to be airtight.
[0131]
The constituent materials of the components that are at least partially arranged in the chamber 5 are selected from materials that are resistant to fixed and / or synthetic chemical reagents, in particular OGN. Examples of constituent materials that may be cited are PTFE, stainless steel, and KALREZ.
[0132]
The external motor 71 is protected from chemical reagents, which avoids the risk of fire or explosion.
[0133]
With respect to the localized supply means 6 and their transfer means 30, the transfer means 30 is for the micro-deposition of localized liquids or reagents on the microwells 4 arranged on the support 1. It should be noted that the station 60 is designed to ensure translational movement along the Y axis.
[0134]
As with the transfer means 7 described above, the transfer means 30 includes a motor 31 connected to one end of a transmission worm screw 34 terminated at another end by a buffer 36. As mentioned above, certain parts of the element and other terminal parts of the element are located outside the chamber 5. Reference should be made to the above description of the means 7 for further details.
[0135]
For a micro-deposition station 60, which can be carried in translation by a worm screw 34 along a guide rail 33, it consists of a carriage with an element 61 supporting a bottle of reagent 62 (only one in FIG. 8). Is shown), and by a rack 64 parallel to the Y-axis and supporting a series of micro-deposition devices 65 (only one is shown in FIG. 8). This rack 64, which is parallel to the Y axis, faces side by side with the element 61 supporting the reagent bottle 62.
[0136]
As mentioned above, the elements of the means 6 and 30 arranged inside the saturation chamber 5 are made of a material which is resistant to the reagents used (eg acetonitrile) and / or Separated from Examples of resistant materials have been given above.
[0137]
Transport of the reagent contained in the bottle 62 to the corresponding micro-deposition device 65 is accomplished by line 66 (only one is shown in FIG. 8). The localizing reagent or liquid is propelled by a neutral gas (eg, argon) flow carried under the pressure supplied via line 67.
[0138]
In a variant, the reagent bottle 62 can be arranged outside the working volume defined by the reaction chamber 5. In that case, ad hoc "fluid" connections would be provided.
[0139]
The distance along the Z-axis between the substrate or support 1 and the micro-deposition device 65 can be determined by using the movement of the carriage 60 and / or 72 along the X and / or the Y-axis to localize the reagent. Are adjusted to address the assembly of the array of microwells 4 defined by the resin grid 3 of the support 1.
[0140]
This addressing by micro-deposition using the apparatus 65 (comprising a reagent bottle 62 and a propellant gas upstream) and movement of the carriages 60, 72 is accomplished by using a memory (computer) (see FIG. (Not shown) are controlled and managed by a central control.
[0141]
9A, 9B, 9C, 9D show details of the delocalized supply means 8, and more particularly its specific elements constituted by the piston 80. As shown in FIG. 7, the piston 80 for the delocalized supply means 8 is movable in translation along the Z-axis. A moving means 40 provided for this purpose comprises a motor 41 connected to a worm screw 44 in a manner similar to the moving means 7 and 30 described above, and is parallel to the Z axis and the worm screw 44. The piston 80 is driven in translation along the Z axis along the two guide rails 43. The lower ends of the guide 43 and the worm screw 44 have buffer blocks 45 and 46. The same is true for the upper end of the guide rail 43. More specifically, reference should be made to the description of the moving means 7 and 30 described above.
[0142]
The piston 80 is integrated with a carriage 81 which cooperates with the moving means 40.
[0143]
The vertical axis Z of the piston 80 is perpendicular to the axis X for the movement of the support 1 and to the axis Y for the movement of the micro deposition station 60. As a result, it is possible to start the moving means 7 and 40 in a manner that aligns the center of the support 1 with the axis of the piston 80 and ensures that the piston 80 covers the support substrate 1.
[0144]
As shown in FIGS. 9A, 9B, 9C and 9D, the lower surface of the piston 80 is provided with an annular O-ring 82, preferably made of KALREZ. This annular O-ring is coaxial with the piston and its diameter is substantially smaller than the diameter of the lower surface of the piston 81.
[0145]
As shown in FIG. 9D, this annular O-ring 82 is between the lower surface 84 of the piston 81 and the surface of the support 1 (whereas the piston 81 is applied by the moving means 40) Interstitial space 83 is defined.
[0146]
In this situation where the piston 81 is horizontal with respect to the support 1, the support of the piston 81 + the annular O-ring 82 + the lower surface 84 of the piston 81 is of very small dimensions, for example a 4 inch diameter (10.16 cm). ) For a substrate having 5 cubic centimeters (cm ³ ) -10cm ³ Is formed.
[0147]
The piston 81 is constituted by a wall 85 having a thick lower end portion defining a hollow inner volume 86.
[0148]
The lower part of the wall 85, which is arranged in a diametric plane, is the outer surface forming the lower surface 84 of the piston 81, as well as the angular positions 0 °, 90 °, 180 ° and 270 °. It has an inner surface 87 provided with peripheral tubular elements. The tubular element 88 places the interior 86 of the piston 81 in communication with the outside and in particular with the interstitial space 83 (as shown in FIG. If placed horizontally). These tubular elements 88 are connected via tubes (not shown) to a delocalized reagent reservoir (also not shown in the figure, and the reservoir 12 and FIG. 3 of the first embodiment). 13 (corresponding to 13). Thus, it is possible to supply the delocalized reagent to the interstitial space 83 having a much reduced volume without interrupting the saturation of the reaction chamber assembly 5. In addition to supplying the delocalized reagent, the tubular element 88 also drains the delocalized reagent and / or dries the substrate by passing a gas stream or by pumping out. obtain.
[0149]
Like the localized supply means 6 and their moving means 30, the means 7 for moving the substrate 1, the delocalized supply means 8 and their moving means 40 comprise the localized OGN synthesis method according to the invention. And / or controlled by a central control unit, which may manage all of the procedures for supply, cleaning, rinsing, draining and drying, etc. used in the context of the printing method.
[0150]
It should be noted that those skilled in the art will be able to devise numerous variations to ensure that the reaction chamber 5 is sealed. In particular, sealed ball races and seals of various shapes may be used, and these mechanical construction elements are described in MEMOTEC, PRODUCTIQUE, conception et desinsins (C. Barrier and R. Bourgeois).
[0151]
In a further aspect, the invention provides a support for supporting an array of OGN sequences as described above.
[0152]
The invention also provides for detecting and / or detecting a target OGN sequence using a bioarray comprising probes made from the OGN sequence that are complementary to the target OGN sequence and that can pair together by base pairing. Or a method of identifying, comprising:
Using a support that supports an array of OGN probes as described above;
And clarifying the probe / target pairing using a "pico green" type fluorescent marker;
A method is provided.
[0153]
"Picogreen" is a molecule that fluoresces in the presence of double-stranded DNA. Picogreen does not require a specific reaction and only has to be brought to the presence of the duplex for 5 minutes before measuring fluorescence. The molecules are then used to detect duplexes, regardless of whether they are fluorescent markers (fluorescein, rhodamine, ...) or intercalators (ethidium bromide, Hoechst 33258). Have a number of advantages over commonly used materials.
[0154]
Therefore, picogreen does not require a labeling reaction, in contrast to the fluorescent probes used in DNA arrays (chips).
[0155]
The linearity of the fluorescent signal is excellent over a wide range of concentrations. Thus, it detects duplexes at concentrations far below what can be measured with conventional intercalators, even in the presence of contaminants (single-stranded DNA, RNA) in the solution being measured. I can do it. The sensitivity of picogreen is up to 400 times better than Hoechest 33258, which is then more sensitive than ethidium bromide.
[0156]
This high sensitivity allows local fluorescence levels to be measured, which cannot be measured with conventional intercalators. Thus, picogreen is more suitable than intercalators for detecting hybridization in high or medium density arrays containing small sized hybridization units.
[0157]
The fluorescent signal is independent of the base composition of the sequence being tested, as opposed to the Hoechst product.
[0158]
"Picogreen" is excited by the same means as fluorescein (excitation at 480 nm (emission at 520 nm)), which makes its use with standard equipment very practical.
[0159]
Finally, the present invention provides for detecting and / or detecting a target OGN sequence using a bioarray comprising probes made from the OGN sequence that are complementary to the target OGN sequence and that can pair by base pairing. Or a method of identifying, comprising:
Using a support that supports an array of OGN probes as described above;
Providing the support with at least one structure comprising at least one semiconductor material Sc, coated on at least one of its faces with at least one insulating layer Is [the layer has an OGN probe on its surface] Being formed into an affinity array;
Contacting the OGN probe with a conductive liquid medium LC containing the target OGN sequence; and
Apply the following operating procedures:
a) selecting unlabeled OGN probe;
b) ensuring that the Sc Fermi level substantially corresponds to or exceeds the intrinsic surface level of Sc;
c) Sc undergoes periodic irradiation with photons having an energy that is ≧ Sc bandgap energy; and
d) The change ΔVfb in the flat band voltage Vfb of Sc is measured directly or indirectly, and this change is directly related to the specific pairing of the complementary ligand of the OGN probe with the target OGN sequence from the conductive medium LC. Triggered by a charge-effect phenomenon that is linked together and essentially, except:
(I) changes resulting from charge and / or charge transfer effects caused by chemical reactions catalyzed by the enzyme (and where some of the detected substance is consumed);
(Ii) and the said at least one tracer product can be revealed by a change in pH or redox potential and / or by a marker [preferably a type of radiation absorption or emission (eg fluorescence, radioactivity, coloration)]. A change in the light response coupled to its appearance in the medium LC;
e) interpreting the signal collected by identifying and / or assaying the target OGN sequence of the LC;
A method is provided.
[0160]
This method comprises an array of OGN sequences prepared according to the methods described above, and as described in PCT application PCT / WO-A-95 / 57157, which is hereby incorporated by reference in its entirety. Relate the method of identification and / or detection of biological material.
[0161]
This association gives rise to a method of detecting and / or identifying target OGN sequences that is particularly effective.
[0162]
The following examples illustrate the operation of the method and apparatus of the present invention, and particularly the first embodiment of the apparatus.
[0163]
(Example)
(Example 1)
(1.1. Reagents and methodology)
Table 1 shows a classification example for the phosphoramidite chemistry used below. Many variations are possible within the scope of the present invention.
[0164]
[Table 1]

[0165]
(1.2. Apparatus)
The apparatus used is that described above with reference to FIG.
[0166]
This device includes an injection system 6 made of six microvalve devices of the type shown in FIG. 3A to inject the reagents A, C and D of Table 1 (one valve for reagent A, reagent D One valve, one for 4 bases in C).
[0167]
The apparatus also comprises six delocalized supply means 10 of the type shown in FIG. 3 for reagents A, B, E, F and G in Table 1.
[0168]
Product A (acetonitrile) can be sent in both localized and delocalized formats. The apparatus also comprises means for drying the substrate with argon gas (ie, reagent H in Table 1).
[0169]
The support is constituted by a silicon or glass plate with 256 lithographic resin wells (epoxy).
[0170]
Each microwell had a surface of 415 μm × 415 μm and a depth of about 415 μm.
[0171]
The volume of the well needs to be filled by injecting the base droplet and the activator droplet without overflow.
[0172]
The microwell fabrication protocol for silicon of this size is as follows:
1. Wash the support (acetone, alcohol ...)
2. Dry with dry argon
3. For example, using a cationically polymerized epoxy resin (e.g., a resin EPON SU8-100 developed by IBM, supplied at different ratios diluted with gamma-butyrolactone), using a spinner to deposit a lithographic resin on a substrate. The speed of the spinner controls the resin thickness from 1 μm to 1000 μm (typically 100 μm); the resins cited as examples are used in making microsystems and Thick vertical wells can be created with form factors up to;
A 400 μm deep microwell was prepared as follows:
・ Slowly applying a single resin layer at a low rotation speed;
Or by sequentially applying 2 to 4 layers and repeating steps 4, 5 and 6 below for each layer,
Made by any of the following:
4. Cure at 90 ° C. for 5-30 minutes, depending on the thickness of the resin and the support;
5. Selective exposure using a UV source (directly with a UV lamp or UV laser using a set of masks). Microjoules per square meter absorbed by the substrate (mJ / m ² ) Energy dose must be adjusted to obtain good resin / substrate adhesion between the different curing and developing steps. Microwell size varies from 5 μm × 5 μm to 1000 μm × 1000 μm (typically 415 μm × 415 μm);
6. Secondary curing for 5-30 minutes at 50-100 ° C., depending on the thickness of the resin and the support;
7. Dissolve the unexposed resin using a solvent (acetone, gamma-butyrolactone, PGMEA ...) with optional ultrasonic activation;
8. Final cure to increase crosslinking of the resin: 5 minutes to 30 minutes at 90 ° C to 150 ° C. This step determines the behavior of the microwell for oligonucleotide synthesis reagents.
9. Wash the support with the sulfochrome mixture for 5 minutes, then rinse with double distilled water;
10. Aqueous silanization by immersion in a GPTS / water mixture (1% v / v) for 45 minutes, then at 125 ° C. for 45 minutes.
[0173]
The parameters of the method were adjusted, resulting in:
The solvents (acetonitrile, dichloromethane, THF, pyridine, etc.) and reagents (iodine, etc.) used in the oligonucleotide synthesis do not cause the decomposition of the resin superstructure or its separation;
• Accelerate the curing of the resin sufficiently so that the superstructure is inert to oligonucleotide synthesis.
[0174]
[Table 2]

[0175]

[0176]
The control of the moving means is adjusted such that the distance between the positions of the nozzles is about 1 mm from the opening of the well 4 during the ejection of the localized liquid. The pressure in the bottle containing the localized reagent is in the following range:
From the minimum value at which the reagent is not completely injected but adheres to the base;
-Up to the maximum value corresponding to the volume of the well.
[0177]
To reduce the risk of contamination from well to well, only every other well is used so that only 64 of the 256 wells in the substrate constitute a node for the OGN array.
[0178]
The memory 15 and the central unit 16 are mounted (in the 3 'to 5' direction) such that an array containing all of the following variations of the following OGN is created:
3'-GAG GAT GGT X ₁ X ₂ X ₃ CCT GCT AGG TAT-5 '
X ₁ , X ₂ And X ₃ Form 64 possible combinations of A, C, G and T. X in a specific case ₁ X ₂ X ₃ = This example was chosen because ACG is a L226 OGN of biological interest.
[0179]
In practice, with respect to steric hindrance during hybridization, it is useful for the OGN to keep room from the substrate. For this purpose, the sequence is extended with a 10C header, for example, resulting in:
3'-CCC CCC CCC CGA GGA TGG TX ₁ X ₂ X ₃ CC TGC TAG GTA TC-5 '
The first and third parts of the array are the following sequences:
CCC CCC CCC CGA GGA TGGT and CC TGC TAG GTA TC were made using a Perkin Elmer Expertise OGN synthesizer modified to use a substrate.
[0180]
Center part X ₁ X ₂ X ₃ Was produced by the apparatus of FIGS. 1 to 4 as a function of FIG. FIG. 10 shows that OGN L226 is in a central position and is surrounded by probes of very different composition, so that hybridization of the ACG pin (pin) is easy to detect. Using such an array, it was possible to detect U226 OGN complementary to L226.
[0181]
(Industrial application)
A preferred application of the invention is to create a support bearing a plurality of polynucleotide sequences (preferably oligonucleotide sequences and / or peptides, which are different from each other and at least one of its faces. Above can be paired with these complements.
[Brief description of the drawings]
This detailed description is made with reference to the accompanying drawings.
FIG.
FIG. 1 is a perspective view of a support on which the OGN array of the present invention is manufactured.
FIG. 2
FIG. 2 is a vertical sectional view taken along line II-II of FIG.
FIG. 3
FIG. 3 is a schematic diagram of a first embodiment of the device of the present invention.
FIG. 3A
FIG. 3A is an improved schematic diagram of a system for ejecting droplets of a localized reagent.
FIG. 4A
FIG. 4A is a detail of FIG. 3 including the reaction chamber, the support and the localized supply means, and moving the support and a portion of the reaction chamber relative to the localized supply means along the X axis. Show.
FIG. 4B
FIG. 4B is a cross-sectional view along line IV-IV of FIG. 4A and shows the movement of the support and a portion of the reaction chamber relative to the localized delivery means along the Y-axis.
FIG. 4C
FIG. 4C repeats the same elements as FIG. 4A, but shows the movement of the lower part of the reaction chamber and the support relative to the localized supply means along the Z-axis.
FIG. 5
FIGS. 5 and 6 are enlarged photographs of the support 1 including a grid 3 of photoresin defining dozens of microwells intended to function as sheets for chemical fixation and / or OGN synthesis reactions. . The difference between FIGS. 5 and 6 lies in the presence of localized reagent droplets in the micro-wells circled in the support of FIG.
FIG. 6
FIGS. 5 and 6 are enlarged photographs of the support 1 including a grid 3 of photoresin defining dozens of microwells intended to serve as sheets for chemical fixation and / or OGN synthesis reactions. . The difference between FIGS. 5 and 6 lies in the presence of localized reagent droplets in the micro-wells circled in the support of FIG.
FIG. 7
FIG. 7 is a general schematic perspective view of the second embodiment of the present apparatus.
FIG. 8
FIG. 8 is a detailed perspective view of FIG. 7 showing the support 1 and its moving means 7 and the localized supply means and their moving means 30.
FIG. 9A
9A, 9B, 9C and 9D show delocalized supply means. FIG. 9A is a front view of the piston 80 of the delocalized supply means 8.
FIG. 9B
9A, 9B, 9C and 9D show delocalized supply means. FIG. 9B is a diametrical cross section through the piston of FIG. 9A.
FIG. 9C
9A, 9B, 9C and 9D show delocalized supply means. FIG. 9C is a top view of the piston 80 of FIGS. 9A and 9B.
FIG. 9D
9A, 9B, 9C and 9D show delocalized supply means. FIG. 9D is a partial diametrical cross section of the piston 80 applied to the support 1.
FIG. 10
FIG. 10 shows an array 64 of 3 mer oligonucleotides each produced in the example.
1 and 2 show a support 1 forming an integral part of the device according to the invention.

Claims (18)

A process for the preparation of a support having a plurality of poly-, preferably oligo-nucleotide and / or peptide sequences on one of its faces, advantageously wherein said different sequences (OGN sequences) are base-paired Can be paired with their complements, and these are obtained by chemical synthesis in the presence of volatile solvents;
The method essentially comprises:
a) using a support (1) [where the surface intended to carry the OGN has an array of reaction wells (4), each well (4) having a predetermined OGN The wells serve as sheets for localized immobilization of sequences and / or chemical synthesis; the wells are exposed to actinic radiation in the area where the photocurable resin is applied and the wells are intended to be defined. By curing the resin and removing the uncured resin to obtain a grid of resin (3) forming the walls defining the wells (4)];
b) placing the support (1) in a reaction chamber (5) and using the vapor of a volatile solvent used in the localized fixation of the OGN and in the chemical synthesis, preferably in the reaction chamber (5) saturating the chamber by circulating a gas stream containing a suitable saturated vapor through it;
c) performing (by increasing) localized immobilization and / or chemical synthesis of the OGN sequence in at least a portion of the well;
And this operation includes the following:
(I) locally and separately supplying reagents and liquid consumables for fixing and / or synthesizing the corresponding OGN sequence to each associated well;
(Ii) collectively (non-locally) supplying common reagents and / or liquid consumables to the relevant wells for reactions intended to be performed in all the wells; and (iii) 2.) Before each operation for localized supply to said well (4), at least a portion of any liquid contained in each well intended to be supplied in a localized manner is removed. Make sure it is done.
Achieved by: 2. The material according to claim 1, wherein the support is selected from the group consisting of glass, quartz, silicon, optionally coated with at least one layer of oxide or nitride. 3. the method of. Treating the surface of the support to create an immobilization site thereon capable of forming a non-labile bond with the head comonomer of the OGN sequence, the treatment preferably prior to creating a microwell array and / or The method according to claim 1 or 2, characterized by or later silanization using an epoxyalkoxysilane. (Meth) acrylates, wherein the photocurable resin can be photocured from the group constituted by positive or negative resins, preferably from a subgroup comprising negative resins, and more preferably by radical and / or cationic route; 4. The method according to claim 1, wherein the method is selected from a class comprising epoxy, polyester and / or polystyrene resins. The saturated gas stream for the reaction chamber contains, in addition to the associated solvent vapor, at least one neutral gas, which gas stream preferably determines the overpressure in the chamber relative to the ambient atmosphere The method according to any of the preceding claims, characterized in that: To ensure localized and delocalized supply to the wells, corresponding means are used, wherein the localized supply means and the support are three-dimensional (X, Y, And Z) are movable with respect to each other; and to immobilize and / or synthesize n OGN sequences, each comprising an x comonomer, on the support:
The composition of the array of n OGN sequences to be produced is x- continuous, corresponding to a series of actions of removal, supply and deposition of reagent liquid, each containing a comonomer and each being localized in the n- well of the support; Storing it in memory by storing the composition in a rung configuration [the liquid determines the properties of the comonomer of the rung Ri = 1- x in the n- well];
And firstly, instructing the localized supply means and the mobile means to perform various series of actions as defined above, and secondly, instructing the delocalized supply means with the fixed And at various times during the OGN sequence synthesis procedure, to remove, transport, and supply the delocalized liquid to the n- well of the support [subsequent to each localized supply, And more generally, a step is performed to drain the liquid present in the reaction chamber];
[The instructions and actions they derive are repeated by incrementing i by 1 in R _{i up to} i = x]
The method according to any one of claims 1 to 5, characterized in that: The liquid is removed in step 3 (iii) of the method, so that the associated wells have a liquid depth of at least 90%, preferably at least 95%, and more preferably at least 98% of their volume. The method according to claim 1, wherein the method does not include: The liquid is dried, advantageously by injecting a gas (preferably a neutral gas) into the reaction chamber and / or by raising the temperature of the reaction chamber and / or the support; Method according to claim 7, characterized in that it is removed by reducing the vapor pressure of the liquid removed from the reaction chamber. Less than:
At least one support (1) [where the surface intended to carry the OGN has an array of reaction wells (4), each well being a localization of a given OGN sequence; Acting as a sheet for fixation and chemical synthesis, the wells apply a photocurable resin, cure the resin by exposure to actinic radiation in the areas intended to define the wells, and By removing the uncured resin to obtain a resin grid (3) that forms the walls defining the wells (4)];
At least one reaction chamber (5) intended to contain the support;
A localization liquid (reagent / consumable) to the reaction well (4), which allows the fixation and synthesis of a given OGN sequence in each of the wells (4) of the support (1) Means for localized supply (6);
Means for moving said localized supply means (6) and / or vice versa with respect to said support (1);
Means for the delocalized supply of a delocalized liquid (product / consumable) common to the reaction intended to be carried out in all or part of said wells to said reaction wells (8) ;
Optional means (40) for moving said delocalized supply means (8);
Means for discharging a delocalized liquid (9, 88); said discharge means (9, 88) being associated with said delocalized supply means (8);
An apparatus (10) for saturating the reaction chamber using the volatile solvent vapor used in the immobilization and synthesis of the localized OGN;
At least one container (11, 62) for the localized liquid;
At least one container (12) for the delocalized liquid;
At least one container (13) for emptying liquid effluent;
-Any device for supplying gas to the chamber;
-Optionally, at least one memory (15) for various OGN sequences immobilized and synthesized on the support; and-optionally reading the memory and generating a signal At least one central unit (16) for controlling the supply of fluid and the movement of said localized supply means and said support relative to each other;
Apparatus for performing the method according to any one of claims 1 to 8, characterized in that it comprises: The support 1 comprises:
At least one substrate plate (2) made of a material selected from the group consisting of glass, quartz, silicon optionally coated with at least one layer of oxide or nitride;
At least one array (3) (preferably a grid) of photo-cured resin integral with one of the faces of the substrate [the array, together with the reaction wells (4), defining a matrix; , Preferably selected from the group consisting of epoxy or (meth) acrylate resins that are photocurable by cationic and / or radical pathways],
The device according to claim 9, comprising: Said localized supply means (6) comprises a system for the release or arrangement of a localized liquid, said system advantageously comprising:
1) Mechanical injection using the following types of equipment:
a) Drop-on-demand using piezoelectric effect;
b) continuous jet interrupted by electrostatic effects;
2) injection under pressure using a microvalve device,
Device according to claim 9 or 10, characterized in that it is selected from: The device (10) for saturating the reaction chamber (5) is designed to allow the circulation of a saturated gas stream comprising the volatile solvent vapor used and at least one neutral gas. Device according to any one of claims 9 to 11, characterized in that it is located. The reaction chamber (5) comprises a sealing bellows _(3), its one end _{(5 2)} of the localization supply means (6) and carry (carry), and its other end _{(5 1} ) Has a reaction site opposite said supply means for receiving said support (1) and comprises a delocalized supply means (8), said end carrying said localized supply means (5 ₂ ) is preferably fixed, and the other end (5 ₁ ) carrying the support ( ₁ ) is preferably three-dimensional x , y , being movable in z .
Apparatus according to any one of claims 9 to 12. The localized supply means (6) is intended to receive the support (1) and to a fixed part of the reaction chamber (5) associated with the delocalized supply means (8) Device according to any of claims 9 to 13, characterized in that it can be moved in three dimensions x , y , z by the action of a moving means (7). Less than:
The support (1) is movable in translation along an axis x using a moving means (7) and the localized supply means (6) is moved using a moving means (30); , Translational along the axis y , said delocalized supply means (8) being movable in translation along the axis z using a moving means (40), x , y , and z are perpendicular to each other;
-At least a part of said moving means (7), (30), (40) is arranged outside said sealed saturable reaction chamber (5), said means (7), (30), (40) outer part of, preferably at least one electric motor _(71), the (3 1), _{(4 1)} and its power supply;
And that it comprises at least one airlock (50) for loading and unloading the support (1);
Apparatus according to any one of claims 9 to 12, characterized in that: A support for supporting an array of OGN sequences as defined in claims 1 and 10. A method for detecting and / or identifying a target OGN sequence using a bioarray comprising probes made from the OGN sequence that is complementary to the target OGN sequence and that can pair together by base pairing. And the following:
Using a support for supporting an array of OGN probes according to claim 16;
And clarifying the probe / target pairing using a "pico green" type fluorescent marker;
The method. A method for detecting and / or identifying a target OGN sequence using a bioarray comprising probes made from the OGN sequence that is complementary to the target OGN sequence and is capable of pairing by base pairing. ,Less than:
Using a support for supporting an array of OGN probes according to claim 16;
Providing the support with at least one structure comprising at least one semiconductor material Sc, coated on at least one of its faces with at least one insulating layer Is [the layer has an OGN probe on its surface] Being formed into an affinity array;
Contacting the OGN probe with a conductive liquid medium LC containing the target OGN sequence; and applying the following operating procedure:
a) selecting unlabeled OGN probe;
b) ensuring that the Sc Fermi level substantially corresponds to or exceeds the intrinsic surface level of Sc;
c) Sc undergoes periodic irradiation with photons having an energy that is ≧ Sc bandgap energy; and d) directly or indirectly measuring the change ΔVfb in the flat band voltage Vfb of Sc, Is triggered by a charge effect phenomenon that is directly and essentially linked to a specific pairing of the complementary ligand of the OGN probe with a target OGN sequence from the conductive medium LC, except:
(I) changes resulting from charge and / or charge transfer effects caused by chemical reactions catalyzed by the enzyme (and where some of the detected substance is consumed);
(Ii) and at least one of the tracer products that can be revealed by a change in pH or redox potential and / or by a marker (preferably a type of radiation absorption or emission (eg, fluorescence, radioactivity, color)). Altering the light response linked to its appearance in the medium LC; and interpreting the signal collected by identifying and / or assaying the target OGN sequence of the LC;
The method.