WO1999051770A1 - Nouveau procede de preparation de puces micromatricielles composees et puces micromatricielles composees ainsi obtenues - Google Patents

Nouveau procede de preparation de puces micromatricielles composees et puces micromatricielles composees ainsi obtenues Download PDF

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Publication number
WO1999051770A1
WO1999051770A1 PCT/CN1999/000013 CN9900013W WO9951770A1 WO 1999051770 A1 WO1999051770 A1 WO 1999051770A1 CN 9900013 W CN9900013 W CN 9900013W WO 9951770 A1 WO9951770 A1 WO 9951770A1
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Prior art keywords
micro
substrate
template
microseal
compound
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PCT/CN1999/000013
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English (en)
French (fr)
Inventor
Zuhong Lu
Lingang Zhang
Jianmin Ma
Chunxiang Xu
Yali Chen
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Zuhong Lu
Lingang Zhang
Jianmin Ma
Chunxiang Xu
Yali Chen
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Application filed by Zuhong Lu, Lingang Zhang, Jianmin Ma, Chunxiang Xu, Yali Chen filed Critical Zuhong Lu
Priority to AU21480/99A priority Critical patent/AU2148099A/en
Priority to US09/647,525 priority patent/US6423552B1/en
Publication of WO1999051770A1 publication Critical patent/WO1999051770A1/zh

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00351—Means for dispensing and evacuation of reagents
    • B01J2219/00382—Stamping
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00497—Features relating to the solid phase supports
    • B01J2219/00527—Sheets
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00585—Parallel processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00596—Solid-phase processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00659—Two-dimensional arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00709—Type of synthesis
    • B01J2219/00711—Light-directed synthesis
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B60/00—Apparatus specially adapted for use in combinatorial chemistry or with libraries
    • C40B60/14—Apparatus specially adapted for use in combinatorial chemistry or with libraries for creating libraries

Definitions

  • the present invention relates to a new method for preparing a compound microarray chip, in particular to a method for preparing a compound microarray chip by a multi-imprint fixed-point synthesis method, and a compound microarray chip prepared by the method.
  • Compound microarray chip refers to the preparation of a group of compound arrays composed of different molecular microunits on the surface of a solid substrate.
  • Compounds mainly refer to biological macromolecular substances including nucleic acids such as DNA, RNA, oligonucleotides, proteins such as enzymes, antibodies, antigens, peptides, etc., and other artificially synthesized biologically active substances such as PNA (peptide nucleic acids, etc.).
  • a compound microarray chip is also called a gene chip.
  • the compound microarray chip is of great significance in biological detection, medical detection, drug screening, gene sequence analysis and compound library synthesis.
  • biology for example, with the continuous development of molecular biology, especially since the implementation of the world-renowned Human Genome Project, data on nucleic acid, protein sequence and structure have grown exponentially.
  • the most challenging work in the next century is how to analyze a large amount of molecular information after the completion of the Human Genome Project, that is, in the post-gene era, to find out the laws and make biology rise from experiments to theories, so as to better Recognize the phenomena of life and revolutionize medical treatment.
  • Biochip technology is to integrate many discontinuous analysis processes involved in life science research, such as sample preparation, chemical reactions, and analytical detection, into the chip by using microelectronics, micromechanics and other processes to make it continuous and integrated. And miniaturization.
  • life science-related fields such as disease diagnosis and treatment, new drug development, forensic identification, food and the environment, and provide a powerful means for obtaining and analyzing biological information.
  • Compound microarray chips are very important and necessary for life science research.
  • the sequence of a biological substance is detected or sequenced by the interaction between an array of known compound molecules on a chip and the biomolecules being measured.
  • nucleic acid detection includes firstly preparing an oligonucleotide molecular probe array, that is, a compound microarray, on a solid-phase carrier, and then hybridizing the gene to be tested with the oligonucleotide molecular probe array, and hybridizing by a computer. The results were analyzed to obtain the information of the tested gene sequence.
  • the key point is the preparation of oligonucleotide molecular probe arrays.
  • the probe array of the chip has high spatial resolution, small synthesis workload, high speed, simple method, and low cost.
  • spray or printing techniques to combine different probe molecules at different positions on the substrate to form a probe array. It is difficult to achieve high spatial resolution by preparing probe arrays by spraying or printing methods, and the synthesis of the probe molecules is performed one by one, and the synthesis workload is large.
  • the other method is the United States
  • Affymetrix proposed a template-based photochemical reaction to synthesize a probe array on a substrate.
  • Using this method to prepare a probe array can achieve a high spatial resolution (40 X 40 ⁇ 2 ), and it is a parallel synthesis during chip synthesis, and the synthesis speed is fast.
  • the yield of the photochemical reaction is low, and there are many side reactions in the reaction, which makes the accuracy of the synthetic probe sequence is not high, and the reagent with a special protective group is needed, and the cost is high. Therefore, it is still used in the preparation of compound microarray chips. There needs to be a better way.
  • the purpose of the present invention is to provide a method for preparing a compound microarray chip, which is simple, reliable, has high spatial resolution, and high accuracy, that is, a microarray chip is prepared by multiple imprint fixed-point synthesis methods.
  • micro-stamps with bumps are prepared according to the micro-array design of the desired compound, and then the corresponding chemical reactants are coated on the prepared micro-stamps according to the pre-design. Finally, the chemically reacted The microseals are sequentially embossed on the same substrate one by one to obtain a compound microarray chip.
  • the present invention has been completed based on the above. In the above method, the position of the chemical reaction on the substrate and the reaction of the chemical groups on the substrate are controlled by the convexo-concave of the seal, so that the chemical reactants are covalently coupled to the substrate, and finally a desired chemical order is formed on the substrate. Microarray chip.
  • the first aspect of the present invention relates to a new method for preparing a compound microarray chip, and more particularly to a method for preparing a compound microarray chip using a multi-imprint fixed-point synthesis method.
  • Another aspect of the present invention relates to a compound microarray chip prepared by the method of the present invention, especially a high-density DNA microarray chip and a high-density PNA microarray core.
  • the method of the present invention is characterized by: (a) designing and preparing a microseal based on the required compound microarray chip; (b) adding a corresponding chemical reactant to the microseal prepared in (a); ( c) According to the designed sequence, the micro-stamps containing the corresponding chemical reactants on the surface in (b) are embossed on the same substrate one by one through the positioning device, so as to form the desired micro-elements containing micro-units of different compounds on the substrate.
  • Array Column chip
  • the method according to the present invention is characterized in that: (a) the micro-seal is processed on a substrate such as a silicon wafer by a method such as photolithography and etching to form a template with a designed concave-convex pattern, and then the liquid polymer raw material is poured into the template. On the template, after the polymerization is cured, the cured polymer is peeled off from the template, or (a) the micro-seal is prepared by photolithography and etching on a substrate such as a silicon wafer to have the designed bump The patterned template is then pressed against the polymer surface under heating.
  • the polymer After cooling, the polymer is prepared by removing the polymer, or the micro-stamp in (a) uses a laser beam, a particle beam, or a microtool According to the designed concave-convex pattern, the polymer material is directly processed on the surface of the polymer material, and is prepared.
  • the material used to prepare the micro-seal in (a) is solid, such as a polymer material such as rubber, or a polymer material such as porous rubber containing microvoids.
  • a catalyst or a bioenzyme for promoting the connection of the compound on the microseal in step (c) to the substrate may be added to the microseal prepared in (a).
  • step (c) the chemical reaction positions imprinted on the same substrate multiple times may be overlapped or may not overlap each other.
  • step (c) in the imprinting process of step (c), by introducing sound, light, heat, electricity or (and) magnetic energy onto the substrate or stamp, the chemical reaction at the imprinting position can be accelerated. .
  • steps (b)-(c) are performed in a vacuum or a gas having no adverse effect on steps (b) and (c), wherein the gas is selected from nitrogen and argon.
  • a method for preparing a microseal is to use, for example, a photolithographic etching method to process a template with a designed concave-convex pattern on a substrate such as a silicon wafer, and then pour the liquid polymer on the template. After the polymer is cured, the cured polymer is peeled off from the template, and the convex and concave pattern on the original template is copied on the surface of the cured polymer to form the micro-seal of the present invention.
  • another method of preparing the microseal of the present invention is to use, for example, photolithography.
  • the etching method is processed on a silicon wafer substrate into a template with a designed concave-convex pattern, and then the template is pressed against the softened polymer surface under heating, and after cooling, the polymer is removed as described in the present invention.
  • Micro seal is a method of preparing the microseal of the present invention.
  • the third method for preparing a microseal is to use a laser beam, a particle beam (including an electron beam or an ion beam), or a micro-tool under computer control according to the designed four-convex pattern directly on the polymer seal material.
  • the surface is processed to make the micro-stamp according to the present invention.
  • the polymer material used for preparing the microseal is a chemical substance such as rubber, which may be a solid polymer or a porous polymer material containing minute pores.
  • a frame made of a material such as metal is used to fix the polymer microseal in order to determine the position of the micro-bumps on the surface of the microseal by the edge of the frame.
  • the micro-seal and the embossed substrate are fixed by a mechanical device, and the relative position of the micro-seal and the substrate is measured to control the embossing position of the micro-seal.
  • the convex portion of the surface of the stamp is accurately embossed in accordance with design requirements Corresponding position on the substrate surface.
  • the chemical reaction positions imprinted multiple times on the same substrate can be overlapped, that is, at the same position, a multi-step chemical reaction can be performed while the sequence of the multi-step chemical reactions can be controlled to avoid mutual or cross-chemical reactions. Reaction; It is also possible to perform a fixed-point chemical reaction without overlapping. After each imprinted fixed-point chemical reaction, the substrate can be cleaned and chemically treated to meet the requirements of subsequent imprinted fixed-point chemical reactions.
  • physical processes such as (super) sound field, light energy, thermal energy, electric field, magnetic field, photoacoustic surface wave, surface excitons (resonance) can be introduced through the substrate or micro-seal during the chemical reaction of the fixed-point imprint.
  • to accelerate the chemical reaction at the imprinting position or to add a catalyst or a biological enzyme to the chemical reactant solution coated or injected into the microstamp to accelerate the chemical reaction at the imprinting position by a chemical method.
  • the embossing fixed-point chemical reaction process, and even the entire preparation process of the microarray chip can be performed in a vacuum or an inert gas such as nitrogen, argon, and the like for embossing chemical reactions.
  • the substrates used to prepare compound microarray chips can be silicon, glass, ceramics, metals, polymers, and other inorganic or organic solid materials, as well as molecular films modified or assembled on the surface of these materials.
  • the surface can be dense, or Can be porous.
  • the present invention also relates to a compound microarray chip prepared by the method of the present invention, wherein the compound microarray chip prepared by the method of the present invention has high spatial resolution, for example, the spatial resolution of the DNA chip prepared by the method of the present invention is 30 X 30 ⁇ 2 and the high degree of integration of the array.
  • the number of DNA chip arrays prepared by the method of the present invention can reach 6.5536 X 10 4 / cm2, and the accuracy rate is high.
  • the accuracy rate of each step synthesis is more than 99.5%, 20-
  • the total accuracy of the mer oligonucleotide is above 90%.
  • FIG. 1 is a schematic diagram of preparing a micro seal using a template in the present invention.
  • FIG. 2 is a schematic view of a compound microarray prepared by the method of the present invention by using four micro-stamps coated with four kinds of chemical reactants A, B, C, and D respectively.
  • FIG. 3 is a schematic diagram of the present invention for preparing a compound microarray by using four micro-seals coated with four chemical reactants A, ⁇ , B, C, and D, respectively, by an overlay embossing method.
  • Figure 4 is the No. 1 stamp for preparing a full array of dinucleotides.
  • Figure 5 is the No. 2 seal for the preparation of a full array of dinucleotides.
  • Figure 6 shows the No. 3 seal for the preparation of a full array of dinucleotides.
  • Figure 7 shows the No. 4 stamp for the preparation of a full array of dinucleotides.
  • Figure 8 is the No. 5 seal for the preparation of a full array of dinucleotides.
  • Figure 9 is the No. 6 seal for the preparation of a full array of dinucleotides.
  • Fig. 10 is the No. 7 seal for preparing the full array of dinucleotides.
  • Figure 11 shows the No. 8 seal for the preparation of a full array of dinucleotides.
  • Figure 12 is a schematic diagram of a full array of dinucleotides on a substrate.
  • Figure 13 Figure 19 is from template preparation, micro-seal preparation to compound microarray preparation Schematic diagram of the chip flow.
  • Figure 13 is a micron-sized photoresist coated on the surface of a smooth substrate such as silicon or glass.
  • FIG. 14 shows exposure through a mask. After development, the designed concave-convex pattern is transferred to the photoresist 3.
  • Figure 15 shows the silicone rubber PDMS raw material poured onto a photoresist template and polymerized to form a PDMS polymer microseal 1.
  • FIG. 16 is a micro seal after the photoresist template is detached from the micro seal 1.
  • Figure 17 shows a coating of chemical reactant 4 on the surface of the microseal.
  • Fig. 18 shows a microseal coated with a chemical reactant 4 embossed on a substrate 6 with a chemical group 5 modified on its surface.
  • FIG. 19 shows the micro-seal 3 being detached after a fixed-point chemical reaction by imprinting, and a micro-array of chemical reaction products is connected to the imprinting position of the substrate.
  • FIG. 20 is a partial electron micrograph of the PDMS micro-seal of the prepared 65336 microarray, 1.28 x 1.28 cm 2 .
  • PDMS Silicon rubber
  • a PDMS stamp was coated with an anhydrous acetonitrile solution of its 5, -OH nucleotides such as dAdp and tetrazole (catalyst) protected with di-p-trimethoxymethyl (DMT). Then, the stamp is pressed on the substrate by a micro-embossing mechanism, and the imprinting position is measured by the micro-embossing mechanism and aligned with the required imprinting points on the substrate.
  • the 3, -OH of deoxytriphosphate is covalently coupled to the substrate.
  • a piezoelectric ultrasonic vibration source is introduced on the substrate.
  • the ultrasonic reaction accelerates the speed of the chemical reaction.
  • the above process is repeated at different positions to form a single nucleotide array.
  • the substrate is stripped of the protective agent DMT on the nucleotide 5, -OH on the substrate with a solution of phenylthiopan (or trichloroacetic acid) in acetonitrile. OH exposed.
  • the stripped DMT solution adjust it to a certain volume, and use DMT-C1 monomer as the standard solution to measure DMT and light absorption value (OD value) at 495nm. According to the ratio of adjacent secondary OD values, the layer can be obtained. Synthetic yield.
  • the second, third, ... layers of nucleotide molecules can be bonded. After synthesizing to twenty layers (that is, a 20-base oligonucleotide), the substrate was treated with 30% ammonia water, and the protective groups on the base and phosphoric acid were removed. Rinse the chip with water and store it in a dry package. At this point, the preparation of the group chip is complete.
  • each gene probe unit is 30 X 30 ⁇ 2, a total of 6.5536 X 10 4 different genes probes LCM second surface; measurement method according OD DMT, which is synthesized in each synthetic efficiency 99.5% The accuracy of the synthetic probe is more than 90%; the preparation time of each layer is about 20 minutes, and the synthesis time of the entire chip is about 7 hours.
  • PNA Peptide nucleic acid
  • oligomeric N-2 (2-aminoethyl glycine) with bases and it is a peptide analog with nucleotide properties.
  • PNA can hybridize with complementary DNA, RA, and PNA, and its hybridization has high thermal stability and high sensitivity to mismatches. Under certain conditions, a single base mismatch can be identified. So use The preparation of high-density gene array chips from PNA sequences can greatly improve the accuracy and sensitivity of gene chip hybridization, and has very important application prospects.
  • the high-density PNA microarray chip is prepared as follows:
  • a piezoelectric ultrasonic vibration source is introduced on the substrate, and the speed of the chemical reaction is accelerated by the action of ultrasonic waves. Then use different seals to coat PNA monomers with different bases, such as Gly-T, Gly-G, Gly-C and pentafluorophenyl ester mixed solution, and repeat the above process to form a single-layer single-base PNA array.
  • the ninhydrin method is used to determine the coupling ratio of the first layer. Repeating the above embossing process, the second, third ... layer of PNA monomer molecules can be bonded.
  • the substrate was treated with a 30% NaOH aqueous solution to remove the protective groups on the bases and linolenic acid. Rinse the chip with water and store it in a dry package. At this point, the preparation of the PNA gene chip is complete.
  • each gene probe unit of the chip is 30 X 30 ⁇ 2 and there are 6.5536 X 10 4 different gene probes on the surface of 1 cm 2. According to the ninhydrin method, the synthesis efficiency of each layer of the chip is above 99.9%. 98% accuracy for synthetic probes the above.
  • the preparation time of each layer is about 1 hour, and the entire chip synthesis time is about 24 hours.

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Description

制备化合物微阵列芯片的新方法
及由该方法制备的化合物微阵列芯片 发明领域
本发明涉及制备化合物微阵列芯片的新方法， 尤其是采用多 次压印定点合成法制备化合物微阵列芯片的方法， 及由该方法 制备的化合物微阵列芯片。
化合物微阵列芯片是指在固体基片表面上制备一组由不同 分子微单元组成的化合物阵列。化合物主要指生物大分子物质包 括核酸如 DNA、 RNA、寡核苷酸等， 蛋白质如酶、抗体、抗原、 多肽等， 以及其它人工合成的生物活性物质如 PNA (肽核酸 等） 。 当化合物具有核酸分子、 性质时， 化合物微阵列芯片又称 之为基因芯片。
发明背景
化合物微阵列芯片在生物检测、 医学检测、 药物筛选、 基因 序列分析和化合物库的合成上有着极其重要的意义。例如在生物 学中， 随着分子生物学的不断发展， 特別是举世瞩目的人类基因 组计划实施以来， 有关核酸、 蛋白质序列和结构的数据呈指数增 长。 而下世纪最富挑战性的工作就是人类基因组计划完成后， 即 在后基因时代， 我们如何对大量的分子信息进行分析， 找出其中 规律， 使生物学由实验上升到理论， 从而更好地认识生命现象, 并使医学治疗产生根本革命。 在医学中， "系统、 器官、 组织、 细胞层次上的第二阶段医学" 正在向 "基因水平上的， DNA~ RNA~蛋白质与蛋白质核酸相互作用， 以及它们与环境 相互作用水平上的第三阶段医学" 转化。 这种在分子层次上进行 的基因诊断与基因治疗， 将根本地认识疾病产生的根源， 并将有 希望根本认识和治疗包括癌症在内的重大疾病。 这些生物学、 医 学的根本变革， 一个根本的前提是基因序列的测定和分析。 能否 有效快速地进行基因测序与分析， 将影响到人类基因组计划的 实施， 从而影响生物学、 医学的进一步发展。 传统基因测序所采 用的方法包括化学反应、 凝胶电泳法等一系列繁杂的步骤， 这些 方法花费时间较长， 且需要操作繁复， 尤其在大规模测序方面费 时、 并且不适宜便携化快速测序。 在对传统基因测序方法进行改 进的过程中， 以基因芯片为代表的生物芯片技术应运而生。 生物 芯片技术是将生命科学研究中所涉及的许多不连续的分析过程， 如样品制备， 化学反应和分析检测等通过采用微电子， 微机械等 工艺集成到芯片中， 使之连续化， 集成化和微型化。 这一技术的 成熟和应用将在下个世纪的疾病诊断和治疗、 新药开发、 司法鉴 定、 食品和环境等生命科学相关领域带来一场革命， 为生物信息 的获取及分析提供强有力的手段。
化合物微阵列芯片对于生命科学研究是非常重要和必要 的。 生物物质的序列是通过芯片上的已知化合物分子阵列与被测 定的生物分子之间的相互作用进行检测或测序。 以核酸检测为 例， 其包括首先在固相载体上制备寡核苷酸分子探针阵列， 即化 合物微阵列， 然后使待测基因与寡核苷酸分子探针阵列进行杂 交， 通过计算机对杂交结果进行分析而获得待测基因序列的信 息。 而在基因芯片的制备中， 其关键点在于寡核苷酸分子探针阵 列的制备。
人们希望芯片的探针阵列空间分辨率高， 并且合成工作量小， 速度快， 方法简单， 成本低。 目前， 有两种制备寡核苷酸探针阵 列的方法。 一种是利用常规固相合成技术分别合成好需要的单个 探针分子， 然后利用喷打或印刷技术将不同的探针分子结合在 基片上的不同位置， 从而形成探针阵列。 利用喷打或印制方法制 备探针阵列很难达到较高的空间分辨率， 并且在探针分子制备 时为逐个合成， 合成工作量大。 而另一种方法则是美国
Affymetrix公司提出的利用模板定域光化学反应， 在基片上合成 探针阵列。 利用这种方法制备探针阵列可达到较高的空间分辨率 (40 X 40μιη2), 而且在片合成时为并行合成， 合成速度快。 但由 于光化学反应产率较低， 反应中副反应较多， 使得合成探针序列 正确率不高， 而且需要具有特殊保护基团的试剂， 成本较高， 因 此， 在化合物微阵列芯片的制备中仍需要有更好的方法。
发明目的
本发明的目的就是提供一种制备方法筒单、 可靠、 空间分辨 率高、 正确率高的化合物微阵列芯片的制备方法， 即采用多次压 印定点合成法制备微阵列芯片。
发明简述
本发明人经长期广泛深入的研究， 现已发现一种制备化合物 微阵列芯片的新方法， 该方法包括：
首先根据所需化合物的微阵列设计制备有凹凸的微印章， 然 后根据预先的设计在制备的各微印章上涂上对应的化学反应物， 最后按照设计的顺序和位置将涂有化学反应物的微印章逐个依 次压印在同一个基片上， 得到化合物微阵列芯片。 本发明基于以 上得以完成。 在上述方法中， 用印章的凸凹控制基片上化学反应 的位置及基片上的化学基团反应， 从而使化学反应物共价偶联 在基片上， 最后在基片上形成所需的化令.物微阵列芯片。
发明详细描述
本发明第一方面涉及的是制备化合物微阵列芯片的新方 法， 尤其是采用多次压印定点合成法制备化合物微阵列芯片的方 法。
本发明再一方面涉及的是由本发明的方法制得的化合物微 阵列芯片 ,尤其是高密度 DNA微阵列芯片和高密度 PNA微阵列 心
根据本发明， 本发明方法的特征在于： （a)根据所需的化合物 微阵列芯片设计和制备微印章； （b) 在 (a)中制备的微印章上加入 含有对应的化学反应物；（c) 按照设计的顺序， 通过定位装置， 将 (b)中表面含有对应的化学反应物的微印章逐个压印在同一个基 片上， 从而在基片上形成所需的含有不同化合物微单元的微阵 列芯片。
根据本发明的方法， 其特征在于： （a)中微印章是用光刻、 腐 蚀等方法在硅片等基片上加工成具有所设计的凹凸图案的模板， 然后将液状聚合物原料倾注在该模板上， 待聚合固化后， 将固化 的聚合物从模板上揭下而制备的，或 (a)中微印章制备是用光刻腐 蚀等方法在硅片等基底上加工成具有所设计的凹凸图案的模板， 然后在加热的状态下将该模板压在聚合物表面， 待冷却后， 取下 聚合物而制备得到的， 或 (a)中微印章是利用激光束， 粒子束， 或 微刀具， 按照所设计的凹凸图案， 直接在聚合物材料表面进行加 工， 制备得到的。
根据本发明方法， 其中用于制备 (a)中微印章的材料是实心 的，如橡胶等聚合物材料， 或是含有微小孔洞的多孔橡胶等聚合 物材料。
根据本发明方法， 其中在步骤 (b)中， 还可往 (a)中制备的微 印章中加入用于促进步骤 (c)中微印章上化合物连接到基片上的 催化剂或生物酶。
根据本发明方法， 其中在步骤 (c)中， 同一个基片上多次压印 的化学反应位置， 可以是重叠的， 也可以彼此不重叠。
根据本发明方法， 其中在步骤 (c)的压印过程中， 通过往基片 或徵印章上引入声、 光、 热、 电或 (和)磁等能量， 可加速压印位 置上的化学反应。
根据本发明方法， 其中步驟 (b)-(c)是在真空或对步骤 (b)和 (c) 无不良作用的气体中进行的， 其中所述气体选自氮气， 氩气。
根据本发明， 更具体讲， 微印章的制备方法是用如光刻腐蚀 方法， 在如硅片基底上加工成具有所设计的凹凸图案的模板， 然 后将液状聚合物倾注在该模板上， 待该聚合物固化后， 将固化的 聚合物从模板上揭下， 该固化的聚合物表面就复制了原先模板 上的凸凹图案， 即制成本发明所述的微印章。
根据本发明， 本发明微印章的另一种制备的方法是用如光刻 腐蚀方法在如硅片基底上加工成具有所设计的凹凸图案的模板， 然后在加热的状态下将该模板压在软化的聚合物表面， 待冷却 后， 取下聚合物即成本发明所述的微印章。
根据本发明， 微印章的第三种制备方法是利用激光束、 粒子 束（包括电子束或离子束)， 或微刀具， 在计算机控制下， 按照设 计的四凸图案， 直接在聚合物印章材料表面进行加工， 制成本发 明所述的微印章。
根据本发明， 制备微印章所用的聚合物材料是如橡胶等化学 物质， 可以是实心的聚合物， 也可以是含有微小孔洞的多孔聚合 物材料。
根据本发明， 在微印章制备过程中， 用一个如金属等材料制 成的框架， 固定聚合物微印章， 以便通过框架的边缘来确定微印 章表面上微凹凸点的位置。 通过机械装置固定微印章和被压印的 基片， 通过测量微印章和基片的相对位置， 控制微印章的压印位 置. 从而使该印章表面凸起部分按照设计要求， 精确地压印在基 片表面的对应位置。
根据本发明， 在同一个基片上多次压印的化学反应位置， 可 以是重叠的， 即在同一位置， 进行多步化学反应同时还可控制多 步化学反应的顺序， 避免产生相互或交叉化学反应； 也可以不重 叠， 进行定点化学反应。 在每次压印定点化学反应后， 可对基片 进行相应的清洗和化学处理， 使之满足后续压印定点化学反应 的要求。
根据本发明， 在压印定点化学反应过程中， 可以通过基片或 微印章引入 (超)声场， 光能， 热能， 电场， 磁场， 光致声表面波， 表面激元 (共振)等物理作用能， 加速压印位置上的化学反应， 也 可以在涂覆或注入微印章的化学反应物溶液中加入催化剂或生 物酶， 通过化学的方法加速压印位置上的化学反应。 压印定点化 学反应过程， 以至整个微阵列芯片的制备过程可在真空或对压 印类的化学反应惰性的气体如氮气、 氩气等环境中进行。 制备化合物微阵列芯片需用的基片可以是硅， 玻璃， 陶瓷， 金属， 聚合物等无机或有机固体材料， 以及在这些材料表面上修 饰或组装的分子膜， 其表面可以是致密的， 也可以是多孔的。
根据本发明， 本发明还涉及由本发明方法制备的化合物微阵 列芯片， 其中按本发明方法制备的化合物微阵列芯片具有高的 空间分辨率， 如用本发明方法制备 DNA芯片的空间分辨率为 30 X 30μπι2，及阵列的集成度高， 如用本发明方法制备的 DNA芯片 的阵列数目可达 6.5536 X 104个 /平方厘米， 正确率高， 每步合成 正确率在 99.5%以上， 20-mer 寡核苷酸的总正确率在 90%以 上。 随着微印章及相应的压印机械装置精度的提高， 上述指标还 可以大幅度提高。
附图说明：
图 1是本发明中利用模板制备微印章示意图。
图 2是本发明利用分别对应涂有 A、 B、 C . D四种化学 反应物的四个微印章， 用多次压印方法制备的化合物微阵列示 意图。
图 3是本发明利用分别对应涂有 A、―. B、 C、 D四种化学 反应物的四个微印章， 用重叠压印的方法制备化合物微阵列示 意图。
图 4是制备二核苷酸全阵列的 1号印章。
图 5是制备二核苷酸全阵列的 2号印章。
图 6是制备二核苷酸全阵列的 3号印章。
图 7是制备二核苷酸全阵列的 4号印章。
图 8是制备二核苷酸全阵列的 5号印章。
图 9是制备二核苷酸全阵列的 6号印章。
图 10是制备二核苷酸全阵列的 7号印章。
图 11是制备二核苷酸全阵列的 8号印章。
图 12是基片上二核苷酸全阵列的示意图。
图 13 图 19是从模板制备、 微印章制备至制备化合物微阵 列芯片的流程示意图。
图 13为在硅或玻璃等平滑基底表面涂覆微米级厚的光刻胶
3。
图 14为通过掩模曝光， 显影后， 把所设计的凹凸图形转移在 光刻胶 3上。
图 15为将硅橡胶 PDMS原料倾注在光刻胶模板上， 聚合固 化成 PDMS聚合物微印章 1 。
图 16为将光刻胶模板从微印章 1上脱开后的微印章。
图 17为在微印章表面涂覆一层化学反应物 4。
图 18为将涂覆有化学反应物 4的微印章压印在表面修饰有 化学基团 5的基片 6上。
图 19为经压印定点化学反应后， 将微印章 3脱开， 在基片的 压印位置上连接有化学反应产物的微阵列。
图 20是制得的 65336微阵列的 PDMS微印章 1.28 χ 1.28cm2 的电子显微镜局部照片。
下面的实施则是对本发明的进一步说明， 但其不意味着对本 发明范围的任何限制。 一,
实施例 1， 高密度 DNA微阵列芯片的制备。
A. 微印章的制备。 在干净的硅片上利用匀胶机涂上一层约 ΙΟμπι 厚的光刻胶置于所设计的光刻掩模板下， 进行曝光， 从而 形成具有表面凹凸的模板。 然后将一小盒放在模板上， 模板与小 盒边缘的位置进行校准， 其误差小于 1μπι。 小盒的下底是空的。 当该小盒放在模板上时， 模板位于小盒内的底部， 在小盒的上部 设有注入孔， 从该孔中注入硅橡胶 (PDMS)原料， 则 PDMS 以模 板为底充满小盒。 待 PDMS 固化后， 将小盒连同里面的 PDMS 从模板上小心地取下， 则 PDMS复制了原模板上的凹凸图案， 此 时微印章即制成。
B. 基片上制备寡核苷酸探针阵列。 将玻片清洗， 干燥后， 分 别放入 APTS (氨基丙基三乙氧基硅烷)的苯溶液中反应 2 小时， 8
在苯中漂洗后再放入琥珀酸的苯溶液中反应 1小时， 从而在玻璃 片表面形成羟基。 整个基因芯片的压印过程中均在氩气的保护之 下进行的。 在 PDMS印章上涂布其 5，-OH已用二对甲氧三苯甲 基 (DMT)保护的核苷酸例如 dAdp和四唑 (催化剂）的无水乙晴溶 液。 然后用微压印机械装置将印章压在基片上， 压印位置由微压 印机械装置测定， 并与基片上所要求的压印点对准。 通过四唑的 作用，脱氧三磷^ ^苷的 3，-OH共价偶联在基片上。 在基片上引 入压电超声振动源。 通过超声波作用， 加速该化学反应的速度。 再利用不同的印章和不同的单核苷酸 (如 dGdp， dCdp, dTdp), 在不同位置上重复上述过程， 形成单核苷酸阵列。 当第一层单核 苷酸已制好后， 将基片用苯硫盼 (或三氯乙酸)的乙腈溶液脱去基 片上核苷酸 5，-OH上的保护剂 DMT， 将 5，-OH暴露。 收集脱去 的 DMT 液， 调节至一定的体积， 以 DMT-C1 单体为标准液于 495nm处检测 DMT和光吸收值 (OD值)， 根据相邻二次 OD值的 比值， 可以获得该层的合成产率。 重复上述压印过程， 可键合上 第二、 三、 ……层核苷酸分子。 合成至二十层 (即 20个碱基长度 的寡核苷酸)后， 用 30%氨水处理基片， >¾去除碱基及磷酸上的 保护基团。 将芯片用水冲净， 干燥封装保存。 至此， 基团芯片制 备完毕。该芯片每个基因探针单元的大小为 30 X 30μιη2, 在 lcm2 表面共有 6.5536 X 104个不同的基因探针； 根据 DMT的 OD测 量方法， 该合成每层的合成效率在 99.5%以上， 合成探针的正确 率为 90%以上； 每一层的制备时间约为 20分钟， 完成整个芯片 的合成时间约为 7小时左右。
实施例 2 , 高密度 PNA微阵列芯片的制备
肽核酸（ peptide nucleic acid, PNA ) 是一种带有碱基的寡 聚 N-2氨基乙基甘氨酸（ N-(2-aminoethyl glycine) ) , 是具有核 苷酸性质的多肽类似物。 ΡΝΑ可以与序列互补的 DNA、 R A 以及 PNA杂交， 并且其杂交具有高的热稳定性和对错配的高灵 敏性。 在一定的条件下， 可以识别单个碱基的错配。 因此， 利用 PNA序列制备高密度基因阵列芯片， 可以大大提高基因芯片的 杂交准确性和灵敏度， 具有十分重要的应用前景。
高密度 PNA微阵列芯片的制备过程为：
A.微印章的制备（同实施例 1中 A )
B.制备四种分别含有胸腺嘧啶， 胸嘧啶， 腺嘌呤和鸟嘌呤四 种碱基可用于 PNA序列合成的单体， 即 N-2-叔丁氧羰基氨基乙 基 胸腺嘧啶 -1-乙酰甘氨酸 (Gly-T), N-2-叔丁氧談基乙基 -NO 胞嘧啶 -1-乙酰甘氨酸 (Gly-C), N-2-叔丁氧羰基乙基 腺嘌呤- 1-乙酰甘氨酸 (Gly-A)和 N-2-叔丁氧羰基乙基 -N-鸟嘌呤 -1-乙酰 甘氨酸 (Gly-G)„
C.基片上制备 PNA探针阵列。 将玻片清洗， 干燥后， 分别 放入 APTES 笨溶液中反应 2 小时， 从而在玻璃片表面形成氨 基。 整个基因芯片的压印过程均在氮气保护下进行的。 在 PDMS 印章上涂布含有碱基的 PNA合成单体， 例如 Gly-A , 以及五氟 苯酯， 然后， 用微压印机械装置将印章压在基片上， 压印位置由 微压印机械装置测定， 并与基片上所要求的压印点对准。 通过五 氟苯酯激活基片上的化学基团， 使 Gly-A的 C端化学键合在基 片上。 在基片上引入压电超声振动源， 通过超声波作用， 加速该 化学反应的速度。 再利用不同的印章， 分别涂布上含不同碱基的 PNA单体如 Gly-T, Gly-G, Gly-C和五氟苯酯混合溶液， 重复上 述过程， 形成单层单碱基 PNA阵列， 当第一层合成完毕后， 用 水合茚三酮法测定第一层的偶合率。 重复上述压印过程， 可键合 上第二、 三…层 PNA单体分子。 合成至二十层（即二十个碱基 长度的寡聚准肽链） 后， 用 30%NaOH 水溶液处理基片， 以去 除碱基及鱗酸上的保护基团。 将芯片用水沖净， 干燥封装保存。 至此， PNA基因芯片制备完毕。
该芯片每个基因探针单元大小为 30 X 30μπι2 ,在 1cm2表面 具有 6.5536 X 104个不同基因探针， 根据水合茚三酮法测量， 该 芯片每层的合成效率在 99.9%以上， 合成探针的正确率为 98 % 以上。 每一层的制备时间约为 1小时， 完成整个芯片合成时间约 为 24小时左右。

Claims

权利要求
1. 一种制备化合物微阵列芯片的方法， 其特征在于： （a)根据 所需的化合物微阵列芯片设计和制备微印章； （b) 在 (a)中制备的 微印章上加入含有对应的化学反应物； （c) 按照设计的顺序， 通 过定位装置， 将 (b)中表面含有对应的化学反应物的微印章逐个 压印在同一个基片上， 从而在基片上形成所需的含有不同化合 物微单元的微阵列芯片。
2. 根据权利要求 1所述的方法， 其特征在于： （a)中微印章是 用光刻、 腐蚀等方法在硅片等基片上加工成具有所设计的凹凸图 案的模板， 然后将液状聚合物原料倾注在该模板上， 待聚合固化 后， 将固化的聚合物从模板上揭下而制备的。
3. 根据权利要求 1所述的方法， 其特征在于： （a)中微印章制 备是用光刻腐蚀等方法在硅片等基底上加工成具有所设计的凹 凸图案的模板， 然后在加热的状态下将该模板压在聚合物表面， 待冷却后， 取下聚合物而制备得到的。
4. 根据权利要求 1所述的方法， 其特征在于： （a)中微印章是 利用激光束， 粒子束， 或微刀具， 按照所设计的凹凸图案， 直接 在聚合物材料表面进行加工， 制备得到的。
5. 根据权利要求 1或 2或 3或 4所述的方法， 其特征在于： 用 于制备 (a)中微印章的材料是实心的， 如橡胶等聚合物材料。
6. 根据权利要求 1或 2或 3或 4所述的方法， 其特征在于： 用 于制备 (a)中微印章的材料是含有微小孔洞的多孔橡胶等聚合物 材料。
7. 根据权利要求 1-6中任一方法， 其中在权利要求 1方法 (b) 中， 还可往 (a)中制备的微印章中加入用于促进权利要求 1方法 (c) 中微印章上化合物连接到基片上的催化剂或生物酶。
8. 根据权利要求 1-7 任一所述的方法， 其特征在于： 在权利 要求 1(c)中， 同一个基片上多次压印的化学反应位置， 可以是重 叠的， 也可以彼此不重叠。
9. 根据权利要求 1或 2或 3或 4所述的方法， 其中在权利要 求 1(c)的压印过程中， 通过往基片或微印章上引入声、 光、 热、 电或 (和)磁等能量， 可加速压印位置上的化学反应。
10. 根据权利要求前述任一方法， 其中在权利要求 1步骤 (b)- (c)是在真空或对步骤 (b)和 (c)无不良作用的气体中进行的。
11. 根据权利要求 10 的方法， 其中所述气体选自氮气， 氩 气。
12. 根据前述任一权利要求的方法得到的化合物微阵列芯 片。
13. 权利要求 12的化合物微阵列芯片是高密度 DNA微阵列 心
14. 权利要求 12的化合物微阵列芯片是高密度 ΡΝΑ微阵列 心 1
PCT/CN1999/000013 1998-04-03 1999-01-29 Nouveau procede de preparation de puces micromatricielles composees et puces micromatricielles composees ainsi obtenues WO1999051770A1 (fr)

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