技术领域technical field
本发明属于生物芯片技术领域，具体涉及一种小分子微阵列及其制备方法。The invention belongs to the technical field of biochips, and in particular relates to a small molecule microarray and a preparation method thereof.
背景技术Background technique
小分子微阵列是指以规则的阵列形式在固体基片上固定小分子形成的高密度的微阵列，具有高通量、大规模、并行性、样品消耗少等突出优点，为研究小分子的相互作用、生物催化过程、蛋白组学以及药物发现等提供了强大的工具。Small molecule microarray refers to a high-density microarray formed by immobilizing small molecules on a solid substrate in the form of a regular array. It has outstanding advantages such as high throughput, large scale, parallelism, and less sample consumption. It provides powerful tools for biological processes, biocatalytic processes, proteomics, and drug discovery.
小分子微阵列将大量的具有不同结构和不同基团的小分子固定在修饰后的芯片基片上，要求将小分子高效率地固定在基片上并且不破坏小分子与靶蛋白结合的特性。目前，选择性共价固定方法和选择性非共价固定方法是制备小分子微阵列最常见的方法，如可以通过环氧化物与酰肼基团的共价反应选择性地固定含有酰肼基团的小分子，或通过抗生物素蛋白链菌素与生物素间的强非共价相互作用选择性地固定含有生物素的小分子。选择性固定方法已经成功地将带有氨基、羟基、巯基、羧基等基团的化合物固定在载体表面，对于小分子阵列的研究起到了重要的作用。但是，选择性固定方法存在的问题是，小分子化合物必须具有某种特定的基团才能连接到基片表面上。固相合成的小分子在合成过程中可以连接上某种基团，但是天然产物、或医药公司等机构拥有的小分子库中的小分子有各种不同的化学结构和基团，因此不能采用选择性固定方法将上述小分子固定，非选择性的固定方法能从一定程度上克服这些缺点。Small molecule microarray immobilizes a large number of small molecules with different structures and different groups on the modified chip substrate, which requires efficient immobilization of small molecules on the substrate without destroying the binding properties of small molecules and target proteins. At present, selective covalent immobilization method and selective non-covalent immobilization method are the most common methods for preparing small molecule microarrays. small molecules, or selectively immobilize small molecules containing biotin through strong non-covalent interactions between streptavidin and biotin. Selective immobilization methods have successfully immobilized compounds with amino, hydroxyl, sulfhydryl, carboxyl and other groups on the surface of carriers, which played an important role in the research of small molecule arrays. However, the problem with the selective immobilization method is that the small molecular compound must have a specific group to be attached to the surface of the substrate. Small molecules synthesized in solid phase can be connected with certain groups during the synthesis process, but small molecules in natural products or small molecule libraries owned by institutions such as pharmaceutical companies have various chemical structures and groups, so they cannot be used Selective immobilization methods fix the above-mentioned small molecules, and non-selective immobilization methods can overcome these shortcomings to a certain extent.
非选择性的固定方法之一为光交联表面化学（Kanoh, N., et al. Immobilization of naturalproducts on glass slides by using a photoaffinity reaction and the detection of protein-small-molecule interactions. Angew.Chem. Int. Ed. Engl. 42, 5584–5587,2003），采用高反应活性的卡宾中间体来固定各类小分子。但是这种非选择性固定方法存在较多的假阳性。另一种非选择性固定方法是采用异氰酸酯基团来固定任意具有亲核基团的小分子（Bradner, J. E.et al., A robust small-molecule microarray platform for screening cell lysates, Chem Biol 13, 493–504, 2006），但是该异氰酸酯表面对羧基、仲醇羟基等基团的固定效率非常低，仅为氨基固定效率的10%。研究高效率地、能够固定多种小分子的微阵列制备方法是小分子微阵列广泛应用的前提与基础。One of the non-selective immobilization methods is photocrosslinking surface chemistry (Kanoh, N., et al. Immobilization of natural products on glass slides by using a photoaffinity reaction and the detection of protein-small-molecule interactions. Angew.Chem. Int . Ed. Engl. 42, 5584–5587, 2003), using highly reactive carbene intermediates to immobilize various small molecules. However, there are many false positives in this non-selective fixation method. Another non-selective immobilization method uses isocyanate groups to immobilize any small molecule with nucleophilic groups (Bradner, J. E. et al., A robust small-molecule microarray platform for screening cell lysates, Chem Biol 13 , 493–504, 2006), but the immobilization efficiency of the isocyanate surface to carboxyl groups, secondary alcohol hydroxyl groups and other groups is very low, only 10% of the immobilization efficiency of amino groups. Research on efficient microarray preparation methods capable of immobilizing various small molecules is the premise and basis for the wide application of small molecule microarrays.
发明内容Contents of the invention
本发明的目的在于提供一种普适的、高效的、将小分子固定在芯片基片上的小分子微阵列及其制备方法。与Bradner等人的制备方法相比，本发明提供的制备方法对羧基、仲醇羟基等与异氰酸酯反应活性较弱的基团的固定效率提高了2~3倍。The purpose of the present invention is to provide a universal and efficient small molecule microarray for immobilizing small molecules on a chip substrate and a preparation method thereof. Compared with the preparation method of Bradner et al., the preparation method provided by the present invention improves the immobilization efficiency of carboxyl groups, secondary alcohol hydroxyl groups and other groups with weaker reactivity with isocyanate by 2 to 3 times.
本发明提供的小分子微阵列的制备方法，具体步骤为：The preparation method of the small molecule microarray provided by the invention, the specific steps are:
（1）将芯片基片表面修饰上亲水骨架聚合物；(1) Modify the surface of the chip substrate with a hydrophilic skeleton polymer;
（2）将亲水骨架聚合物终端修饰为异氰酸酯基团；(2) Modify the end of the hydrophilic backbone polymer with isocyanate groups;
（3）将小分子溶液点印到所处理的芯片上，芯片基片干燥后进行后处理，得到小分子微阵列。(3) Spot-print the small molecule solution onto the processed chip, and post-process the chip substrate after drying to obtain a small molecule microarray.
本发明中，所述的异氰酸酯基团是多官能异氰酸酯化合物，一部分官能团用于将多官能异氰酸酯共价连接于亲水骨架聚合物上，剩余官能团用于提供自由的异氰酸酯基团。In the present invention, the isocyanate group is a polyfunctional isocyanate compound, a part of the functional groups are used to covalently link the polyfunctional isocyanate to the hydrophilic backbone polymer, and the remaining functional groups are used to provide free isocyanate groups.
本发明中，所述的多官能异氰酸酯化合物是芳香族聚异氰酸酯化合物。相比脂肪族异氰酸酯化合物，芳香族异氰酸酯化合物反应活性更强，更易于羟基、羧基等反应。因此，采用芳香族异氰酸酯化合物修饰的芯片基片，能够与具有亲核基团（氨基、羟基、巯基、羧基等）的小分子更高效地共价结合，从而提高固定效率。In the present invention, the polyfunctional isocyanate compound is an aromatic polyisocyanate compound. Compared with aliphatic isocyanate compounds, aromatic isocyanate compounds are more reactive and easier to react with hydroxyl and carboxyl groups. Therefore, the chip substrate modified with aromatic isocyanate compounds can be more efficiently covalently bonded to small molecules with nucleophilic groups (amino, hydroxyl, mercapto, carboxyl, etc.), thereby improving the immobilization efficiency.
本发明中，所述的芳香族聚异氰酸酯化合物是对苯二异氰酸酯、甲苯二异氰酸酯、二苯甲烷二异氰酸酯、萘二异氰酸酯、苯二亚甲基二异氰酸酯以及它们的混合物。在芳香族聚异氰酸酯中优选对苯二异氰酸酯。In the present invention, the aromatic polyisocyanate compound is p-phenylene diisocyanate, toluene diisocyanate, diphenylmethane diisocyanate, naphthalene diisocyanate, xylylene diisocyanate and mixtures thereof. Among the aromatic polyisocyanates, p-phenylene diisocyanate is preferred.
本发明中，所述的亲水骨架聚合物为芴甲氧羰基保护的多元醇。亲水骨架化合物具有两个功能，其一是使固定的小分子距离固体芯片基片表面一定距离，降低基片表面阻挡小分子结位点的可能性。其二是提供亲水表面，有效抑制非特异性吸附。In the present invention, the hydrophilic skeleton polymer is polyhydric alcohol protected by fluorenyl moxycarbonyl group. The hydrophilic skeleton compound has two functions. One is to keep the immobilized small molecules at a certain distance from the surface of the solid chip substrate, reducing the possibility that the surface of the substrate blocks the junction sites of small molecules. The second is to provide a hydrophilic surface to effectively inhibit non-specific adsorption.
本发明中，所述的芴甲氧羰基保护的多元醇是聚乙二醇（PEG）、聚丙二醇（PPG）或其共聚物。优选聚乙二醇。In the present invention, the polyhydric alcohol protected by the fluorenyl moxycarbonyl group is polyethylene glycol (PEG), polypropylene glycol (PPG) or a copolymer thereof. Polyethylene glycol is preferred.
本发明中，所述的多元醇的链长n为1 ~ 40。In the present invention, the chain length n of described polyhydric alcohol is 1～40.
本发明中，所述的后处理为加热后处理。加热处理能够加快异氰酸酯基团与亲核基团的反应速率，与采用在吡啶蒸汽中催化处理的后处理方法相比，可以获得更高的固定效率，并有效解决了吡啶蒸汽催化处理造成的小分子点样点弥散的问题。In the present invention, the post-treatment is heating post-treatment. Heat treatment can speed up the reaction rate of isocyanate groups and nucleophilic groups. Compared with the post-treatment method of catalytic treatment in pyridine steam, it can obtain higher immobilization efficiency and effectively solve the small problem caused by pyridine steam catalytic treatment. The problem of dispersion of molecular spots.
本发明中，所述的加热后处理是40℃ ~ 50 ℃在空气中加热处理或40℃ ~ 50 ℃在氮气中加热处理。In the present invention, the post-heating treatment is heating in air at 40°C to 50°C or heating in nitrogen at 40°C to 50°C.
本发明中，所述的芯片基片为玻璃、硅片、塑料、石英、凝胶或尼龙膜。基片表面一般为氨基基团，可以通过与羧基的反应来共价连接亲水骨架聚合物。In the present invention, the chip substrate is glass, silicon chip, plastic, quartz, gel or nylon film. There are generally amino groups on the surface of the substrate, which can be covalently linked to the hydrophilic backbone polymer by reacting with carboxyl groups.
作为典型例子，本发明制备方法的具体操作过程如下：As a typical example, the specific operation process of the preparation method of the present invention is as follows:
一、制备亲水骨架聚合物溶液，溶剂为二甲基甲酰胺，骨架聚合物浓度为100uM ~ 10mM；六氟磷酸苯并三唑-1-基-氧基三吡咯烷基浓度为0.2mM ~ 20mM； N，N-二异丙基乙胺浓度为2mM ~ 200mM；将芯片基片浸泡在所制备的溶液中，室温搅拌反应5 ~ 20个小时，在芯片基片表面修饰带芴甲氧羰基保护的骨架聚合物；1. Prepare a hydrophilic backbone polymer solution, the solvent is dimethylformamide, the concentration of the backbone polymer is 100uM ~ 10mM; the concentration of benzotriazol-1-yl-oxytripyrrolidinyl hexafluorophosphate is 0.2mM ~ 20mM; The concentration of N, N-diisopropylethylamine is 2mM ~ 200mM; soak the chip substrate in the prepared solution, stir and react at room temperature for 5 ~ 20 hours, and modify the surface of the chip substrate with fluorenyl moxycarbonyl Protected backbone polymer;
二、制备去保护溶液，溶剂为二甲基甲酰胺，反应试剂为哌啶，哌啶与二甲基甲酰胺的体积比为0.001 ~ 0.1；将步骤一处理的芯片基片浸泡在哌啶溶液中，室温搅拌反应5 ~ 24个小时，使芯片基片表面去保护为自由氨基基团；2. Prepare a deprotection solution, the solvent is dimethylformamide, the reaction reagent is piperidine, and the volume ratio of piperidine to dimethylformamide is 0.001 ~ 0.1; soak the chip substrate processed in step 1 in the piperidine solution , stirring and reacting at room temperature for 5 to 24 hours, so that the surface of the chip substrate is deprotected as a free amino group;
三、制备芳香族聚异氰酸酯化合物溶液，溶剂为二甲基甲酰胺，芳香族聚异氰酸酯化合物浓度为6mM ~ 600mM；将步骤二处理的芯片基片浸泡在芳香族聚异氰酸酯化合物溶液中，室温搅拌反应0.1 ~ 10个小时，使芯片基片表面修饰为异氰酸酯基团；3. Prepare an aromatic polyisocyanate compound solution, the solvent is dimethylformamide, and the concentration of the aromatic polyisocyanate compound is 6mM ~ 600mM; soak the chip substrate treated in step 2 in the aromatic polyisocyanate compound solution, and stir at room temperature for reaction 0.1 to 10 hours to modify the surface of the chip substrate with isocyanate groups;
四、制备小分子溶液，溶剂为二甲基亚砜，小分子溶液浓度为10μM ~ 100mM；将小分子溶液采用接触式点样机点印在步骤三制备的芯片基片上；干燥芯片基片后，将芯片基片进行后处理，后处理时间为5 ~ 40个小时，得到小分子微阵列。4. Prepare a small molecule solution, the solvent is dimethyl sulfoxide, and the concentration of the small molecule solution is 10 μM ~ 100mM; use a contact spotting machine to print the small molecule solution on the chip substrate prepared in step 3; after drying the chip substrate, The chip substrate is post-processed, and the post-processing time is 5 to 40 hours to obtain a small molecule microarray.
本发明还提供一种小分子微阵列，包括：The present invention also provides a small molecule microarray, comprising:
（1）芯片基片作为固体支持物；(1) The chip substrate is used as a solid support;
（2）芯片基片表面连接一层亲水骨架聚合物；(2) A layer of hydrophilic skeleton polymer is connected to the surface of the chip substrate;
（3）亲水骨架聚合物表面连接一层多官能异氰酸酯化合物，所述多官能异氰酸酯化合物通过异氰酸酯共价结合亲水骨架聚合物，并提供游离异氰酸酯基团；(3) A layer of multifunctional isocyanate compound is attached to the surface of the hydrophilic backbone polymer, and the multifunctional isocyanate compound is covalently bonded to the hydrophilic backbone polymer through isocyanate to provide free isocyanate groups;
（4）异氰酸酯基团表面共价连接小分子，所述的小分子按阵列格式离散分布在所处理的芯片基片上。(4) Small molecules are covalently linked to the surface of the isocyanate group, and the small molecules are discretely distributed in an array format on the processed chip substrate.
本发明中，所述的异氰酸酯基团是多官能异氰酸酯化合物。In the present invention, the isocyanate group is a polyfunctional isocyanate compound.
本发明中，所述的多官能异氰酸酯化合物是芳香族聚异氰酸酯化合物。In the present invention, the polyfunctional isocyanate compound is an aromatic polyisocyanate compound.
本发明中，所述的芳香族聚异氰酸酯化合物为对苯二异氰酸酯、甲苯二异氰酸酯、二苯甲烷二异氰酸酯、萘二异氰酸酯、苯二亚甲基二异氰酸酯以及它们的混合物。在芳香族聚异氰酸酯中优选对苯二异氰酸酯。In the present invention, the aromatic polyisocyanate compound is p-phenylene diisocyanate, toluene diisocyanate, diphenylmethane diisocyanate, naphthalene diisocyanate, xylylene diisocyanate and mixtures thereof. Among the aromatic polyisocyanates, p-phenylene diisocyanate is preferred.
本发明中，所述的亲水骨架聚合物为芴甲氧羰基保护的多元醇。In the present invention, the hydrophilic skeleton polymer is polyhydric alcohol protected by fluorenyl moxycarbonyl group.
本发明中，所述的芴甲氧羰基保护的多元醇是聚乙二醇（PEG）、聚丙二醇（PPG）或其共聚物。优选聚乙二醇。In the present invention, the polyhydric alcohol protected by the fluorenyl moxycarbonyl group is polyethylene glycol (PEG), polypropylene glycol (PPG) or a copolymer thereof. Polyethylene glycol is preferred.
本发明中，所述的多元醇的链长n为1 ~ 40。In the present invention, the chain length n of described polyhydric alcohol is 1～40.
本发明中，所述的芯片基片为玻璃、硅片、塑料、石英、凝胶或尼龙膜。In the present invention, the chip substrate is glass, silicon chip, plastic, quartz, gel or nylon film.
本发明提出的小分子微阵列及其制备方法，基于异氰酸酯基团与氨基、羟基等多种亲核基团间的反应，通过采用芳香族异氰酸酯化合物和加热高温后处理方法，将多种小分子高效率地固定在芯片基片上。尤其对于与异氰酸酯基团反应较弱的羟基和羧基，固定效率提高2~3倍。本发明制备的小分子微阵列，具有制备简单，小分子固定效率高，抗非特异性吸附能力强，通量高，样品消耗少等优点，在高通量药物筛选方面具有广阔的应用前景。The small molecule microarray proposed by the present invention and its preparation method are based on the reaction between isocyanate groups and various nucleophilic groups such as amino groups and hydroxyl groups. By using aromatic isocyanate compounds and high-temperature post-treatment methods, various small molecules Efficiently fixed on the chip substrate. Especially for the hydroxyl and carboxyl groups that react weakly with isocyanate groups, the immobilization efficiency is increased by 2~3 times. The small molecule microarray prepared by the invention has the advantages of simple preparation, high small molecule immobilization efficiency, strong anti-nonspecific adsorption ability, high throughput, less sample consumption, etc., and has broad application prospects in high-throughput drug screening.
附图说明Description of drawings
图1小分子微阵列制备方法流程示意图。Fig. 1 Schematic diagram of the preparation method of small molecule microarray.
图2基于对苯二异氰酸酯和45℃加热后处理条件制备的小分子微阵列中的生物素抗体的表面质量密度图像。Figure 2 The surface mass density images of biotin antibodies in small molecule microarrays prepared based on p-phenylene diisocyanate and post-heating conditions at 45°C.
图3基于对苯二异氰酸酯和45℃加热后处理条件制备的小分子微阵列中各个生物素分子的固定效率。Fig. 3 The immobilization efficiency of individual biotin molecules in small molecule microarrays prepared based on p-phenylene diisocyanate and post-heating conditions at 45°C.
具体实施方式Detailed ways
下面通过具体实施例进一步描述本发明：Below further describe the present invention by specific embodiment:
实施例1：以二甲基甲酰胺为溶剂，制备芴甲氧羰酰基氨基-六聚乙二醇-羧酸溶液，其浓度为1mM,该溶液还含有六氟磷酸苯并三唑-1-基-氧基三吡咯烷基（其浓度为2mM）、N，N-二异丙基乙胺（其浓度为20mM）。将芯片基片浸泡在甲氧羰酰基氨基-六聚乙二醇-羧酸溶液中，室温搅拌反应10个小时。以二甲基甲酰胺为溶剂对处理后的芯片基片进行清洗。清洗后的芯片基片放入哌啶去保护溶液中，溶剂为二甲基甲酰胺，哌啶与二甲基甲酰胺的体积比为0.01，室温搅拌反应12个小时。使用二甲基甲酰胺清洗芯片基片并将其放入对苯二异氰酸酯溶液中，该溶液采用四氢呋喃为溶剂，对苯二异氰酸酯的浓度为60mM，室温搅拌反应1个小时。反应后的芯片基片依次在二甲基甲酰胺溶剂和四氢呋喃溶剂中彻底清洗。晾干清洗后的芯片基片，并用氮气吹干芯片基片。经过如上处理的芯片基片放在真空干燥器中保存。Example 1: Using dimethylformamide as a solvent, prepare a solution of fluorenylmethoxycarbonylamino-hexaethylene glycol-carboxylic acid, the concentration of which is 1mM, and the solution also contains benzotriazole-1-hexafluorophosphate yl-oxytripyrrolidinyl (concentration of 2mM), N,N-diisopropylethylamine (concentration of 20mM). Soak the chip substrate in the methoxycarbonylamino-hexaethylene glycol-carboxylic acid solution, and react with stirring at room temperature for 10 hours. The treated chip substrate was cleaned with dimethylformamide as solvent. The cleaned chip substrate is put into piperidine deprotection solution, the solvent is dimethylformamide, the volume ratio of piperidine to dimethylformamide is 0.01, and the reaction is stirred at room temperature for 12 hours. Clean the chip substrate with dimethylformamide and put it into p-phenylene diisocyanate solution, the solution uses tetrahydrofuran as a solvent, the concentration of p-phenylene diisocyanate is 60mM, and stir at room temperature for 1 hour. After the reaction, the chip substrate was thoroughly cleaned in dimethylformamide solvent and tetrahydrofuran solvent in sequence. Dry the chip substrate after cleaning, and blow dry the chip substrate with nitrogen. The chip substrates treated as above were stored in a vacuum desiccator.
制备小分子溶液，溶剂为二甲基亚砜或者二甲基亚砜与水的混合物。小分子分别为具有氨基的生物素（生物素-1），具有对硝基苯酯的生物素（生物素-2），具有酰肼的生物素（生物素-3），具有羟基的生物素（生物素-4）和生物素（生物素-5）。溶液浓度为1mM, 4mM, 8mM 和10mM。使用接触式点沿样机将各个生物素溶液依次点印在异氰酸酯修饰的芯片基片上。等待芯片基片上的溶剂（二甲基亚砜或二甲基亚砜与水的混合物）完全挥发后，将芯片基片放在45℃的恒温箱中12个小时，完成芯片的后处理过程。经过后处理的芯片基片放置于-20℃环境中保存。以上为小分子微阵列的制备过程。To prepare small molecule solutions, the solvent is dimethyl sulfoxide or a mixture of dimethyl sulfoxide and water. The small molecules are biotin with amino group (biotin-1), biotin with p-nitrophenyl ester (biotin-2), biotin with hydrazide (biotin-3), biotin with hydroxyl group (biotin-4) and biotin (biotin-5). The solution concentrations are 1mM, 4mM, 8mM and 10mM. Each biotin solution was sequentially printed on the isocyanate-modified chip substrate using a contact dot edge prototype. After waiting for the solvent on the chip substrate (dimethyl sulfoxide or a mixture of dimethyl sulfoxide and water) to evaporate completely, put the chip substrate in a thermostat at 45°C for 12 hours to complete the post-processing process of the chip. The post-processed chip substrate was stored in a -20°C environment. The above is the preparation process of the small molecule microarray.
为了研究异氰酸酯修饰的芯片基片对具有亲核基团的小分子的固定效率，将上述小分子微阵列与生物素抗体进行反应，生物素抗体的表面质量密度如图2所示，不同的生物素分子的固定效率如图3所示。In order to study the immobilization efficiency of isocyanate-modified chip substrates on small molecules with nucleophilic groups, the above-mentioned small molecule microarray was reacted with biotin antibodies. The surface mass density of biotin antibodies is shown in Figure 2. Different organisms The immobilization efficiency of prime molecules is shown in Figure 3.
结果表明，采用六聚乙二醇为骨架聚合物，以对苯二异氰酸酯为终端分子，采用45℃后处理条件，能够有效地制备小分子微阵列。其中，羧基的表面固定效率为30%左右，是采用六亚甲基二异氰酸酯以及催化后处理过的基片的羧基表面固定效率的3倍左右。该方法制备简单，固定效率高，抗非特异性吸附能力，保证了高质量的小分子微阵列。The results show that small molecule microarrays can be effectively prepared by using hexaethylene glycol as the backbone polymer, using p-phenylene diisocyanate as the terminal molecule, and adopting post-treatment conditions at 45°C. Among them, the surface immobilization efficiency of carboxyl groups is about 30%, which is about 3 times that of the surface immobilization efficiency of carboxyl groups on substrates treated with hexamethylene diisocyanate and catalytic post-treatment. The method is simple to prepare, has high immobilization efficiency and non-specific adsorption resistance, and ensures high-quality small molecule microarrays.
实施例2：以二甲基甲酰胺为溶剂，制备芴甲氧羰酰基氨基-十二聚乙二醇-羧酸溶液，其浓度为1mM,该溶液还含有六氟磷酸苯并三唑-1-基-氧基三吡咯烷基（其浓度为2mM,N）、N-二异丙基乙胺（其浓度为20mM）。将芯片基片浸泡在芴甲氧羰酰基氨基-六聚乙二醇-羧酸溶液中，室温搅拌反应5个小时。以二甲基甲酰胺为溶剂对处理后的芯片基片进行清洗。清洗后的芯片基片放入哌啶去保护溶液中，溶剂为二甲基甲酰胺，哌啶与二甲基甲酰胺的体积比为0.01，室温搅拌反应5个小时。使用二甲基甲酰胺清洗芯片基片并将其放入甲苯二异氰酸酯溶液中，该溶液采用二甲基甲酰胺为溶剂，甲苯二异氰酸酯的浓度为60mM，室温搅拌反应0.5小时。反应后的芯片基片依次在二甲基甲酰胺溶剂和四氢呋喃溶剂中彻底清洗。晾干清洗后的芯片基片，并继续用氮气吹干芯片基片。经过如上处理的芯片基片放在真空干燥器中保存。Example 2: Using dimethylformamide as a solvent, prepare a solution of fluorenylmethoxycarbonylamino-dodecapolyethylene glycol-carboxylic acid, the concentration of which is 1mM, and the solution also contains benzotriazole-1 hexafluorophosphate -yl-oxytripyrrolidinyl (concentration of 2mM, N), N-diisopropylethylamine (concentration of 20mM). Soak the chip substrate in the fluorenylmethoxycarbonylamino-hexaethylene glycol-carboxylic acid solution, and react with stirring at room temperature for 5 hours. The treated chip substrate was cleaned with dimethylformamide as solvent. The cleaned chip substrate is put into piperidine deprotection solution, the solvent is dimethylformamide, the volume ratio of piperidine to dimethylformamide is 0.01, and the reaction is stirred at room temperature for 5 hours. Clean the chip substrate with dimethylformamide and put it into toluene diisocyanate solution, the solution uses dimethylformamide as a solvent, the concentration of toluene diisocyanate is 60mM, and stir at room temperature for 0.5 hour. After the reaction, the chip substrate was thoroughly cleaned in dimethylformamide solvent and tetrahydrofuran solvent in sequence. Dry the chip substrate after cleaning, and continue to dry the chip substrate with nitrogen. The chip substrates treated as above were stored in a vacuum desiccator.
制备小分子溶液，溶剂为二甲基亚砜或者二甲基亚砜与水的混合物。小分子分别为具有氨基的生物素（生物素-1），具有对硝基苯酯的生物素（生物素-2），具有酰肼的生物素（生物素-3），具有羟基的生物素（生物素-4）和生物素（生物素-5）。溶液浓度为1mM, 4mM, 8mM 和10mM。使用接触式点沿样机将各个小分子依次点印在异氰酸酯修饰的芯片基片上。等待芯片基片上的溶剂（二甲基亚砜或二甲基亚砜与水的混合物）完全挥发后，将芯片基片放在40℃的恒温箱中12个小时，完成芯片的后处理过程。经过后处理的芯片基片放置于-20℃环境中保存。以上为小分子微阵列的制备过程。To prepare small molecule solutions, the solvent is dimethyl sulfoxide or a mixture of dimethyl sulfoxide and water. The small molecules are biotin with amino group (biotin-1), biotin with p-nitrophenyl ester (biotin-2), biotin with hydrazide (biotin-3), biotin with hydroxyl group (biotin-4) and biotin (biotin-5). The solution concentrations are 1mM, 4mM, 8mM and 10mM. Each small molecule is sequentially printed on the isocyanate-modified chip substrate using a contact dot edge prototype. After waiting for the solvent on the chip substrate (dimethyl sulfoxide or a mixture of dimethyl sulfoxide and water) to evaporate completely, put the chip substrate in a thermostat at 40°C for 12 hours to complete the post-processing process of the chip. The post-processed chip substrate was stored in a -20°C environment. The above is the preparation process of the small molecule microarray.
以上所述仅为本发明的较佳实施例，凡依本发明申请专利范围所做的均等变化与修饰，皆应属本发明的涵盖范围。The above descriptions are only preferred embodiments of the present invention, and all equivalent changes and modifications made according to the scope of the patent application of the present invention shall fall within the scope of the present invention.
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| CN201510343984.8ACN104931694B (en) | 2015-06-22 | 2015-06-22 | A kind of small-molecular micro-array based on isocyanates and preparation method thereof |
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