WO2015006934A1 - 一种小分子药物筛选芯片、其构建方法及应用 - Google Patents
一种小分子药物筛选芯片、其构建方法及应用 Download PDFInfo
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- WO2015006934A1 WO2015006934A1 PCT/CN2013/079504 CN2013079504W WO2015006934A1 WO 2015006934 A1 WO2015006934 A1 WO 2015006934A1 CN 2013079504 W CN2013079504 W CN 2013079504W WO 2015006934 A1 WO2015006934 A1 WO 2015006934A1
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- Prior art keywords
- small molecule
- drug screening
- molecule drug
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- surface modification
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- 229940126586 small molecule drug Drugs 0.000 title claims abstract description 31
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
-
- 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
- B01J2219/00612—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports the surface being inorganic
-
- 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
- B01J2219/00614—Delimitation of the attachment areas
- B01J2219/00617—Delimitation of the attachment areas by chemical means
- B01J2219/00619—Delimitation of the attachment areas by chemical means using hydrophilic or hydrophobic regions
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- 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
- B01J2219/00632—Introduction of reactive groups to the surface
- B01J2219/00637—Introduction of reactive groups to the surface by coating it with another layer
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
- C40B50/14—Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support
- C40B50/18—Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support using a particular method of attachment to the solid support
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2610/00—Assays involving self-assembled monolayers [SAMs]
Definitions
- the present invention relates to the field of biochip technology, and in particular, to a small molecule drug screening chip, a construction method thereof and an application thereof.
- Biochips are new methods and technologies developed by DNA microarrays in the last two decades. They are characterized by high throughput, low cost and parallel experiments. They provide DNA sequencing, biochemical detection, environmental monitoring and drug screening. Effective research methods. With the development of this technology, its detection methods can not only be screened by fluorescent or radioactive labeling, but also combined with unlabeled characterization methods such as quartz crystal microbalance, ellipsometric imaging and surface plasmon resonance imaging. The interaction between the two provides more kinetic information. Small molecule drug screening chips, as a member of the biochip family, have recently made great progress in drug screening. This technology allows small molecule drugs to be immobilized on the surface of the substrate, providing a high-throughput target molecular screening platform for natural compound products and artificially designed synthetic small molecules.
- Non-covalent immobilization is the modification of a tag molecule on the inactive side chain of a small molecule that can be selectively non-covalently bound by a substance that recognizes the tag on the surface of the substrate to construct a small molecule chip. This method not only changes the structure of small molecules, but also greatly increases the workload of small molecule and tag synthesis.
- the covalent immobilization is carried out by covalent attachment to an activated carboxyl group, an activated amino group, a maleimide group or the like on the surface of the substrate by a reactive group such as an amino group, a carboxyl group or a thiol group of the small molecule.
- This method is limited to many amino, carboxyl, and sulfhydryl groups, which are the active centers of small molecules. Experiments are prone to false negative results, and due to the diversity of small molecules, it is difficult for a single chip to satisfy a variety of small molecules. Screening.
- the inventors have constructed a small molecule drug screening chip through a large amount of experiments and creative labor, and the small molecule drug screening chip can realize most non-selective and effective immobilization of small molecules, and has an inherent structure that does not destroy small molecules, and is small. Excellent molecular weight and high molecular activity.
- the present invention provides a small molecule drug screening chip comprising a substrate layer, a surface modification layer formed by surface modification of the base layer, and a small molecule non-selectively adsorbed on the surface modification layer .
- the manner of surface modification includes non-selective adsorption, covalent attachment, initiation polymerization, self-assembly, and the like.
- the self-assembly comprises a silicon germanium thiol self-assembly of a glass surface, a silicon germanium thiol self-assembly of a silicon surface, and a thiol self-assembly of a metal surface, the initiation polymerization including initiation polymerization of a metal surface, and the like.
- the surface modification layer has a surface structure and/or a terminal group to achieve non-selective adsorption of the small molecule.
- the surface structure comprises a two-dimensional structure and a three-dimensional structure, the terminal group comprising a hydroxyl group, an amino group, a carboxyl group, a maleimide group, an epoxy group and a carbonyl group; the hydroxyl end group is preferably a poly group The ethylene glycol hydroxyl terminus; the amino terminal group is preferably a polyethylene glycol amino terminus.
- a non-selectively adsorbed small molecule drug screening chip that initiates a polymeric three-dimensional surface structure on a gold substrate construction surface is provided.
- the material of the base layer is a composite material of any one or at least two of glass, silicon, silicon dioxide, quartz, metal and polymer.
- a small molecule drug screening chip having a silica as a base material is provided, the surface of which is a silicon germanium amino surface as a site for non-selective adsorption with small molecules.
- the present invention provides a method for constructing a small molecule drug screening chip according to the first aspect, the method comprising surface modifying a base layer of the chip to form a surface modification layer to make the small molecule non-selective a step of adsorbing on the surface modification layer.
- the method further comprises a step of small molecule spotting, vacuum drying, cryopreservation, incubation, and rapid drying.
- the cryopreservation is stored below ambient temperature; in one embodiment of the invention, it is stored overnight in a refrigerator at -20 °C.
- the incubation is carried out on the surface of the small molecule chip by agglomerating a certain layer of water from the cold or hot process.
- the water layer which is rapidly dried to the surface of the small molecule chip is quickly dried, and usually, a blow drying oven, an incandescent lamp, and ultraviolet light irradiation can be used, but is not limited thereto.
- the present invention provides the use of the small molecule drug screening chip of the first aspect for detecting the interaction of a small molecule with a target protein.
- the application specifically refers to: the small molecule drug screening chip combined with fluorescent label scanning, surface plasmon resonance, radioactive element label scanning, enzyme-linked immunolabeling, quartz crystal microbalance or ellipsometry to detect small molecules and Application in the interaction of target proteins.
- the beneficial effects of the present invention are as follows: The present invention achieves non-selective and effective immobilization of most small molecules by non-selective adsorption of small molecule drugs by a surface modification layer on a chip, and has an inherent structure that does not destroy small molecules. The experiment proves that: the small molecule drug screening chip of the invention has the advantages of large immobilization of small molecules and high activity of small molecules.
- Figure 1 is a schematic view showing the chemical structure of the surface of an amino glass chip used in Example 1 of the present invention.
- Example 2 is a fluorescence diagram showing the change of non-selective adsorption amount of small molecules based on fluorescent label scanning under different cleaning conditions according to Example 1 of the present invention.
- Fig. 3 is a graph showing the change of non-selective adsorption amount of a small molecule (ImM Rhodamine B, solvent: dimethyl sulfoxide DMSO) based on fluorescent label scanning according to Example 1 of the present invention under different washing conditions.
- Fig. 4 is a graph showing the change of non-selective adsorption amount of a small molecule (I mM Rhodamine B, solvent: water) based on fluorescent label scanning under different washing conditions according to Example 1 of the present invention.
- Fig. 5 is a graph showing the change of non-selective adsorption amount of small molecules (ImM Cy3, O.lmM Cy5, solvent: water) based on fluorescent label scanning under different washing conditions according to Example 1 of the present invention.
- Figure 6 is a FKBP12 plasmid map of Example 2 of the present invention.
- FIG. 7 is a fluorescence diagram of a spotting sequence and a detection result of a non-selective adsorption small molecule array based on fluorescent label scanning according to Embodiment 2 of the present invention, wherein A, B, C, D, E, and F are biotin, respectively. , FK506, HIS peptide (HIS peptide), FLAG peptide (FLAG peptide), cyclosporine A (Cyclosporine A) and digoxin (Digoxin) test results.
- HIS peptide HIS peptide
- FLAG peptide FLAG peptide
- Cyclosporine A cyclosporine A
- Digoxin digoxin
- Fig. 8 is a schematic view showing the surface-initiated polymerization three-dimensional surface structure of the gold substrate according to Embodiment 3 of the present invention.
- Fig. 9 is a graph showing the results of biotin (Biotm) detection by non-selective adsorption small molecule array based on surface plasmon resonance imaging according to Example 3 of the present invention.
- Example 1 Non-selective adsorption amount of small molecules based on fluorescent label scanning under different cleaning conditions
- This embodiment provides a small molecule adsorption amount of a small molecule drug screening chip constructed by a non-selective adsorption method as a function of cleaning conditions.
- Two commonly used dye molecules, Rhodamme B and Cy3-Cy5 were spotted onto the surface of an amino glass chip as shown in FIG.
- the specific method is: ultrasonic cleaning of the amino glass chip in aqueous solution for 10 minutes, spotting, specific information is as follows: Rhodamine B (ImM solvent: DMSO), Rhodamine B (ImM solvent: water) and cy3-cy5 ( Cy3: ImM, Cy5: O.lmM dissolves 1": water) is printed using a TOYOBO chip printer with a 200 ⁇ m diameter needle. After printing, it was allowed to stand in a vacuum drying vessel for 20 minutes, sealed and stored in a refrigerator at -20 ° C overnight; the next day, after taking out the package to room temperature, it was sufficiently dried in a blast oven at 50 ° C. Then carry out the following cleaning experiments: 1) Ultrasonic cleaning in Milli Q (water) solution for 5 minutes at room temperature, centrifugal drying, detection at Genepix 635 nm and 532 nm wavelength, this process is repeated and recorded 5 times;
- Example 2 Identification of small molecules immobilized by non-selective adsorption based on fluorescent label scanning method
- This example provides the use of fluorescent label scanning methods to identify the activity of a variety of small molecules immobilized by non-selective adsorption. Seven different small molecules were spotted onto the surface of an amino glass chip (same as in Example 1). The specific method is: ultrasonic cleaning of the amino glass chip in aqueous solution for 10 minutes, spotting, specific information is as follows: rhodamine B (lmM), biotin (Biotin, 5mM), FK506 (5mM), HIS peptide (HIS The peptide, lmM), FLAG polypeptide (FLAG peptide, 5 mM), cyclosporine A (5 mM) and digoxin (5 mM) were printed using a TOYOBO chip printer with a 200 ⁇ m diameter needle.
- A detecting biotin
- B detecting FK506
- C detecting HIS peptide
- D detecting FLAG polypeptide
- E detecting cyclosporine A
- F detecting digoxin
- a 20-fold diluted FKBP12 expressed by Cellfree Science's wheat germ expression system (in which the structure of the FKBP12 plasmid is shown in Figure 6) was added to the surface of the chip.
- the cell lysate mixture was 50 ⁇ M (solvent as a milk sealant) at 30°.
- the reaction was carried out for 1 hour in an incubator of C, and it was taken out and washed 3 times with TBST for 5 minutes each time.
- Add 10 g/mL of Mouse Anti-his antibody (solvent as milk sealant) to the surface of the chip, react for 1 hour in an incubator at 30 ° C, and remove it by TBST for 3 times for 5 minutes each time.
- the binding sites of the corresponding small molecules on the six chips showed strong fluorescence signals, indicating that the small molecules had a large immobilization amount and a high activity.
- the corresponding small molecules to be detected on the six chips only bind to the corresponding antibodies, indicating that the specificity is good, and can meet the requirement of detecting a plurality of small molecules on the same chip, that is, the embodiment shows that the present invention can realize most of the small molecule non-selection. Effectively fixed.
- Example 3 Non-selective adsorption of small molecule arrays based on surface plasmon resonance imaging
- This example provides a method for non-selectively adsorbing small molecule drug screening chips that initiate a polymeric three-dimensional surface construction by constructing a surface on a gold substrate that has a strong resistance to protein non-specific adsorption.
- the test object is the interaction between small molecules and proteins (non-antibody), including the interaction between biotin and streptavidin, FK506 and FKBP12 proteins, and Rapamycin and FKBP12 proteins.
- a structurally similar bromine initiator thiol and PEG-3-hydroxy thiol were used to construct molecular self-assembly on the surface of the gold piece using SPR experiments.
- the ratio of the bromine initiator thiol to PEG-3-hydroxythiol was 1:99 (total amount of I mM), and the reaction was carried out at normal temperature for 16 hours.
- a three-dimensional surface chemistry having a polyethylene glycol hydroxyl group as a branch (the structure is shown in Fig. 8) was constructed, and then ultrasonic cleaning was carried out using an aqueous solution.
- the spotting method, vacuum drying, cryopreservation, and re-drying method were the same as in Example 2. Finally, install it on Plexera's SPRi detection unit and perform the following tests:
- a cell-free expression system mixture of FKBP12 (solvent PBST (containing 0.05% Tween20) was diluted 20-fold at a flow rate of 2 L/s.
- the reaction included a 180s baseline, a 200s binding process, a 400s dissociation process, and a 200s phosphoric acid (1:200 v/v MilliQ water) regeneration process.
- the experimental results are shown in Figure 10.
- the interaction between FK506 and FKBP12 protein, Rapamycin and FKBP12 protein is strongly signaled; while biotin, HIS peptide, FLAG peptide and digoxin have no response signal. (In Figure 10, the four curves are at a level where the standard response is zero).
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Abstract
一种小分子药物筛选芯片、其构建方法及应用。小分子药物筛选芯片包括基底层，对基底层进行表面改性生成的表面修饰层，以及非选择性吸附于表面修饰层上的小分子。构建方法包括对芯片的基底层进行表面改性生成表面修饰层以使小分子非选择性吸附于表面吸附层上的步骤。可以实现大部分小分子非选择性的有效固定，并尽量不破坏小分子的固有结构，且能够实现小分子固定量大、活性高的特性，可以用于检测小分子与靶标蛋白的相互作用。
Description
一种小分子药物筛选芯片、 其构建方法及应用 技术领域
本发明涉及生物芯片技术领域， 尤其涉及一种小分子药物筛选芯片、 其构 建方法及应用。
背景技术
生物芯片是最近二十年由 DNA微阵列发展而来的新方法、 新技术， 具有高 通量、 低成本和平行实验等特点， 为 DNA测序、 生化检测、 环境监控以及药物 筛选等多方面提供了有效的研究手段。 随着这项技术的发展， 其检测手段不仅 可以通过荧光或放射性等物质标记进行筛选， 而且已经和石英晶体微天平、 椭 偏成像和表面等离子共振成像等无标签表征手段相结合， 为生物分子之间的相 互作用提供了更多丰富的动力学信息。 小分子药物筛选芯片作为生物芯片大家 族的一名成员， 最近在药物筛选方面有了很大的发展。 这种技术将小分子药物 通过固定在基底表面上， 为天然化合产物和人工设计合成的小分子提供高通量 的靶标分子筛选平台。
小分子药物筛选芯片目前可以按照小分子的固定方式分为非共价固定和共 价固定。 非共价固定是在小分子的非活性的侧链上修饰一个标签分子， 该标签 分子可以通过基底表面识别该标签的物质进行选择性的非共价结合， 从而构建 小分子芯片。 这种方法不仅改变了小分子的结构， 而且大大增加了小分子与标 签合成的工作量。 共价固定是通过小分子的氨基、 羧基、 巯基等活性基团， 与 基底表面的活化羧基、 活化氨基、 马来酰亚胺基团等形成共价连接完成共价固 定。 这种方法局限于很多氨基、 羧基、 巯基正是小分子的活性中心， 实验容易 出现假阴性的结果， 而且由于小分子的多样性， 一张芯片很难满足多种小分子
的筛选。 日本理化学研究所的长田裕之课题组报道了光交联小分子阵列的方法， 它使用卡宾基团在紫外线剌激下， 可以和小分子任意一个 C-H或 N-H基团发生 反应， 从而非选择性地普适性地完成小分子的固定。 这种方法虽然解决了普适 性小分子的固定问题， 但是破坏了小分子固有的结构， 且一些紫外耐受能力差 的小分子容易丧失活性。
因此， 亟需一种构建小分子药物筛选芯片的新方法， 以实现大部分小分子 非选择性地有效固定， 并尽量不破坏小分子的固有结构， 且能够实现小分子固 定量较大、 活性较高等特性。
发明内容
本发明人经过大量实验和创造性劳动， 构建了一种小分子药物筛选芯片， 该小分子药物筛选芯片可以实现大部分小分子非选择性地有效固定， 且具备不 破坏小分子的固有结构、 小分子固定量大、 小分子活性高等优异特性。
本发明提供以下技术方案：
在第一方面， 本发明提供一种小分子药物筛选芯片， 包括基底层， 对所述 基底层进行表面改性生成的表面修饰层， 以及非选择性吸附于所述表面修饰层 上的小分子。
优选地， 所述表面改性的方式包括非选择性吸附、 共价连接、 引发聚合和 自组装等。
进一歩优选， 所述自组装包括玻璃表面的硅垸硫醇自组装、 硅表面的硅垸 硫醇自组装和金属表面的硫醇自组装等， 所述引发聚合包括金属表面的引发聚 合等。
优选地， 所述表面修饰层具有表面结构和 /或末端基团， 以实现所述小分子 的非选择性吸附。
进一歩优选， 所述表面结构包括二维结构和三维结构， 所述末端基团包括 羟基、 氨基、 羧基、 马来酰亚胺基、 环氧基和羰基； 所述羟基末端基团优选为 聚乙二醇羟基末端； 所述氨基末端基团优选为聚乙二醇氨基末端。
在本发明一个实施方案中， 提供一种在金基底构建表面引发聚合三维表面 结构的非选择性吸附小分子药物筛选芯片。
优选地， 所述基底层的材料为玻璃、 硅、 二氧化硅、 石英、 金属和聚合物 等中任意一种或至少两种组成的复合材料。
在本发明一个实施方案中， 提供一种以二氧化硅为基底层材料的小分子药 物筛选芯片， 其表面为硅垸氨基表面， 作为与小分子非选择性吸附的场所。
在第二方面， 本发明提供一种在第一方面所述的小分子药物筛选芯片的构 建方法， 所述方法包括对芯片的基底层进行表面改性生成表面修饰层以使小分 子非选择性吸附于所述表面修饰层上的歩骤。
优选地， 所述方法还包括小分子点样、 真空干燥、 低温保存、 孵育及迅速 干燥的歩骤。
优选地， 所述低温保存为低于室温的环境保存； 在本发明一个实施方案中， 在 -20°C的冰箱中保存过夜。 所述孵育为小分子芯片表面由冷即热过程中凝集一 定的水层于表面。 迅速干燥为小分子芯片表面的水层迅速干燥， 通常可采用鼓 风干燥箱、 白炽灯和紫外光照射等方式， 但不限于此。
在第三方面， 本发明提供一种在第一方面所述的小分子药物筛选芯片在检 测小分子与靶标蛋白的相互作用中的应用。
优选地， 所述应用具体是指： 所述小分子药物筛选芯片结合荧光标记扫描、 表面等离子共振、 放射性元素标记扫描、 酶联免疫标记、 石英晶体微天平或椭 偏成像技术在检测小分子与靶标蛋白的相互作用中的应用。
本发明的有益效果为： 本发明通过芯片上的表面修饰层对小分子药物的非 选择性吸附实现大部分小分子非选择性的有效固定， 且具备不破坏小分子的固 有结构。 实验证明： 本发明小分子药物筛选芯片具有小分子固定量大、 小分子 活性高等优异特性。
附图说明
图 1为本发明实施例 1所用氨基玻璃芯片表面化学结构示意图。
图 2为本发明实施例 1 的基于荧光标记扫描的小分子在不同清洗条件下的 非选择性吸附量变化荧光图。
图 3为本发明实施例 1的基于荧光标记扫描的小分子 （ImM罗丹明 B， 溶 剂： 二甲基亚砜 DMSO) 在不同清洗条件下的非选择性吸附量变化曲线图。
图 4为本发明实施例 1的基于荧光标记扫描的小分子 （ImM罗丹明 B， 溶 剂： 水） 在不同清洗条件下的非选择性吸附量变化曲线图。
图 5为本发明实施例 1的基于荧光标记扫描的小分子 （ImM Cy3， O.lmM Cy5， 溶剂： 水） 在不同清洗条件下的非选择性吸附量变化曲线图。
图 6为本发明实施例 2的 FKBP12质粒图谱。
图 7为本发明实施例 2的基于荧光标记扫描的非选择性吸附小分子阵列的 点样顺序和检测结果荧光图， 其中 A、 B、 C、 D、 E和 F分别为生物素（Biotin)、 FK506、 HIS 多肽 （HIS peptide )、 FLAG 多肽 （FLAG peptide )、 环孢素 A (Cyclosporine A) 和地高辛 （Digoxin) 的检测结果。
图 8为本发明实施例 3的金基底构建表面引发聚合三维表面结构示意图。 图 9为本发明实施例 3的基于表面等离子共振成像的非选择性吸附小分子 阵列检测生物素 （Biotm) 的结果曲线图。
图 10为本发明实施例 3的基于表面等离子共振成像的非选择性吸附小分子
阵列检测 FK506和雷帕霉素 （Rapamycin) 的结果曲线图。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述。 本领域技术人员将 会理解， 下面的实施例仅用于说明本发明， 而不应视为限定本发明的范围。 实 施例中未注明具体技术或条件者， 按照本领域内的文献所描述的技术或条件， 或者按照产品说明书进行。 所用试剂或仪器未注明生产厂商者， 均为可以通过 市购获得的常规产品。
实施例 1 :基于荧光标记扫描的小分子在不同清洗条件下的非选择性吸附量 变化
2)室温条件下， 乙醇溶液中超声清洗 5分钟， 离心干燥，在 Genepix 635nm 和 532nm波长下检测， 此过程重复并记录 5次；
3 )室温条件下， 二甲基亚砜溶液中超声清洗 5分钟， 离心干燥， 在 Genepix 635nm和 532nm波长下检测， 此过程重复并记录 5次；
4 )室温条件下，二甲基亚砜溶液中震荡过夜（ 18小时），离心干燥，在 Genepix 635nm和 532nm波长下检测。
实验结果： 罗丹明 B和 Cy3-Cy5小分子通过非选择性吸附在芯片表面的吸 附量变化如图 2至图 5所示。 结果显示： 本实施例中罗丹明 B和 Cy3-Cy5在氨 基玻璃芯片表面的吸附量较大， 并且吸附力强； 虽然经过 Milli Q溶液、 乙醇溶 液和二甲基亚砜溶液分别 5次 （每次 5分钟） 清洗， 并且经过二甲基亚砜溶液 中震荡过夜处理， 仍有较多的小分子吸附在芯片表面上， 并且渐趋稳定状态， 尤其是图 3所示的罗丹明 B ( ImM溶剂： DMSO) 点样的情况下， 小分子的吸 附量更大、 稳定程度更高。
实施例 2: 基于荧光标记扫描方法鉴定通过非选择性吸附固定的小分子
HN NH
Biotin
F 506
CyclosporineA Digoxin
本实施例提供使用荧光标记扫描方法鉴定多种通过非选择性吸附固定的小 分子的活性。 将七种不同的小分子点样到氨基玻璃芯片 （同实施例 1 )表面。 具 体做法是： 将氨基玻璃芯片在水溶液下超声清洗 10分钟， 进行点样， 具体点样 信息如下： 罗丹明 B ( lmM)、 生物素 （Biotin, 5mM)、 FK506 ( 5mM)、 HIS 多肽 （HIS peptide, lmM)、 FLAG 多肽 （FLAG peptide, 5mM)、 环孢素 A (Cycrosporine A， 5mM) 和地高辛 （Digoxin, 5mM)， 使用 TOYOBO芯片打 印仪 200μπι直径的针头打印。打印后， 在真空干燥器皿中静置 30分钟， 密封并 于 -20°C冰箱中保存过夜； 次日， 取出开封至室温后， 在白炽灯下充分干燥。 通 过 lmg/mL牛奶 （溶剂为 TBST, 含 0.05% Tween20) 封闭芯片 1小时后， 使用 TBST清洗表面 3次，每次 5分钟。芯片标记为 A (检测生物素）， B (检测 FK506) ,
C (检测 HIS多肽）， D (检测 FLAG多肽）， E (检测环孢素 A) 和 F (检测地 高辛） 六个芯片。
分别对 A~F六个芯片进行如下所示的反应流程：
A: 在芯片表面加入 lO g/mL的 Cy5标记的链霉亲和素 5(^L (溶剂为牛奶 封闭剂）， 于 30°C的恒温箱中反应 1小时， 取出后使用 TBST清洗 3次， 每次 5 分钟。 然后， 使用 GenePix扫描 635nm波长 （强度 800) 和 532nm波长 （强度 500)。
B: 在芯片表面加入 20倍稀释的通过 Cellfree Science公司麦胚表达体系表 达的 FKBP12 (其中 FKBP12质粒的结构如图 6所示）细胞裂解混合液 50μΙ^ (溶 剂为牛奶封闭剂）， 于 30°C的恒温箱中反应 1小时， 取出使用 TBST清洗 3次， 每次 5分钟。在芯片表面加入 lO g/mL的 Mouse Anti-his抗体（溶剂为牛奶封闭 剂）， 于 30°C的恒温箱中反应 1小时， 取出使用 TBST清洗 3次， 每次 5分钟。 在芯片表面加入 lO g/mL的 633nm荧光标记的 Goat Anti Mouse抗体（GE公司 购买， 溶剂为牛奶封闭剂）， 于 30°C的恒温箱中反应 1小时， 取出使用 TBST清 洗 3次，每次 5分钟。然后，使用 GenePix扫描 635nm波长（强度 800)和 532nm 波长 （强度 500)。
C: 在芯片表面加入 lO g/mL的 Mouse Anti-his抗体（溶剂为牛奶封闭剂）， 于 30°C的恒温箱中反应 1小时， 取出使用 TBST清洗 3次， 每次 5分钟。 在芯 片表面加入 lO g/mL的 633nm荧光标记的 Goat Anti Mouse抗体（GE公司购买， 溶剂为牛奶封闭剂），于 30°C的恒温箱中反应 1小时，取出使用 TBST清洗 3次， 每次 5分钟。 然后， 使用 GenePix扫描 635nm波长 （强度 800) 和 532nm波长 (强度 500)。
D: 在芯片表面加入 lO g/mL的 Rabbit Anti-FLAG抗体 （Sigma公司购买，
溶剂为牛奶封闭剂），于 30°C的恒温箱中反应 1小时，取出使用 TBST清洗 3次， 每次 5分钟。 在芯片表面加入 lO g/mL的 633nm荧光标记的 Goat Anti Rabbit 抗体（溶剂为牛奶封闭剂）， 于 30°C的恒温箱中反应 1小时， 取出使用 TBST清 洗 3次，每次 5分钟。然后，使用 GenePix扫描 635nm波长（强度 800)和 532nm 波长 （强度 500)。
E: 在芯片表面加入 54 g/mL的 Mouse Anti-Cycrosporine A抗体 (Hytech公 司购买， 溶剂为牛奶封闭剂）， 于 30°C的恒温箱中反应 1小时， 取出使用 TBST 清洗 3次，每次 5分钟。在芯片表面加入 lO g/mL的 633nm荧光标记的 Goat Anti Mouse抗体 （溶剂为牛奶封闭剂）， 于 30°C的恒温箱中反应 1 小时， 取出使用 TBST清洗 3次， 每次 5分钟。然后， 使用 GenePix扫描 635nm波长（强度 800) 和 532nm波长 （强度 500)。
F:在芯片表面加入 54 g/mL的 Rabbit Anti-Digoxin抗体（Sigma公司购买， 溶剂为牛奶封闭剂），于 30°C的恒温箱中反应 1小时，取出使用 TBST清洗 3次， 每次 5分钟。 在芯片表面加入 lO g/mL的 633nm荧光标记的 Goat Anti Rabbit 抗体（溶剂为牛奶封闭剂）， 于 30°C的恒温箱中反应 1小时， 取出使用 TBST清 洗 3次，每次 5分钟。然后，使用 GenePix扫描 635nm波长（强度 800)和 532nm 波长 （强度 500)。
结果如图 7所示， 六个芯片上相应待检测小分子的结合位点呈现较强的荧 光信号， 说明小分子固定量较大、 活性较高。 此外， 六个芯片上相应待检测小 分子仅与相应抗体结合， 说明其特异性好， 能满足同一芯片检测多种小分子的 需求， 即该实施例证明本发明可以实现大部分小分子非选择性地有效固定。
实施例 3 : 基于表面等离子共振成像的非选择性吸附小分子阵列
Rapamycin
本实施例提供在通过金基底构建表面引发聚合三维表面构建的非选择性吸 附小分子药物筛选芯片的方法， 该表面具有很强的抗蛋白质非特异性吸附的特 点。 其检测对象是小分子与蛋白质 （非抗体） 之间的相互作用， 包括生物素与 链霉亲和素、 FK506与 FKBP12蛋白、 雷帕霉素 （Rapamycin) 与 FKBP12蛋白 之间的相互作用。将结构类似的溴引发剂硫醇和 PEG-3-羟基硫醇在使用 SPR实 验的金片表面构建分子自组装。溴引发剂硫醇和 PEG-3-羟基硫醇比例为 1 :99(总 量为 ImM), 常温下反应 16小时。 清洗后引发表面聚合反应， 构建以聚乙二醇 羟基作为支链的三维表面化学 （其结构如图 8所示） 后， 使用水溶液进行超声 清洗。 点样方法、 真空干燥、 低温保存以及再干燥方法与实施例 2 相同。 最后 安装至 Plexera公司的 SPRi检测装置上进行如下检测：
( 1 ) 鉴定生物素：
l g/mL链霉亲和素稀释于 PBST (含 0.05% Tween20) 中， 流速为 2 L/s。 反应包括 180s基线、 200s结合过程、 400s解离过程、 200s的磷酸（ 1 :200 v/v MilliQ Water) 重生过程。 实验结果如图 9所示， 生物素与链霉亲和素之间的相互作用 有很强的信号； 而链霉亲和素与 FK506、 HIS多肽、 FLAG多肽、 雷帕霉素、 地 高辛没有相互作用显示出的信号 （在图 9中表现为 曲线处于标准响应为 0 的水平）。
(2) 鉴定 FK506和雷帕霉素：
20倍稀释表达 FKBP12的无细胞表达体系混合液（溶剂为 PBST (含 0.05% Tween20))，流速为 2 L/s。反应包括 180s基线、 200s结合过程、 400s解离过程、 200s的磷酸 （1 :200 v/v MilliQ 水） 重生过程。 实验结果如图 10所示， FK506 与 FKBP12蛋白、 雷帕霉素 （Rapamycin) 与 FKBP12蛋白之间的相互作用有很 强的信号； 而生物素、 HIS多肽、 FLAG多肽和地高辛没有响应信号 （在图 10 中表现为 4条曲线处于标准响应为 0的水平）。
本领域技术人员将会理解： 尽管本发明的具体实施方式已经得到详细的描 述， 根据已经公开的所有教导， 可以对那些细节进行各种修改和替换， 这些改 变均在本发明的保护范围之内。 本发明的全部范围由所附权利要求及其任何等 同物给出。
Claims
1、 一种小分子药物筛选芯片， 其特征在于， 包括基底层， 对所述基底层进 行表面改性生成的表面修饰层， 以及非选择性吸附于所述表面修饰层上的小分 子。
2、 根据权利要求 1所述的小分子药物筛选芯片， 其特征在于， 所述表面改 性的方式包括非选择性吸附、 共价连接、 引发聚合和自组装。
3、 根据权利要求 2所述的小分子药物筛选芯片， 其特征在于， 所述自组装 包括玻璃表面的硅垸硫醇自组装、 硅表面的硅垸硫醇自组装和金属表面的硫醇 自组装， 所述引发聚合包括金属表面的引发聚合。
4、 根据权利要求 1至 3任一项所述的小分子药物筛选芯片， 其特征在于， 所述表面修饰层具有表面结构和 /或末端基团， 以实现所述小分子的非选择性吸 附。
5、 根据权利要求 4所述的小分子药物筛选芯片， 其特征在于， 所述表面结 构包括二维结构和三维结构， 所述末端基团包括羟基、 氨基、 羧基、 马来酰亚 胺基、 环氧基和羰基； 所述羟基末端基团优选为聚乙二醇羟基末端； 所述氨基 末端基团优选为聚乙二醇氨基末端。
6、 根据权利要求 1至 3任一项所述的小分子药物筛选芯片， 其特征在于， 所述基底层的材料为玻璃、 硅、 二氧化硅、 石英、 金属和聚合物中任意一种或 至少两种组成的复合材料。
7、一种权利要求 1至 6任一项所述的小分子药物筛选芯片的构建方法， 其 特征在于， 所述方法包括对芯片的基底层进行表面改性生成表面修饰层以使小 分子非选择性吸附于所述表面修饰层上的歩骤。
8、 根据权利要求 7所述的小分子药物筛选芯片的构建方法， 其特征在于， 所述方法还包括小分子点样、 真空干燥、 低温保存、 孵育及迅速干燥的歩骤。
9、一种权利要求 1至 6任一项所述的小分子药物筛选芯片在检测小分子与 靶标蛋白的相互作用中的应用。
10、 根据权利要求 9所述的应用， 其特征在于， 所述小分子药物筛选芯片 结合荧光标记扫描、 表面等离子共振、 放射性元素标记扫描、 酶联免疫标记、 石英晶体微天平或椭偏成像技术在检测小分子与靶标蛋白的相互作用中的应 用。
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