表面光聚合反应制备三维基因芯片及蛋白微阵列研究

发布时间：2018-06-18 21:07

本文选题：光接枝 + 基因芯片　；参考：《北京化工大学》2016年硕士论文

【摘要】：生物芯片具有装置微型、自动、高度并行和高通量等特点,在临床诊断、新药筛选、个体化医疗及生物工程研究等领域有广阔的应用前景。20世纪90年代以来,生物芯片的制作工艺和检测手段已经有了飞速发展,但因涉及学科多,仍然面临巨大的挑战。如制备过程复杂、成本高,处理程序多导致重复性不理想等,这些问题制约了生物芯片的广泛应用。研发更为先进的生物分子固定技术,对于发展高效的微阵列芯片技术意义重大。本论文以光引发表面接枝聚合为基础,发展了两种制备表面三维接枝结构的新方法,进而制备了三维结构的基因芯片和复杂结构的蛋白质微阵列。实现了DNA探针和蛋白质分子在载体表面的高密度有效固定,并对三维基因芯片在脑胶质瘤诊断中的应用进行了初步研究。本文主要的研究工作及结果：1.以商用载玻片为基材,通过乙烯基三氯硅烷偶联剂处理引入双键,再在双键化表面以甲基丙烯酸缩水甘油酯(GMA)与聚乙二醇二丙烯酸酯(PEGDA)为混合单体运用紫外光接枝法,制备具有一定厚度且表面含有环氧基团的三维基片。利用氨基与环氧基团的开环反应,将荧光分子(Cy3)标记且末端为氨基的DNA探针固定在基片上。通过改变环氧基团在三维交联层中的含量、交联层厚度及DNA探针点样液浓度,可以调节DNA探针的固定效率和密度。与传统的二维基因芯片相比,三维芯片的探针固定效率由30%提高到80%左右,固定密度也同时提高。芯片的杂交结果证明三维基材表面可成功实现核酸探针分子与靶基因的杂交。2.将三维基因芯片技术应用于脑胶质瘤疾病辅助诊断用基因芯片的研制。首先设计并完成了脑胶质瘤样本基因的荧光标记过程并进行了优化,确定了最佳的扩增时间、标记时间以及模板cDNA、随机引物和Klenow酶等的用量。进而设计了脑胶质瘤辅助诊断用基因芯片的探针序列并将探针点制成阵列,制备了包含240条探针的基因芯片。芯片与标记样品杂交后的荧光扫描结果表明所设计的探针可与标记样品成功杂交,并显示了较好的特异性和灵敏度。3.以低密度聚乙烯薄膜(LDPE)为基材,采用可见光引发接枝技术制备了具有层级结构的蛋白质微阵列。首先在紫外光照射下,异丙基硫杂蒽酮(ITX)通过夺氢-偶联反应将光敏休眠基引入LDPE表面。在可见光照射下休眠基能够断开,产生表面自由基并引发双官能团单体PEGDA的交联聚合,将PEG交联网络结构接枝在基材表面。以丙烯酸钠和PEGDA为混合单体在接枝PEG交联层表面进行二次接枝,红外光谱及图案化接枝的显微镜照片证明一次接枝后的表面仍含有活性的休眠基,可再次引发接枝发生聚合。基于上述特点,利用光掩膜依顺序进行多次接枝的方法,可在基材表面形成具有多层次复杂结构的3D蛋白质微阵列。原子力显微镜高度测试表明不论是没有接枝的LDPE-ITXSP区域还是在第一层接枝区域,第二层图案的增长速率是一致的,可以保证第二层接枝图案的完整性。以此方法为基础,通过加入活性单体成功实现在特定的区域内多层分隔固定不同的蛋白质分子。
[Abstract]:Biochip has the characteristics of micro, automatic, high parallel and high throughput. It has broad application prospects in the fields of clinical diagnosis, new drug screening, individualized medical and bioengineering research. Since the 90s of.20 century, the manufacturing technology and detection methods of biochips have developed rapidly, but they are still faced with a large number of subjects. Big challenges, such as complex preparation process, high cost, and many processing programs resulting in unrepeatability and so on, these problems restrict the wide application of biochip. Developing more advanced biomolecular fixation technology is of great significance for the development of efficient microarray technology. This paper has developed two on the basis of photoinduced surface grafting polymerization. A new method of preparing the three-dimensional graft structure of the surface was prepared, and the three dimensional structure gene chip and the complex structure protein microarray were prepared. The high density and effective immobilization of the DNA probe and protein molecules on the surface of the carrier was realized. The application of the three-dimensional gene chip in the diagnosis of glioma was preliminarily studied. The work and results are as follows: 1. use the commercial carrier slide as the base material, use the vinyl three chlorosilane coupling agent to treat the double bond, and then use the UV grafting method on the double bonding surface with the glycidyl methacrylate (GMA) and the polyethylene glycol two acrylate (PEGDA) as the mixed monomers, to prepare three of the epoxy group with a certain thickness and the surface containing the epoxy group. Using the open ring reaction of the amino group and the epoxy group, the DNA probe labeled by the fluorescent molecule (Cy3) and the end of the amino group is immobilized on the substrate. The fixed efficiency and density of the DNA probe can be adjusted by changing the content of the epoxy group in the three-dimensional cross-linking layer, the thickness of the crosslinking layer and the concentration of the DNA probe sample solution. The immobilization efficiency of the probe was increased from 30% to 80%, and the fixed density was also increased at the same time. The hybridization results of the chip showed that the hybridization of nucleic acid probe molecules and target genes on the surface of the three dimensional substrate was successful and the 3D gene chip technology was applied to the development of the gene chip for the diagnosis of glioma diseases. The fluorescence labeling process of the brain glioma sample gene was completed and optimized. The optimum amplification time, marking time, the template cDNA, the random primer and the Klenow enzyme were determined. Then the probe sequence of the gene chip for glioma assisted diagnosis was designed and the probe points were made into arrays. 240 probes were prepared. The fluorescence scanning results after hybridization with the labeled samples showed that the designed probe could successfully hybridized with the labeled sample, and showed the good specificity and sensitivity.3. with low density polyethylene film (LDPE) as the base material and the first level structure of protein microarray was prepared by visible light initiation grafting technique. Under ultraviolet light, isopropyl thio anthrone (ITX) is introduced to the surface of LDPE by the coupling reaction of hydrogen and hydrogen. Under visible light, the dormant base can be disconnected, the surface free radical is broken, the free radical of the surface is produced and the cross-linking polymerization of the difunctional monomer PEGDA is initiated, and the PEG crosslinking network structure is grafted on the substrate surface. Sodium acrylate and PEGDA are mixed. Two graft copolymers are grafted on the surface of PEG cross linking layer on the surface of the graft. The infrared spectra and patterned grafting microscope photographs prove that the surface of the graft still contains the active dormancy base, which can lead to graft polymerization again. Based on the above characteristics, the multi grafting method of the photomask in sequence can be used to form a lot of material on the substrate surface. 3D protein microarrays with complex hierarchical structures. The atomic force microscopy (AFM) height test shows that the growth rate of the second layer pattern is consistent regardless of the graft LDPE-ITXSP region or the first layer graft region, which can ensure the integrity of the grafted pattern. Based on this method, the active monomer is successfully implemented by adding active monomers. In different regions, different proteins are separated by different layers.
【学位授予单位】：北京化工大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q78;TQ316.312

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