本文選題：光接枝 + 基因芯片��； 參考：《北京化工大學(xué)》2016年碩士論文

【摘要】：生物芯片具有裝置微型、自動(dòng)、高度并行和高通量等特點(diǎn),在臨床診斷、新藥篩選、個(gè)體化醫(yī)療及生物工程研究等領(lǐng)域有廣闊的應(yīng)用前景。20世紀(jì)90年代以來,生物芯片的制作工藝和檢測(cè)手段已經(jīng)有了飛速發(fā)展,但因涉及學(xué)科多,仍然面臨巨大的挑戰(zhàn)。如制備過程復(fù)雜、成本高,處理程序多導(dǎo)致重復(fù)性不理想等,這些問題制約了生物芯片的廣泛應(yīng)用。研發(fā)更為先進(jìn)的生物分子固定技術(shù),對(duì)于發(fā)展高效的微陣列芯片技術(shù)意義重大。本論文以光引發(fā)表面接枝聚合為基礎(chǔ),發(fā)展了兩種制備表面三維接枝結(jié)構(gòu)的新方法,進(jìn)而制備了三維結(jié)構(gòu)的基因芯片和復(fù)雜結(jié)構(gòu)的蛋白質(zhì)微陣列。實(shí)現(xiàn)了DNA探針和蛋白質(zhì)分子在載體表面的高密度有效固定,并對(duì)三維基因芯片在腦膠質(zhì)瘤診斷中的應(yīng)用進(jìn)行了初步研究。本文主要的研究工作及結(jié)果：1.以商用載玻片為基材,通過乙烯基三氯硅烷偶聯(lián)劑處理引入雙鍵,再在雙鍵化表面以甲基丙烯酸縮水甘油酯(GMA)與聚乙二醇二丙烯酸酯(PEGDA)為混合單體運(yùn)用紫外光接枝法,制備具有一定厚度且表面含有環(huán)氧基團(tuán)的三維基片。利用氨基與環(huán)氧基團(tuán)的開環(huán)反應(yīng),將熒光分子(Cy3)標(biāo)記且末端為氨基的DNA探針固定在基片上。通過改變環(huán)氧基團(tuán)在三維交聯(lián)層中的含量、交聯(lián)層厚度及DNA探針點(diǎn)樣液濃度,可以調(diào)節(jié)DNA探針的固定效率和密度。與傳統(tǒng)的二維基因芯片相比,三維芯片的探針固定效率由30%提高到80%左右,固定密度也同時(shí)提高。芯片的雜交結(jié)果證明三維基材表面可成功實(shí)現(xiàn)核酸探針分子與靶基因的雜交。2.將三維基因芯片技術(shù)應(yīng)用于腦膠質(zhì)瘤疾病輔助診斷用基因芯片的研制。首先設(shè)計(jì)并完成了腦膠質(zhì)瘤樣本基因的熒光標(biāo)記過程并進(jìn)行了優(yōu)化,確定了最佳的擴(kuò)增時(shí)間、標(biāo)記時(shí)間以及模板cDNA、隨機(jī)引物和Klenow酶等的用量。進(jìn)而設(shè)計(jì)了腦膠質(zhì)瘤輔助診斷用基因芯片的探針序列并將探針點(diǎn)制成陣列,制備了包含240條探針的基因芯片。芯片與標(biāo)記樣品雜交后的熒光掃描結(jié)果表明所設(shè)計(jì)的探針可與標(biāo)記樣品成功雜交,并顯示了較好的特異性和靈敏度。3.以低密度聚乙烯薄膜(LDPE)為基材,采用可見光引發(fā)接枝技術(shù)制備了具有層級(jí)結(jié)構(gòu)的蛋白質(zhì)微陣列。首先在紫外光照射下,異丙基硫雜蒽酮(ITX)通過奪氫-偶聯(lián)反應(yīng)將光敏休眠基引入LDPE表面。在可見光照射下休眠基能夠斷開,產(chǎn)生表面自由基并引發(fā)雙官能團(tuán)單體PEGDA的交聯(lián)聚合,將PEG交聯(lián)網(wǎng)絡(luò)結(jié)構(gòu)接枝在基材表面。以丙烯酸鈉和PEGDA為混合單體在接枝PEG交聯(lián)層表面進(jìn)行二次接枝,紅外光譜及圖案化接枝的顯微鏡照片證明一次接枝后的表面仍含有活性的休眠基,可再次引發(fā)接枝發(fā)生聚合�；谏鲜鎏攸c(diǎn),利用光掩膜依順序進(jìn)行多次接枝的方法,可在基材表面形成具有多層次復(fù)雜結(jié)構(gòu)的3D蛋白質(zhì)微陣列。原子力顯微鏡高度測(cè)試表明不論是沒有接枝的LDPE-ITXSP區(qū)域還是在第一層接枝區(qū)域,第二層圖案的增長速率是一致的,可以保證第二層接枝圖案的完整性。以此方法為基礎(chǔ),通過加入活性單體成功實(shí)現(xiàn)在特定的區(qū)域內(nèi)多層分隔固定不同的蛋白質(zhì)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.
【學(xué)位授予單位】：北京化工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q78;TQ316.312

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