二氧化硅包覆的量子点荧光编码微球用于G4-DNA多元检测研究

发布时间：2018-08-26 21:27

【摘要】：G-四联体(G4-DNA)是DNA的一种特殊二级结构。它是一条富含G碱基的短链,4个G碱基通过Hoogsteen氢键自组装成G-四分体,多个G-四分体通过π-π 堆积作用形成G-四联体。人类基因组中含有高达376000个G4-DNA结构,广泛的存在于染色体端粒区域、基因的启动子区域、免疫蛋白开关区域以及外显子区域当中。G4-DNA结构参与了诸如细胞的增殖、基因的表达与调控、蛋白质的翻译等生物功能,因此对G4-DNA的高灵敏度、高准确度、快速的分析与相关疾病的诊断方法具有重要的研究意义。近年来,荧光编码微球技术取得了很大的进展,可广泛应用于核酸、蛋白质、糖类等生物组分的高通量、高灵敏度、快速的分析。其中量子点荧光编码微球既利用了量子点优良的光学性质,又使得编码更为多样解码更为方便。然而在大多数情况下其检测灵敏度还无法达到临床分析的要求,需要进一步集成信号放大技术。滚环扩增是一种高效的核酸检测信号放大方法,具有成本较低、操作简单、重复性好和无需改变温度的优点。本论文通过在二氧化硅包覆的量子点荧光编码微球表面进行滚环扩增反应,实现了对人类基因组不同区域的G4-DNA的高灵敏、多元检测。论文主要研究内容如下:1.使用高温热注射法合成了发射峰分别为513 nm(绿色)和582 nm(橙色)的CdSe@ZnS量子点。使用分散聚合法合成了聚苯乙烯微球种子,在此基础上使用种子聚合法合成了 PSDM介孔微球。使用“溶胀-挥发”法包裹橙色和绿色两种量子点形成量子点荧光编码微球。利用Stober法制备了二氧化硅包覆的量子点编码微球(Qbead@Si02)。所构建的Qbead@Si02具有优良的荧光稳定性。2.发展出了 Qbead@Si02-RCA分析法用于G4-DNA的高灵敏检测,检测限达到XXfM。该分析法的主要操作步骤包括:通过羰基二亚胺反应在Qbead@Si02-COOH表面偶联capture probe序列,通过杂交固定Padlock DNA序列;靶标G4-DNA与Padlock DNA杂交,PadlockDNA在T4连接酶作用下封闭成环,并进一步在酶催化作用下复制扩增;ZnPc对RCA产物进行染色;通过流式细胞术对ZnPc荧光信号进行定量检测。3.基于三个Qbead@Si02荧光编码,利用Qbead@Si02-RCA分析法进一步实现了对三种G4-DNA序列的同时检测,检测特异性较好。
[Abstract]:G-quadruple (G4-DNA) is a special secondary structure of DNA. It is a short chain rich in G bases. Four G bases are self-assembled into G- tetrads by Hoogsteen hydrogen bonds, and many G- tetrads form G- tetrads by 蟺-蟺 stacking. The human genome contains as much as 376,000 G4-DNA structures, which are widely found in the telomere region of the chromosome, the promoter region of the gene, the switching region of the immune protein and the exon region, in which the. G4-DNA structure is involved in, for example, cell proliferation. Gene expression and regulation, protein translation and other biological functions, so G4-DNA high sensitivity, high accuracy, rapid analysis and diagnosis of related diseases have important significance. In recent years, fluorescent coding microspheres have made great progress and can be widely used in high throughput, high sensitivity and fast analysis of biological components such as nucleic acid, protein, sugar and so on. The quantum dot fluorescence coding microspheres not only make use of the excellent optical properties of quantum dots, but also make it more convenient to encode various codes and decode. However, in most cases, the detection sensitivity can not meet the requirements of clinical analysis, and further integration of signal amplification technology is needed. Rolling amplification is an efficient amplification method for nucleic acid detection signal, which has the advantages of low cost, simple operation, good repeatability and no need to change temperature. In this paper, the high sensitivity and multivariate detection of G4-DNA in different regions of the human genome was achieved by using a roller-ring amplification reaction on the surface of silica coated quantum dot fluorescence coded microspheres. The main contents of this paper are as follows: 1: 1. CdSe@ZnS quantum dots with emission peaks of 513 nm (green) and 582 nm (orange) were synthesized by high temperature thermal injection. The seeds of polystyrene microspheres were synthesized by dispersion polymerization, and PSDM mesoporous microspheres were synthesized by seed polymerization. Two kinds of quantum dots, orange and green, were encapsulated by "swelling and volatilization" method to form fluorescent coded microspheres of quantum dots. SiO2 coated quantum dot coded microspheres (Qbead@Si02) were prepared by Stober method. The constructed Qbead@Si02 has excellent fluorescence stability. 2. 2. A highly sensitive detection method for G4-DNA by Qbead@Si02-RCA analysis was developed, and the detection limit was up to XXfM.. The main operation steps of this method include: coupling capture probe sequence on Qbead@Si02-COOH surface by carbonyl diimide reaction, fixing Padlock DNA sequence by hybridization, and closing the ring of target G4-DNA and Padlock DNA hybridization Padlock DNA under the action of T4 ligase. Furthermore, the RCA products were stained with RCA products and the fluorescence signals of ZnPc were quantitatively detected by flow cytometry. Based on the three Qbead@Si02 fluorescence codes, the detection of three kinds of G4-DNA sequences at the same time was further realized by Qbead@Si02-RCA analysis, and the detection specificity was good.
【学位授予单位】：东南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R318

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