生物标志物p53与miRNA的表面等离子体激元共振检测

发布时间：2018-06-12 02:26

  本文选题：表面等离子体激元共振 + p53蛋白　； 参考：《中南大学》2010年硕士论文

【摘要】： 表面等离子体激元共振(SPR)是一种基于物质相互作用引起芯片表面折射率或厚度变化而进行检测的光学技术,它是研究生物分子间相互作用的有力工具,具有实时、灵敏度高、免标记以及样品无需纯化等优点,在蛋白及核酸等生物分子检测方面具有广阔的应用前景。本文利用SPR技术的优势对两种生物标志物p53蛋白和miRNA进行了检测。 首先,利用双通道SPR技术实现了对癌细胞溶胞液中野生型及总p53蛋白(野生型与突变型p53蛋白的总和)的高灵敏检测。将捕获野生型p53的一致性双链DNA以及特异性识别总p53蛋白的单克隆抗体分别组装到SPR芯片的两个通道上,通过流动注射p53蛋白的溶液,从而检测野生型及总p53蛋白的浓度水平。由于p53蛋白和一致性双链DNA以及单克隆抗体具有高度的亲和性,所以可检测极低浓度的p53蛋白水平。野生型及总p53蛋白所对应的SPR信号的差减可直接反应不同类型癌细胞中p53的突变量。同一SPR芯片上野生型与突变型p53蛋白的同时测定消除了以往不同方法、不同技术检测的不确定性。此外,该方法简单、无需对样品进行标记,克服了传统的酶联免疫吸附分析操作步骤烦琐、且使用酶标抗体的缺陷,为临床上癌症的预警及早期诊断提供了理论依据。 miRNA是一组含有19-25个核苷酸的非编码小RNA。利用SPR技术免标记、高灵敏的特点,采用类似免疫竞争的方式,即表面固定的miRNA探针与miRNA靶基因及生物素标记的miRNA (biotin-miRNA)发生竞争杂交反应,随后流动注射纳米金粒子标记的抗生素蛋白。生物素与抗生素蛋白之间的特异性相互作用将纳米金粒子引入到传感器表面。随着miRNA浓度的增加,纳米金粒子放大所产生的SPR信号随之降低,从而建立对miRNA靶基因进行定量的分析方法。
[Abstract]:Surface Plasmon Resonance (SPR) is an optical technique based on the change of refractive index or thickness of chip surface caused by material interaction. It is a powerful tool for studying biomolecular interaction. It has real time and high sensitivity. The advantages of free labeling and no purification of samples have a wide application prospect in the detection of protein and nucleic acid and other biomolecules. In this paper, two biomarkers, p53 protein and miRNA, were detected by using the advantages of SPR. Two-channel SPR technique was used to detect wild and total p53 proteins (the sum of wild-type and mutant p53 proteins) in the cytoplasm of cancer cells. The identical double strand DNA of wild type p53 and monoclonal antibody which specifically recognize total p53 protein were assembled into two channels of SPR chip respectively. The concentration of wild type p53 protein and total p53 protein were detected by flow injection of p53 protein solution. Due to the high affinity of p53 protein and conformance double strand DNA and monoclonal antibody, very low concentration of p53 protein can be detected. The decrease of SPR signal corresponding to wild type and total p53 protein can directly reflect the mutation amount of p53 in different types of cancer cells. The simultaneous determination of wild type and mutant p53 protein on the same SPR chip eliminates the uncertainty of different methods and techniques in the past. In addition, the method is simple and does not need to label the sample. It overcomes the disadvantages of the traditional enzyme-linked immunosorbent assay (Elisa) and the use of enzyme-labeled antibodies. MiRNA is a group of small noncoding RNAs containing 19-25 nucleotides. By using SPR technique, which is highly sensitive and highly sensitive, competitive hybridization reaction between the surface fixed miRNA probes and miRNA target genes and biotin-miRNAs labeled with biotin-miRNAs was carried out in a similar way to immune-competitive competition. Then flow injection of gold nanoparticles labeled antibiotic protein. The specific interaction between biotin and antibiotic proteins brings gold nanoparticles onto the surface of the sensor. With the increase of miRNA concentration, the SPR signal produced by the amplification of gold nanoparticles decreased, and a quantitative analysis method of miRNA target gene was established.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R346

【引证文献】

相关硕士学位论文 前1条

1 汪莎莎;动物性食品中泰乐菌素、泰万菌素、替米考星残留检测研究[D];华中农业大学;2012年

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