本文選題：表面等離子體激元共振 + p53蛋白　； 參考：《中南大學(xué)》2010年碩士論文

【摘要】： 表面等離子體激元共振(SPR)是一種基于物質(zhì)相互作用引起芯片表面折射率或厚度變化而進(jìn)行檢測的光學(xué)技術(shù),它是研究生物分子間相互作用的有力工具,具有實(shí)時(shí)、靈敏度高、免標(biāo)記以及樣品無需純化等優(yōu)點(diǎn),在蛋白及核酸等生物分子檢測方面具有廣闊的應(yīng)用前景。本文利用SPR技術(shù)的優(yōu)勢對兩種生物標(biāo)志物p53蛋白和miRNA進(jìn)行了檢測。 首先,利用雙通道SPR技術(shù)實(shí)現(xiàn)了對癌細(xì)胞溶胞液中野生型及總p53蛋白(野生型與突變型p53蛋白的總和)的高靈敏檢測。將捕獲野生型p53的一致性雙鏈DNA以及特異性識別總p53蛋白的單克隆抗體分別組裝到SPR芯片的兩個(gè)通道上,通過流動注射p53蛋白的溶液,從而檢測野生型及總p53蛋白的濃度水平。由于p53蛋白和一致性雙鏈DNA以及單克隆抗體具有高度的親和性,所以可檢測極低濃度的p53蛋白水平。野生型及總p53蛋白所對應(yīng)的SPR信號的差減可直接反應(yīng)不同類型癌細(xì)胞中p53的突變量。同一SPR芯片上野生型與突變型p53蛋白的同時(shí)測定消除了以往不同方法、不同技術(shù)檢測的不確定性。此外,該方法簡單、無需對樣品進(jìn)行標(biāo)記,克服了傳統(tǒng)的酶聯(lián)免疫吸附分析操作步驟煩瑣、且使用酶標(biāo)抗體的缺陷,為臨床上癌癥的預(yù)警及早期診斷提供了理論依據(jù)。 miRNA是一組含有19-25個(gè)核苷酸的非編碼小RNA。利用SPR技術(shù)免標(biāo)記、高靈敏的特點(diǎn),采用類似免疫競爭的方式,即表面固定的miRNA探針與miRNA靶基因及生物素標(biāo)記的miRNA (biotin-miRNA)發(fā)生競爭雜交反應(yīng),隨后流動注射納米金粒子標(biāo)記的抗生素蛋白。生物素與抗生素蛋白之間的特異性相互作用將納米金粒子引入到傳感器表面。隨著miRNA濃度的增加,納米金粒子放大所產(chǎn)生的SPR信號隨之降低,從而建立對miRNA靶基因進(jìn)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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R346

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 汪莎莎;動物性食品中泰樂菌素、泰萬菌素、替米考星殘留檢測研究[D];華中農(nóng)業(yè)大學(xué);2012年

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本文編號：2007857