本文關(guān)鍵詞：基于酶促循環(huán)放大反應(yīng)及納米材料檢測ATP和microRNA　出處：《青島科技大學》2017年碩士論文　論文類型：學位論文

  更多相關(guān)文章： ATP microRNA-21 酶促循環(huán)放大反應(yīng) 聚多巴胺納米微球 金納米粒子

【摘要】：惡性腫瘤,也就是人們所說的“癌癥”,是嚴重危害人體健康的疾病之一。如今,隨著癌癥研究的不斷深入,癌癥的檢測手段也越來越多,其中對腫瘤標志物的檢測顯的至關(guān)重要。腫瘤標志物是惡性腫瘤細胞產(chǎn)生的異常物質(zhì),或者是宿主受到腫瘤的刺激而產(chǎn)生的物質(zhì),腫瘤標志物的檢測對癌癥的確診具有很大的輔助作用。為了檢測腫瘤標志物,生物傳感器作為一種先進的檢測方法,在生化分析方面得到廣泛應(yīng)用和迅速發(fā)展。本文就是在堿基互補配對原則、DNA酶的催化性能以及納米材料的特殊性質(zhì)基礎(chǔ)上,構(gòu)造出多個DNA生物傳感器,來檢測惡性腫瘤細胞中的腫瘤標志物,實現(xiàn)了腫瘤相關(guān)基因和生物分子的高選擇性、高靈敏性檢測。主要研究內(nèi)容包括以下幾個方面:1、基于ATP與ATP適體的特異性結(jié)合、Klenow聚合酶和Exo III外切酶催化的酶促循環(huán)放大反應(yīng)以及聚多巴胺納米微球的特殊性能來檢測ATP。設(shè)計了一個聚多巴胺納米微球生物傳感器檢測ATP的方案,反應(yīng)包括兩次酶促循環(huán)放大過程。靶標ATP存在時,可以和適體進行特異性結(jié)合,使Reporter DNA鏈游離下來,并在Klenow聚合酶的催化作用下發(fā)生循環(huán)放大反應(yīng),產(chǎn)生更多的Reporter DNA鏈,Reporter DNA鏈可以與吸附在聚多巴胺納米微球上修飾了FAM熒光基團的DNA鏈雜交,使其脫離聚多巴胺納米微球的表面,熒光得到恢復,后經(jīng)過Exo III外切酶催化的循環(huán)放大反應(yīng),產(chǎn)生更多脫離聚多巴胺納米微球表面的熒光基團,最終對反應(yīng)體系的熒光進行分析檢測,達到高選擇性、高靈敏性檢測ATP的目的。2、基于Klenow聚合酶催化的酶促循環(huán)放大反應(yīng)和金納米粒子的特殊性能可視化的檢測ATP。靶標ATP存在時,可以和適體進行特異性結(jié)合,使Linker DNA鏈游離下來,并在Klenow聚合酶的催化作用下發(fā)生循環(huán)放大反應(yīng),產(chǎn)生更多的Linker DNA鏈,Linker DNA鏈可以作為橋梁鏈,通過DNA鏈間的雜交作用將分別修飾有兩種不同DNA鏈的金納米粒子連接起來,引起金納米粒子聚沉,顏色產(chǎn)生由紅到紫的變化,達到可視化檢測ATP的目的。3、基于Klenow聚合酶和Nb.BsmI內(nèi)切酶催化的酶促循環(huán)放大反應(yīng)以及分子信標實現(xiàn)高靈敏的檢測micro RNA-21。反應(yīng)包括兩次酶促循環(huán)放大過程。當靶標microRNA-21存在時,可以引發(fā)Klenow聚合酶催化的酶促循環(huán)放大反應(yīng),產(chǎn)生的特殊DNA鏈可以進一步與分子信標雜交,產(chǎn)生Nb.BsmI內(nèi)切酶的酶切位點,引發(fā)Nb.BsmI內(nèi)切酶催化的酶促循環(huán)放大反應(yīng),分子信標中的熒光基團和猝滅基團被分離,熒光得到恢復,最終通過檢測反應(yīng)體系的熒光,達到高靈敏檢測microRNA-21的目的。
[Abstract]:Malignant tumor, which is called "cancer", is one of the diseases that seriously harm the health of the human body. Nowadays, with the development of cancer research, there are more and more detection methods for cancer, and the detection of tumor markers is very important. Tumor markers are abnormal substances produced by malignant tumor cells, or substances produced by stimulation of tumors. The detection of tumor markers plays a great role in the diagnosis of cancer. In order to detect tumor markers, biosensors, as an advanced detection method, have been widely used and developed rapidly in biochemical analysis. This paper is on the catalytic performance of complementary base pairing principle, DNA enzyme and the special properties of nano materials on the basis of using a DNA biosensor to detect malignant tumor cells in tumor markers, to achieve a high selectivity and high sensitivity detection of tumor related genes and biological molecules. The main contents include the following aspects: 1, based on ATP and ATP, combined with specific Klenow polymerase and Exo III exonuclease catalyzed enzymatic amplification reaction and special performance of circulating polydopamine nanoparticles to detect ATP. A dopamine nano microsphere biosensor was designed for the detection of ATP, and the reaction included two cycles. The target ATP, and aptamers specific binding to Reporter, DNA chain free down, and occurs in the catalytic action of Klenow polymerase under cyclic amplification reaction, generate more Reporter DNA chain, Reporter chain and DNA chain DNA can be adsorbed on the hybrid nanoparticles on poly dopamine modified FAM fluorophore. The surface from the polydopamine nanoparticles, fluorescence recovery after Exo III exonuclease catalytic cycle amplification reaction, generate more from the fluorophore polydopamine nanoparticles on the surface of the final fluorescence on the reaction system of detection, high selectivity and high sensitivity to the detection of ATP. 2. The Klenow polymerase catalyzed enzymatic cyclic amplification reaction and the specific performance of gold nanoparticles were visualized for the detection of ATP. The target ATP, and aptamers specific binding to Linker, DNA chain free down, and occurs in the catalytic action of Klenow polymerase under cyclic amplification reaction, generate more Linker DNA chain, Linker DNA can be used as chain bridge chain, through hybridization between DNA chains were modified with two different the DNA chain of gold nanoparticles connected by gold nanoparticles aggregation, color changes from red to purple, to achieve the visual detection of ATP to. 3. High sensitivity detection of micro RNA-21 based on Klenow polymerase and Nb.BsmI endonuclease catalyzed cyclic amplification and molecular beacon. The reaction includes two times of enzymatic cyclic amplification. When the target microRNA-21, can trigger Klenow polymerase catalyzed enzymatic cycling amplification reaction, the special DNA chain can be further hybridized with molecular beacon, Nb.BsmI endonuclease cleavage sites, Nb.BsmI enzyme catalytic triggered enzymatic cycling amplification reaction, fluorescent molecular beacon and the fluorescence quenching group was isolated, recovered finally, through the fluorescence detection system, to achieve the purpose of microRNA-21 high sensitive detection.
【學位授予單位】：青島科技大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R730.4;TP212.3;O657.3

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