基于酶促循环放大反应及纳米材料检测ATP和microRNA

发布时间：2017-12-28 05:10

本文关键词：基于酶促循环放大反应及纳米材料检测ATP和microRNA　出处：《青岛科技大学》2017年硕士论文　论文类型：学位论文

【摘要】：恶性肿瘤,也就是人们所说的“癌症”,是严重危害人体健康的疾病之一。如今,随着癌症研究的不断深入,癌症的检测手段也越来越多,其中对肿瘤标志物的检测显的至关重要。肿瘤标志物是恶性肿瘤细胞产生的异常物质,或者是宿主受到肿瘤的刺激而产生的物质,肿瘤标志物的检测对癌症的确诊具有很大的辅助作用。为了检测肿瘤标志物,生物传感器作为一种先进的检测方法,在生化分析方面得到广泛应用和迅速发展。本文就是在碱基互补配对原则、DNA酶的催化性能以及纳米材料的特殊性质基础上,构造出多个DNA生物传感器,来检测恶性肿瘤细胞中的肿瘤标志物,实现了肿瘤相关基因和生物分子的高选择性、高灵敏性检测。主要研究内容包括以下几个方面:1、基于ATP与ATP适体的特异性结合、Klenow聚合酶和Exo III外切酶催化的酶促循环放大反应以及聚多巴胺纳米微球的特殊性能来检测ATP。设计了一个聚多巴胺纳米微球生物传感器检测ATP的方案,反应包括两次酶促循环放大过程。靶标ATP存在时,可以和适体进行特异性结合,使Reporter DNA链游离下来,并在Klenow聚合酶的催化作用下发生循环放大反应,产生更多的Reporter DNA链,Reporter DNA链可以与吸附在聚多巴胺纳米微球上修饰了FAM荧光基团的DNA链杂交,使其脱离聚多巴胺纳米微球的表面,荧光得到恢复,后经过Exo III外切酶催化的循环放大反应,产生更多脱离聚多巴胺纳米微球表面的荧光基团,最终对反应体系的荧光进行分析检测,达到高选择性、高灵敏性检测ATP的目的。2、基于Klenow聚合酶催化的酶促循环放大反应和金纳米粒子的特殊性能可视化的检测ATP。靶标ATP存在时,可以和适体进行特异性结合,使Linker DNA链游离下来,并在Klenow聚合酶的催化作用下发生循环放大反应,产生更多的Linker DNA链,Linker DNA链可以作为桥梁链,通过DNA链间的杂交作用将分别修饰有两种不同DNA链的金纳米粒子连接起来,引起金纳米粒子聚沉,颜色产生由红到紫的变化,达到可视化检测ATP的目的。3、基于Klenow聚合酶和Nb.BsmI内切酶催化的酶促循环放大反应以及分子信标实现高灵敏的检测micro RNA-21。反应包括两次酶促循环放大过程。当靶标microRNA-21存在时,可以引发Klenow聚合酶催化的酶促循环放大反应,产生的特殊DNA链可以进一步与分子信标杂交,产生Nb.BsmI内切酶的酶切位点,引发Nb.BsmI内切酶催化的酶促循环放大反应,分子信标中的荧光基团和猝灭基团被分离,荧光得到恢复,最终通过检测反应体系的荧光,达到高灵敏检测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.
【学位授予单位】：青岛科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R730.4;TP212.3;O657.3

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