基于核酸酶的高灵敏度和高选择性的铀的生物传感方法研究

发布时间：2018-03-17 01:22

  本文选题：铀　切入点：DNA酶　出处：《南华大学》2015年硕士论文　论文类型：学位论文

【摘要】：铀作为核工业发展的主要原材料,在利用过程中会带来放射性核素污染问题,由此带来的危害主要表现在两个方面:化学毒性和辐射毒性。化学毒性表现在对肾脏、免疫系统、神经系统、生殖生育等影响,辐射毒性则是DNA致突和致癌作用。铀的现行检测方法中,存在检测仪器昂贵,或是检测限高、干扰较大等问题。所以,建立简便、选择性好、灵敏度高的检测方法对于环境中铀污染物的有效监测、防患于未然是极其重要的。本文第二章建立了核酸外切酶III辅助底物碎片循环信号放大超灵敏检测铀酰离子的新方法。铀酰离子能特异性地与DNA酶作用,诱导DNA酶的构象发生变化,释放出ss DNA。加入MB之后,游离出的ss DNA与分子信标杂交形成双链,加入ExoⅢ后,外切酶Ⅲ对特定识别位点进行剪切,打开的MB被剪切为单个碱基,而ss DNA不被剪切并与下一个MB杂交,引起二次循环裂解,导致荧光信号积聚,经过多次循环,可使荧光信号放大。在优化出的最佳实验条件下,铀酰离子浓度范围为8.1×10-12mol/L~9.5×10-11mol/L时,体系的荧光变化值呈现较好的线性关系,线性回归方程为ΔF=162.3c(10-11mol/L)+22.01,相关系数r=0.990。根据空白管的标准偏差Sb和标准曲线的斜率k算出LOD为2.4p M。该方法选择性好,灵敏度高于迄今为止已报道的方法。本文第三章建立了非标记DNAzyme构象改变荧光法检测铀酰离子的新方法。实验表明,在p H 5.5 MES缓冲溶液中,当体系中不存在铀酰离子时,SYBR GreenⅠ(SG)能通过嵌入和小沟结合方式与脱氧核酶作用,导致荧光增强;当铀酰离子存在时,DNAzyme的r A碱基处断裂,游离出单链,此时SG与单链DNA作用减弱,荧光信号降低,且体系的荧光强度变化值与铀酰离子浓度在1.73×10 9~4.40×10 8 mol/L范围内呈良好线性关系,线性回归方程为ΔF=108.99c(×10-8mol/L)+79.22,相关系数r=0.990。根据空白管的标准偏差Sb和标准曲线的斜率k算出LOD为5.2×10 10 mol/L。该方法操作简单、选择性好,灵敏度高。本文第四章基于DNAzyme-MB-GO系统,提出了检测铀酰离子的生物传感策略。研究表明,铀酰离子能特异性地与DNAzyme作用,诱导DNA酶的构象发生变化,释放出单链DNA(ss DNA),加入单标记的分子信标(MB),游离出的ss DNA与MB杂交形成双链,随后加入氧化石墨烯(GO),此时未被杂交的分子信标将会被氧化石墨烯吸附,导致荧光背景信号降低。铀酰离子浓度范围为1.35×10 9~2.5×10 8 mol/L和1.03×10 8~1.75×10 7 mol/L时,体系的荧光变化值呈现较好的线性关系,线性回归方程分别为ΔF=20.19c(×10-8 mol/L)+0.13和ΔF=26.59c(×10-7mol/L)+57.36;相关系数分别为r=0.980和r=0.993。根据空白管的标准偏差Sb和标准曲线的斜率k算出LOD为4.06×10 10mol/L。
[Abstract]:Uranium, as the main raw material for the development of the nuclear industry, will bring about the problem of radionuclide contamination in the process of utilization. The harm caused by it is mainly manifested in two aspects: chemical toxicity and radiation toxicity. Chemical toxicity is manifested in the kidney and immune system. The effects of nervous system, reproduction and fertility, radiation toxicity are caused by DNA and carcinogenicity. Among the current methods for the detection of uranium, there are some problems, such as expensive detection instruments, high detection limits, large interference, etc. Therefore, it is easy to establish, good selectivity, and so on. Highly sensitive methods for the effective monitoring of uranium contaminants in the environment, In the second chapter, a new method for the detection of uranyl ion by amplification of cyclic signal of nucleic acid exonuclease (III) assisted substrate fragment is established. Uranyl ion can specifically interact with DNA enzyme. The conformation of DNA was induced to change, and ss DNA was released. After adding MB, the free ss DNA was hybridized with molecular beacon to form double strands. After adding Exo 鈪，

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