基于电化学检测的DNA分子逻辑门及逻辑运算器的构建

发布时间：2018-03-18 08:37

  本文选题：DNA分子逻辑门　切入点：DNA探针　出处：《宁夏大学》2017年硕士论文　论文类型：学位论文

【摘要】：近年来,以半导体材料为基础的集成电路由于其集成度接近理论的极限,科学家们正在积极开发新型计算机,DNA计算机被认为是最具有发展潜力的计算机。众所周知,计算机的构建以逻辑运算为基础,要建立和发展DNA计算机,分子逻辑门技术是一条不可逾越的必经之路。由于电化学检测方法具有简单、快速、灵敏、选择性好等优点,备受研究者们的青睐。本论文以几种常见食源性致病菌的特征DNA序列和生物小分子为目标物,应用电化学检测技术,提出了一系列DNA分子逻辑门,并且构建了半加器和半减器两种逻辑运算器,为基于DNA分子的逻辑电路提供重要的理论支持,并为核酸和生物小分子的检测提供了可选择的方法。(1)在第一章绪论部分介绍了分子逻辑门及逻辑运算器的相关概念并阐述了国内外研究进展,提出了本轮的研究内容和创新点。(2)在第二章提出了基于目标DNA与双标记探针竞争结合模式构建的“NOR”型DNA分子逻辑门用于沙门氏菌(Sal)DNA和志贺氏菌(Shi)DNA的检测。本实验以二茂铁(Fc)为电化学指示剂,首先利用自组装技术将巯基标记的S1探针修饰在金电极表面,设计两端标记Fc的DP探针和S1探针部分互补杂交。当目标物Sal DNA和Shi DNA任何一个存在时,由于碱基互补配对原则和临近表面杂交作用使DP脱离电极表面。基于目标物存在前后Fc的电化学信号的变化实现对Sal DNA和Shi DNA的高选择性、高灵敏性检测。由实验结果可得,该方法对Sal DNA和Shi DNA的检测范围均在1.00 nmol/L～1000.00 nmol/L之间,检出限分别为0.52 nmol/L和0.72 nmol/L(S/N=3)。该工作以Sal DNA和Shi DNA为输入,以IFc为输出,构建“NOR”型DNA分子逻辑门。(3)在第三章提出了基于Y构型的“OR”型DNA分子逻辑门用于SalDNA和大肠杆菌(E.coli)DNA的检测。本实验以Fc为电化学指示剂,首先将三条相互部分互补杂交的S1、S2和P1探针通过巯基自组装在金电极表面形成Y构型,基于碱基互补配对原则和临近表面杂交反应构建了一个简单、灵敏、特异性检测Sal DNA和E.coli DNA的生物传感器。由实验结果可知,该方法对Sal DNA和E.coli DNA的检测范围均在10.00 nmol/L～1000.00 nmol/L,检出限分别为6.42 nmol/L 和 1.58 nmol/L(S/N=3)。以 Sal DNA 和 E.coli DNA 为输入,以 Fc 信号变化值 △IFc为输出,构建了“OR”型DNA分子逻辑门。(4)在第四章提出了基于发夹结构探针的用于李斯特杆菌(List)DNA和E.coli DNA分析的半加器的构建。本实验以Fc和亚甲基蓝(MB)为电化学指示剂,首先将分别标记了 Fc和MB的发夹结构探针S-1、S-2通过巯基自组装在金电极表面,构建了 DNA生物传感器用于List DNA和E.coli DNA的同时智能分析。S-1和S-2的环部分别和List DNA、E.coli DNA完全互补杂交,当List DNA或E.coli DNA存在时,只有一个发夹结构被打开,List DNA和E.coli DNA都存在时,两发夹结构同时打开。基于目标物存在前后Fc和MB在电极表面位置的变化致使相应的电流发生变化,从而实现对List DNA和E.coliDNA的快速、灵敏、高选择性分析。分析实验结果可得,该方法对List DNA和E.coli DNA的检测范围均在20.00 nmol/L～1000.00 nmol/L,检出限分别为1.98 nmol/L和8.99 nmol/L(S/N=3)。以ListDNA和E.coli DNA作为输入,以两条探针电流变化之和∑△I(∑△I=△IMB+△IFc)和信号之比Y(Y=△IFc/△IMB或△IMB/△IFc)作为输出,同时构建了“AND”型和“XOR”型DNA分子逻辑门。融合这两种逻辑门,构建了具有逻辑运算功能的DNA半加器。(5)在第五章提出了用于分析SalDNA和三磷酸腺苷(ATP)的分子半减器的构建。本实验以Fc和MB为电化学指示剂,首先利用巯基自组装技术将分别将标有Fc和MB的发夹结构探针L-p、ATP的适体AP修饰在金电极表面,AP探针结合了与之完全互补配对C-AP。设计L-p和目标物Sal DNA特异性杂交,当Sal存在时使L-P的环部结构形成刚性双链,ATP的存在可以和AP形成复合物置换出C-AP,拉近MB和电极表面的距离。基于目标物存在前后,Fc和MB与电极表面距离的变化致使相应的电流发生变化,可以实现Sal DNA和ATP的高灵敏性和高特异性分析。分析实验结果可得,SalDNA检测范围为10.00 nmol/L～1000.00 nmol/L,检出限为2.19nmol/L(S/N=3);ATP 的检测范围:10.00nmol/L～1000.00nmol/L,检出限为 3.88nmol/L(S/N=3)。以List DNA和ATP作为输入,以两条探针电流之和ΣI(ΣI=IMB+IFc)、之比Y(Y=△IFc/△IMB或△IMB/△IFc)作为输出,同时构建了“INHIBIT”型和“XOR”型DNA分子逻辑门。融合这两种逻辑门,构建了具有逻辑运算功能的DNA半减器。
[Abstract]:In recent years, based on semiconductor materials and integrated circuits because of its integration is close to the theoretical limit, scientists are actively developing a new type of computer, DNA computer is considered to be the most potential for development of computer. As everyone knows, the computer to construct logic as the basis, to the establishment and development of DNA computer, molecular logic gate technology is the only way which must be passed an impassable. As the electrochemical detection method is simple, rapid, sensitive, good selectivity, has attracted researchers' attention. In this paper, several common foodborne pathogens and biological characteristics of DNA sequence of small molecules as object, application of electrochemical detection technology, put forward a series of DNA molecular logic gate, and construction half adder and half subtractor two logic unit, to provide important theoretical support for logic circuit based on DNA molecules and nucleic acid and biological small Provide alternative method of sub test. (1) in the first chapter introduces the related concepts of molecular logic gate and logic device and describes the study progress at home and abroad, puts forward the research content and innovation points of this round. (2) proposed in the second chapter based on the target of DNA and double labeled probe competition with the model of "NOR" type DNA molecular logic gate (Sal) for Salmonella and Shigella DNA (Shi) DNA detection. In this experiment, two ferrocene (Fc) as the electrochemical indicator, using self assembly technology of S1 probe labeled thiol modified on the electrode surface, both ends of mark design the Fc DP probe and S1 probe complementary hybridization. When the target is Sal DNA and Shi DNA the existence of any one, because of complementary base pairing and near surface hybridization DP from the electrode surface. The electrochemical signal changes before and after the existing object based on Fc To achieve high selectivity for Sal DNA and Shi DNA, high sensitivity detection. From the experimental results, the method of Sal DNA and Shi DNA detection range is between 1 nmol/L and 1000 nmol/L, the detection limits were 0.52 nmol/L and 0.72 nmol/L (S/N=3). The Sal DNA and Shi DNA to work as input in IFc, as output, construction of the "NOR" type DNA molecular logic gate. (3) proposed in the third chapter based on the Y configuration of "OR" type DNA molecular logic gate for SalDNA and Escherichia coli DNA (E.coli) detection. In this experiment, Fc as the electrochemical indicator, the three mutually complementary hybridization S1, S2 and P1 probe Y configuration on the surface of the gold electrode by self-assembled thiol, complementary base pairing and near surface hybridization reaction to construct a simple and sensitive biosensor based on specific detection of Sal, DNA and E.coli DNA. The experimental results show that the method of Sal DNA and E.coli DNA detection range was from 10 nmol/L to 1000 nmol/L, the detection limits were 6.42 nmol/L and 1.58 nmol/L (S/N=3). Sal DNA and E.coli with DNA as input, Fc signal changes in the value of delta IFc as output, the construction of "OR" type DNA molecular logic gate (4) in fourth. Chapter of the hairpin probe for Lester was based on (List) to construct a half adder analysis of DNA and E.coli DNA. In this experiment, Fc and methylene blue (MB) as the electrochemical indicator, first labeled the hairpin probe S-1 Fc and MB S-2, the self-assembled on gold electrode surface. DNA List and E.coli DNA biosensor for DNA and intelligent analysis of ring part.S-1 and S-2 respectively, and List DNA was constructed. E.coli DNA complementary hybridization, List DNA or E.coli DNA when there is only one hairpin structure was opened, List DNA and E.coli DNA There are two, hairpin structure opened at the same time. The target of Fc and MB before and after the change in the position of the electrode surface in the corresponding current changes based on List and E.coliDNA DNA to realize the fast, sensitive, high selective analysis. Analysis of the experimental results, the method of List DNA and E.coli DNA detection range in 20 nmol/L ~ 1000 nmol/L, the detection limits were 1.98 nmol/L and 8.99 nmol/L (S/N=3). The ListDNA and E.coli DNA as input to two probe current changes and I (sigma delta sigma delta I= Delta IMB+ Delta IFc) and the ratio of signal Y (Y= Delta IFc/ delta or delta IMB/ Delta IMB IFc) as the output, and constructs the "AND" and "XOR" type DNA molecular logic gate. The fusion of these two logic gates, with the construction of logic functions DNA half adder. (5) proposed in the fifth chapter for the analysis of SalDNA and adenosine triphosphate (ATP) molecule half subtracter The construction. In this experiment, Fc and MB as electrochemical indicator, hairpin probe L-p first using thiol self-assembled technology will be labeled with Fc and MB, aptamer AP ATP immobilized on the electrode surface, AP probe combined with complementary and matching design of L-p C-AP. and Sal DNA target specific hybridization when the presence of Sal L-P ring structure to form a rigid double chain, the presence of ATP and AP formed a complex replacement of C-AP, between MB and the electrode surface. Based on the distance before and after the object exists, Fc and MB and changes of electrode surface distance in the corresponding current change, analysis can achieve Sal DNA and ATP with high sensitivity and specificity. The analysis of the experimental results, the SalDNA detection range of 10 nmol/L to 1000 nmol/L, the detection limit is 2.19nmol/L (S/N=3); the detection range of ATP: 10.00nmol/L ~ 1000.00nmol/L, the detection limit is 3.88nmol/L (S/N=3). 浠�List DNA鍜孉TP浣滀负杈撳叆,浠ヤ袱鏉℃帰閽堢數娴佷箣鍜屛�I(危I=IMB+IFc),涔嬫瘮Y(Y=鈻矷Fc/鈻矷MB鎴栤柍IMB/鈻矷Fc)浣滀负杈撳嚭,鍚屾椂鏋勫缓浜嗏，

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