【作者】 李伟；

【导师】 李雪梅；

【作者基本信息】 青岛科技大学 ， 有机化学， 2010， 硕士

【摘要】 本论文利用核酸适体与目标的特异结合作用,开展基于核酸适体的DNA生物传感器、分子机器及逻辑门的研究,用于生命活性小分子——三磷酸腺苷、单磷酸腺苷,蛋白质——凝血酶、腺苷脱氨酶的检测。本工作主要研究了基于凝血酶和硫代三聚氰酸-金纳米粒子（TCA-AuNP）网状结构双放大效应的化学发光DNA生物传感器、以蛋白质和小分子作为激发物的双功能DNA分子机器以及以单磷酸腺苷（AMP）和腺苷脱氨酶（AD）为激发物的电致发光逻辑门。本论文共分为五章:第一章,概述了DNA生物传感器的设计原理、分类,介绍了DNA生物传感器的研究现状以及分子机器的研究进展。第二章,研究了凝血酶和硫代三聚氰酸-金纳米粒子网状结构的双重放大作用:首先是凝血酶通过与其适体的特异性结合而标记到PbS纳米粒子上,其次是TCA-AuNP网状结构使每个夹心式结构上能覆盖更多的Au纳米粒子。在此基础利用Luminol-Au³⁺体系进行化学发光检测,在Luminol-Au³⁺体系的最适发光条件下,对靶DNA的检测范围为2.0×10^-16mol/L到3.5×10^-14mol/L,检测限能达到1.0×10^-16mol/L。第三章,研究了以蛋白质和小分子作为激发物的双功能DNA分子机器的研究,六条单链DNA通过碱基互补形成镊子, C和D分别包含两种不同目标适体结构,E和F其两端修饰了电化学发光分子和猝灭基团作为镊子闭合或打开的指示信号。此时镊子处于闭合状态,电化学发光分子和猝灭基团距离比较近,使发光猝灭。当加入核酸适体的目标,使其与C和D链反应,能将后者从分子机器主体上剥离下来,自身变为适体-目标形式的燃烧废物,使得E和F链打开,从而发光增强。当再次加入C和D链时,使其分别与A股和B股的自由碱基杂合配对,就使得这个分子机器呈现关闭状态,这时镊子是闭合的。在这里,目标充当“燃料”的作用,实现其循环工作。核酸适体突破了传统意义上关于核酸只是遗传信息存储和转运载体的认识,利用其结构的多样性可实现类似抗体的分子配体或分子探针的功能。由于适体适用范围广,可用于不具免疫原性的小分子和生物体内不稳定或强毒性的靶标物的检测。第四章,研究了以AMP和AD为激发物的电致发光逻辑门,由于适体具有多样性和复杂性,并且能与其配体发生特异性结合,所以可以用于逻辑门的构建。将修饰了电化学发光分子和猝灭基团的生物大分子、细胞等物质的核酸适体固定在电极上,当加入生物大分子、细胞时,适体结构发生变化,使发光基团与猝灭基团的位置发生变化,从而使逻辑门的输出信号发生变化。本实验设计的逻辑门操作是建立在AMP与其适体的特异性结合以及AD对AMP脱氨基作用的基础上的。加入AMP能与其适体发生特异性结合;加入AD可以催化AMP脱氨基,改变AMP的结构,破坏其与适体的结合。最终,利用电致化学发光分析输出信号。本实验进行了进一步的改进,通过猝灭基团的引入,降低了背景干扰,提高了检测的灵敏度。结论部分,对全文内容进行了总结。更多还原

【Abstract】 This thesis studied on DNA biosensors, molecular machines and logic gates based on nucleic aptamers owing to its specific binding to the targets, which can be used to the detect of bioactive small moleculars—Adenosine triphosphate（ATP） and Adenosine monophosphate（AMP）, proteins—thrombin and Adenosine Deaminase. This present work mainly studied on chemiluminescence DNA biosensors based on dual-amplification of thrombin and thiocyanuric acid-gold nanoparticle network, Logic-Based Dual-Functional DNA Tweezers with Protein and Small Molecules as Mechanical Activators and Eleetrogenerated chemilumineseence logic gate with Adenosine monophosphate AMP and AD as activators.The whole thesis can be divided into five chapters:Chapter 1 This chapter introduced the design principle and classification of DNA biosensors, and also introduced the present research of the DNA biosensor and the recent advances in the study of molecular machines. Chapter 2 The study on chemiluminescence DNA biosensors based on dual-amplification of thrombin and thiocyanuric acid-gold nanoparticle network.This thesis studied the dual-amplification efforts of thrombin and thiocyanuric acid-gold nanoparticle network, first, the thrombin labeled on the PbS NPs through the specific interaction with its aptamers; second, TCA–AuNP network introducing more AuNPs capped for each sandwich format. The CL detection of DNA was taken out with the use of Luminol-Au³⁺ system. On the optimum of Luminol-Au³⁺ system, the target DNA can be detected in the range of 2.0×10^-16M to 3.5×10^-14M, with a limit of detection （LOD） as low as 1.0×10^-16 M.Chapter 3 The study on Logic-Based Dual-Functional DNA Tweezers with Protein and Small Molecules as Mechanical Activators. The tweezers contained two symmetrical components, and six DNA strands （A to F） were hybridized to form the closed state. In strands C and D, there is a 8-base recognition region that is complementary to its aptamer. Strand E and F are labelled at the 5’ and 3’ ends with fluorophore and its quencher as functional components. In the closed state the fluorophore and quencher are close to each other and fluorophore can be quenched efficiently. In the strands C and D, there is a recognition region that is complementary to its input, driving the tweezers to open state and releaseing two inert double-stranded waste products. So the two strands E and F are far away from each other and the quench efficiency is low, so the CL signal increased in intensity relative to that for the closed state. When the strands C and D were added again, they complementary with strands A and B, driving the tweezer to closed state. In this process, the target moleculars were served as the fuel to achieve the cycling operation of the tweezer. The nucleic aptamers changed the traditional recognation of nucliec acid, which was used to storage and carry the genetic information. Due to their diversified structures, aptamers can achieve the similar fuction as the ligands of antibody and molecular probes. The aptamers have many potential applications, such as the detection of small moleculars without immunization and the targets which are unstable or toxic.Chapter 4 The study on Eleetrogenerated Chemilumineseence logic gate with AMP and AD as activators. Nucleic acid aptamers are important matrrials for the construction of logic gate, because their divesity and complexity. They can bind specifically with their ligands. The nucleic aptamers labelled with fluorophore and quencher were immoblized on the electrodes. When the targets were added in, the distance between fluorophore and quencher was changed, so the output signals of the logic gate also changed, The logic gate designed in this thesis was based on specific recognization of AMP to its aptamer and the deamation of AD. When AMP was added, it binded with its aptamer spcifically; the addition of AD catalytics the deamation of AMP, which changed the structure of AMP, therefor, the AMP would not bind with its aptamer. Finally, the output signal of the logic gate was detected by Eleetrogenerated chemilumineseence analysis. A further improvment was taken out by the introduction of the quencher. By this way, the backgroud noise was decreased and the sensitivity was improved efficiently. Chapter 5 was the conclusion of the whole work.更多还原