基于纳米金的新型荧光法检测DNA

发布时间：2018-03-08 05:33

  本文选题：纳米金　切入点：DNA　出处：《山东大学》2011年硕士论文　论文类型：学位论文

【摘要】：基因是遗传的物质基础,控制着人类的生老病死。基因检测对一些疾病的诊断和预防具有重要的意义。乳腺癌是女性最常见的恶性肿瘤之一,严重威胁着女性的健康。乳腺癌易感基因(BRCA1)在乳腺细胞周期的调控、基因转录调节和维持基因稳定性中起着重要的作用。BRCA1的突变则会削弱这些功能,从而增加乳腺癌发病的危险度。因此对该基因进行检测对乳腺癌的早期诊断和癌症预测具有重要的意义。 近年来,纳米材料的迅速发展为分子生物学提供了新的途径。纳米材料的特殊性质如表面效应、小尺寸效应、量子效应和宏观量子隧道效应等使其在疾病诊断、环境监控、药物研究及法医鉴定等领域起着重要的作用。纳米金作为一种重要的纳米材料,在DNA的检测中被作为检测探针、固定载体和荧光猝灭剂广泛使用。 方法的灵敏度和简便性是DNA检测中的重要部分。本论文建立了两种不同的DNA荧光检测方法,两种方法均利用单双链DNA在纳米金表面不同的吸附性质和纳米金作为荧光猝灭剂的特性,探索提高DNA检测灵敏度和实现快速检测的新途径。 论文共分为三个部分： 一、概述了DNA的结构及常见的检测方法。着重介绍了DNA荧光探针、纳米金的特性、合成方法及其在DNA检测中的应用。 二、建立了一种基于纳米金猝灭罗丹明B荧光的免标记法检测DNA。该方法同时利用了单双链DNA在纳米金表面的不同吸附性质和不同聚集程度的纳米金对染料猝灭能力不同的性质,基于靶目标的加入引起的体系荧光信号的增强,实现了DNA的高灵敏检测。在最佳实验条件下,该方法的检出限为2.9×10-13 molL-1。该方法无需进行任何标记,检测过程简单快速。 三、建立了一种基于纳米金和核酸外切酶Ⅲ的循环荧光放大法检测DNA。该方法同样将纳米金作为猝灭剂,不仅利用了单双链DNA在纳米金表面不同的吸附性质,同时还引入了核酸外切酶Ⅲ对双链DNA的水解作用实现了荧光信号的放大,从而提高DNA检测的灵敏度。一方面,纳米金的引入简化了降低体系的背景信号的过程；另一方面在核酸外切酶的作用下,靶目标被释放后重新参与杂交,使得体系的荧光信号从恢复到增强,实现DNA的信号放大检测。在最佳实验条件下,在2.5×10-11mol L-1～1.0×10-9mol L-1的浓度范围内,体系的△F值和目标DNA的浓度呈现良好的线性关系,检测限为1.0×10-11mol L-1。
[Abstract]:Gene is the material basis of heredity and controls the birth, old and death of human beings. Gene detection is of great significance in the diagnosis and prevention of some diseases. Breast cancer is one of the most common malignant tumors in women. Breast cancer susceptibility gene BRCA1) plays an important role in the regulation of breast cell cycle, gene transcription regulation and maintaining gene stability. The mutation of BRCA1 weakens these functions. Therefore, detection of this gene is of great significance for early diagnosis and cancer prediction of breast cancer. In recent years, the rapid development of nanomaterials has provided a new way for molecular biology. The special properties of nanomaterials such as surface effect, small size effect, quantum effect and macroscopic quantum tunneling effect make them in disease diagnosis and environmental monitoring. As an important nanomaterial, gold nanoparticles are widely used as probe, stationary carrier and fluorescence quenching agent in the detection of DNA. The sensitivity and simplicity of the method is an important part of DNA detection. In this paper, two different DNA fluorescence detection methods were established. The two methods use the different adsorption properties of single and double stranded DNA on the surface of gold nanoparticles and the characteristics of gold nanoparticles as fluorescence quenching agents to explore a new way to improve the detection sensitivity of DNA and to realize rapid detection. The thesis is divided into three parts:. The main contents are as follows: 1. The structure and common detection methods of DNA are summarized, especially the fluorescent probe of DNA, the characteristics of nanocrystalline gold, the synthetic method and its application in the detection of DNA. II. An immunolabelling method based on the fluorescence quenching of Rhodamine B by nanocrystalline gold was developed. The different adsorption properties of single and double stranded DNA on the surface of gold nanoparticles and the different properties of the quenching ability of gold nanoparticles with different aggregation degree on the dye surface were used in this method. The high sensitivity detection of DNA is realized based on the enhancement of fluorescence signal caused by the addition of target. Under the optimum experimental conditions, the detection limit of this method is 2.9 脳 10 ~ (-13) mol ~ (-1) mol 路L ~ (-1). This method does not need any labeling, and the detection process is simple and rapid. Thirdly, a cyclic fluorescence amplification method based on nanocrystalline gold and nucleic acid exonuclease 鈪，

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