基于DNA自组装的纳米金比色检测DNA新方法研究
发布时间：2019-01-09 11:17
【摘要】：DNA是生物体重要的遗传物质,通过对DNA的检测能实现对疾病的早期诊断和预防,不同物种的鉴定和环境的监测保护等,因此,对DNA的检测具有十分重要的意义。本论文以DNA检测为主要目标,从DNA无酶自组装信号放大出发,提出了一种新型无酶的指数式发卡型DNA自组装反应,并研究通过多种方式将该反应应用于DNA检测中。首先,在杂交链式反应的基础上,本论文提出了一种新型的指数式发卡自组装反应,该反应只需要四条发卡型DNA,加入引发链之后,在自由能的驱动下自发进行组装,在较短的时间内生成一个较大的DNA聚合体。通过琼脂糖凝胶电泳验证了该反应的可行性；通过原子力显微镜进一步表征了生成产物的形态。在优化发卡型DNA的浓度和反应时间后,形成了规则的纳米微孔网状结构,孔径分布在200 nin到400 nm之间。该纳米微孔网状结构可以作为一种新型的DNA纳米材料。其次,以该纳米材料作为信号放大的载体,结合荧光成像单分子检测技术和纳米金比色检测技术,将该反应应用于DNA的检测中。实验结果显示纳米金比色检测技术能有效的消除背景信号的影响,实现对DNA的灵敏检测。在最佳反应条件下,靶标浓度在25-10000pM之间时,比色检测结果成良好的线性关系,该检测方法能在15 min内实现肉眼检测25 pM的靶标DNA,该检测限比传统的纳米金比色检测方法降低了约400倍,同时通过紫外可见光分光光度计可检测11.2 pM的靶标DNA。另外,通过对不同错配靶标的检测,证明了该方法对单碱基突变具有良好的特异性。本论文通过DNA自组装构建了一种DNA纳米材料,并将该纳米材料应用于纳米金比色检测DNA中,该检测方法反应迅速,耗时短,灵敏度高,反应结果可通过肉眼直接观察,操作简单,因此可潜在的应用于实时现场快速检测。
[Abstract]:DNA is an important genetic material of organism. The detection of DNA can realize the early diagnosis and prevention of disease, the identification of different species and the monitoring and protection of environment, etc. Therefore, the detection of DNA is of great significance. Based on the amplification of DNA self-assembly signal without enzyme, a new type of exponential DNA self-assembly reaction without enzyme was proposed in this paper, and the reaction was applied to DNA detection in many ways. Firstly, based on the hybrid chain reaction, a novel exponential hairpin self-assembly reaction is proposed. The reaction requires only four hairpin type DNA, to be added to the initiation chain, and then self-assembled under the driving of free energy. A large DNA polymer is formed in a short time. The feasibility of the reaction was verified by agarose gel electrophoresis, and the morphology of the product was further characterized by atomic force microscope (AFM). After optimizing the concentration and reaction time of card type DNA, a regular nano-pore network structure was formed, with pore size ranging from 200 nin to 400 nm. The nanoporous network structure can be used as a new type of DNA nanomaterials. Secondly, the nanomaterials were used as the carrier of signal amplification, combined with fluorescence imaging monolayer detection technology and nano-gold colorimetric detection technology, the reaction was applied to the detection of DNA. The experimental results show that the nanocrystalline gold colorimetry can effectively eliminate the influence of background signal and realize the sensitive detection of DNA. Under the optimum reaction conditions, the results of colorimetric detection show a good linear relationship when the concentration of target is between 25-10000pM. This detection method can detect 25 pM target DNA, with naked eyes within 15 min. The detection limit of this method is about 400 times lower than that of traditional nanocrystalline gold colorimetry, and the target DNA. of 11.2 pM can be detected by UV-Vis spectrophotometer. In addition, the detection of different mismatch targets proves that this method has good specificity for single base mutation. In this paper, a kind of DNA nanomaterials was constructed by DNA self-assembly, and the nanomaterials were applied to the determination of gold nanocrystalline colorimetric DNA. The method is rapid, time-consuming and sensitive, and the reaction results can be observed directly by naked eye. It is easy to operate, so it can potentially be used in real-time field fast 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】：2015
【分类号】：O657.3;TB383.1
本文编号：2405550
[Abstract]:DNA is an important genetic material of organism. The detection of DNA can realize the early diagnosis and prevention of disease, the identification of different species and the monitoring and protection of environment, etc. Therefore, the detection of DNA is of great significance. Based on the amplification of DNA self-assembly signal without enzyme, a new type of exponential DNA self-assembly reaction without enzyme was proposed in this paper, and the reaction was applied to DNA detection in many ways. Firstly, based on the hybrid chain reaction, a novel exponential hairpin self-assembly reaction is proposed. The reaction requires only four hairpin type DNA, to be added to the initiation chain, and then self-assembled under the driving of free energy. A large DNA polymer is formed in a short time. The feasibility of the reaction was verified by agarose gel electrophoresis, and the morphology of the product was further characterized by atomic force microscope (AFM). After optimizing the concentration and reaction time of card type DNA, a regular nano-pore network structure was formed, with pore size ranging from 200 nin to 400 nm. The nanoporous network structure can be used as a new type of DNA nanomaterials. Secondly, the nanomaterials were used as the carrier of signal amplification, combined with fluorescence imaging monolayer detection technology and nano-gold colorimetric detection technology, the reaction was applied to the detection of DNA. The experimental results show that the nanocrystalline gold colorimetry can effectively eliminate the influence of background signal and realize the sensitive detection of DNA. Under the optimum reaction conditions, the results of colorimetric detection show a good linear relationship when the concentration of target is between 25-10000pM. This detection method can detect 25 pM target DNA, with naked eyes within 15 min. The detection limit of this method is about 400 times lower than that of traditional nanocrystalline gold colorimetry, and the target DNA. of 11.2 pM can be detected by UV-Vis spectrophotometer. In addition, the detection of different mismatch targets proves that this method has good specificity for single base mutation. In this paper, a kind of DNA nanomaterials was constructed by DNA self-assembly, and the nanomaterials were applied to the determination of gold nanocrystalline colorimetric DNA. The method is rapid, time-consuming and sensitive, and the reaction results can be observed directly by naked eye. It is easy to operate, so it can potentially be used in real-time field fast 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】：2015
【分类号】：O657.3;TB383.1
【参考文献】
相关期刊论文 前1条
1 陈德强,夏安东;双光子激光扫描荧光显微镜及其应用[J];物理;2000年04期
，本文编号：2405550
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