纳米生物界面精确调控及在生物传感中的应用

发布时间：2018-03-11 00:00

本文选题：纳米生物界面　切入点：静态调控　出处：《中国科学院研究生院(上海应用物理研究所)》2016年博士论文　论文类型：学位论文

【摘要】：纳米生物界面精确调控可以很好地实现器件的构建并发挥其良好的生物传感性能,DNA纳米技术基于碱基互补配对的特性可以很好地实现对距离、结构、大小和构象的精确调控。本研究主要基于DNA纳米技术构建了一系列可以精确调控的纳米生物界面并利用其实现了对目前缺乏有效检测手段的前列腺癌靶标小分子的精确检测。最后在构建纳米生物界面的基础上,实现了对细胞表面的精确组装并成功地实现了可视化定量检测乳腺癌细胞。具体内容如下:一、对纳米生物界面进行了静态结构的构建和调控,利用DNA纳米技术的可编辑性同时引入DNA折纸技术构建了二维(2D)的纳米生物界面,并运用该界面实现了对两种生物大分子之间距离的精确调控,可以从10nm到70nm不等。在2D纳米生物界面构建基础上,我们又利用DNA四面体技术构建了三维(3D)的纳米生物界面,实现了对生物大分子从准纳米到纳米距离的精确调控。在成功实现对组装分子从2D到3D的距离调控基础上,又设计了五种不同构象的四面体,构建了五种不同构象的纳米生物界面并发现不同构象的纳米生物界面确实会影响其生物传感性能。二、在成功实现对纳米生物界面的静态调控后,我们又对纳米生物界面实现了动态的调节。借鉴生物酶研究中的别构效应,基于静态构象调控的基础,引入带有茎环结构的四面体并通过加入刺激因子,包括响应链(effector)、抑制链、p H、杂交时间等调控四面体构象改变从而实现对纳米生物界面的动态调控。同时发现纳米生物界面的动态调控可以影响相同探针和靶序列的结合能力从而影响生物传感性能,我们利用构象改变导致的探针和靶序列结合能力的不同实现了对靶序列捕获和释放的精确调控。三、在成功实现精确调控纳米生物界面从2D到3D、从静态到动态、从距离到构象改变的基础上,利用设计的纳米生物界面来构建生物传感器。肌氨酸作为前列腺癌检测的潜在靶标小分子由于分子量小、体内含量低等特点导致目前仍然缺乏高灵敏度、高特异性的手段来检测。我们利用肌氨酸氧化酶、辣根过氧化物酶在肌氨酸存在时可以发生级联反应从而提高检测的特异性,并且运用构建的纳米生物界面可以精确调控酶之间的距离,同时结合电化学技术实现了对肌氨酸的高灵敏检测,检测限可以达到50n M,最后运用该方法进行了大量前列腺癌、前列腺肿大、正常男性血清样本的检测。四、在纳米生物界面构建技术基础上,我们利用DNA适配体和乳腺癌细胞MCF-7表面高表达的上皮细胞粘附因子(Ep CAM)结合,实现了在细胞界面组装DNA的技术。然后利用DNA杂交技术将引发链杂交到细胞表面,这样带有引发链的细胞就可以引发3D的杂交链式反应从而形成DNA水凝胶,在这过程中加入DNA保护的纳米金就可以实现对癌细胞定量、可视化的检测。
[Abstract]:The precise regulation of nanoscale biological interface can realize the construction of devices and give full play to their good biosensor performance. DNA nanotechnology based on the characteristics of base complementary pairing can well realize the distance and structure of the devices. In this study, a series of accurately regulated nanoscale biological interfaces were constructed based on DNA nanotechnology and used to realize the detection of prostate cancer target small molecules that lack effective detection methods. Finally, based on the construction of nanoscale biological interface, The precise assembly of the cell surface and the visual quantitative detection of breast cancer cells have been successfully realized. The main contents are as follows: firstly, the static structure of the nanoscale biological interface is constructed and regulated. Using the editable property of DNA nanotechnology and the introduction of DNA origami technology, the two-dimensional biologic interface was constructed, and the precise control of the distance between the two biomolecules was realized by the interface. It can range from 10 nm to 70 nm. On the basis of 2D nanoscale biological interface construction, we have used DNA tetrahedron technology to construct three dimensional (3D) nanoscale biological interface. On the basis of the successful distance control of the assembled molecules from 2D to 3D, five different conformation tetrahedrons have been designed. Five nanoscale biological interfaces with different conformations were constructed, and it was found that different conformational nanoscale biological interfaces did affect their biosensor performance. Secondly, after the static regulation of nanoscale biological interfaces was successfully realized, Based on the static conformational regulation, the tetrahedron with stem ring structure was introduced and the stimulant factor was added. It includes response chain effector, inhibition of chain Hand, hybridization time and so on to control tetrahedron conformation to realize dynamic regulation of nano-biological interface. At the same time, it is found that the dynamic regulation of nano-biological interface can affect the same probe and target sequence. Binding ability to affect biosensor performance, We make use of the different binding ability of probe and target sequence caused by conformation change to realize the accurate regulation of target sequence capture and release. Third, in the successful implementation of precise regulation of nano-biological interface from 2D to 3D, from static to dynamic, Based on the distance to conformation change, the biosensor was constructed by using the designed nanoscale biological interface. Creatine is used as a potential target for prostate cancer detection because of its small molecular weight. Because of the low content in the body, there is still a lack of high sensitivity and high specificity for detection. We use sarcosine oxidase and horseradish peroxidase to produce cascade reaction in the presence of sarcosine to improve the specificity of the detection. At the same time, the high sensitivity detection of sarcosine can be realized with electrochemical technology, and the detection limit can reach 50nM. finally, a large number of prostate cancer was carried out by this method. Detection of benign prostatic enlargement and normal male serum samples. Fourthly, based on the technique of constructing nanoscale biological interface, we combine DNA aptamer with epithelial cell adhesion factor (Ep CAM), which is highly expressed on MCF-7 surface of breast cancer cells. The technique of assembling DNA at the cell interface is realized, and then using DNA hybridization, the initiation chain is hybridized onto the cell surface, so that the cells with the initiation chain can initiate a 3D hybridization chain reaction to form a DNA hydrogel. DNA-protected nanocrystalline gold can be used to detect cancer cells quantitatively and visually.
【学位授予单位】：中国科学院研究生院(上海应用物理研究所)
【学位级别】：博士
【学位授予年份】：2016
【分类号】：TB383.1;TP212

【参考文献】