纳米界面上DNA分子反应活性的研究及其在生物传感中的应用

发布时间：2018-06-04 22:48

本文选题：纳米材料 + 核苷酸　；参考：《中国科学院研究生院（上海应用物理研究所）》2015年博士论文

【摘要】：纳米生物材料领域是纳米科学中的一个核心领域，其技术的发展引发着新型材料的制造以及生物医学领域的蓬勃发展。纳米生物技术通过将生物分子连接到纳米材料上，可以将纳米材料的优良特性及生物分子的识别能力结合在一起，构建出多种新型的功能性材料，在生物传感、药物传递及靶向治疗、材料科学及DNA纳米技术等方面都有重要的应用。该领域的关键问题之一是如何更有效地将生物分子（如DNA、蛋白质等）与纳米材料进行连接，并且在此过程中仍很好地保持生物分子的活性，形成高效的分子识别界面。研究生物分子在纳米界面上的反应活性对于更好发挥生物纳米材料的性能有着重要的意义。本论文的研究工作主要包括DNA在纳米界面上反应活性的系统研究以及新型的DNA功能化纳米结构体系的构建。主要内容如下：（1）基于课题组之前发展的新型嵌段DNA-纳米金偶联体制备方法，构建了一系列密度和构型可控的DNA-纳米金偶联体,并以此为基础对DNA在纳米界面上分子识别的热力学和动力学等物理化学性质及生物分子活性进行深入的探讨和分析，研究了DNA自组装方法、DNA分子的构型及组装密度等如何影响DNA在纳米界面上的稳定性和分子识别过程的反应活性，建立理论模型。和传统的巯基化学修饰方法相比，双嵌段寡聚核苷酸DNA-纳米金体系独立调控DNA在纳米金表面上的密度而不影响其构型，因此所制备的DNA-纳米金偶联体非常适合作为模型证实、修正和阐释DNA在纳米界面上分子识别的物理化学性质，用于单因素调节分析，有望为生物纳米体系的构建提供理论基础。（2）在双嵌段DNA-纳米金体系中，将DNA酶（DNAzyme）作为功能嵌段，构建了一系列组装密度和形貌可控的DNAzyme-AuNPs复合物，对DNAzyme在纳米金上的反应活性进行研究。DNAzyme的组装密度可以通过改变poly A的长度来调节，我们比较了纳米金界面上不同组装密度的双嵌段DNAzyme和通过巯基自组装的DNAzyme对铅离子的响应能力以及酶的催化能力，并与均相溶液体系进行了比较。这项研究对8-17DNAzyme在纳米金表面的催化过程和作用机制进行了更为系统的研究，可能为生物传感提供一个活性更高、特异性更好的酶催化平台。（3）利用胞嘧啶碱基与纳米银之间的吸附能力，发展了一种新的组装纳米银-寡聚核苷酸复合探针的方法。这种方法通过富含胞嘧啶的序列将DNA自组装于纳米银上，自组装得到的复合探针在含盐及高温环境下仍能保持稳定，并且通过这种方法组装的探针在表面上拥有比巯基方法组装的探针更好的分子识别能力。此外，我们可以通过改变胞嘧啶碱基的个数调节寡聚核苷酸在纳米银上的组装密度，提高纳米银-DNA复合探针的稳定性。这种组装方法无需对寡聚核苷酸进行修饰，降低了自组装的成本且易于操作。所组装的复合探针具备优良的光学特性，可能在光学及生物传感领域具有很大的应用潜力。（4）提出了一种制备纳米尺度花状结构的超微金电极的新方法。将微米级碳纤维进行火焰刻蚀至纳米级，在其表面进行电化学沉积电泳漆，，加热烘烤处理后，通过电化学沉积纳米金得到纳米花状超微金电极。扫描电镜结果表明该方法可以得到尺寸在100μm左右，表面花刺在纳米级别的纳米花状超微金电极。通过将纳米花状超微金电极与核酸适体技术及电化学检测技术相结合，开发了一种基于核酸适体构象变化的超微电极传感器。将其应用于生物分子（如可卡因）的检测中，响应速度快，特异性好。该体系有望应用于单细胞及其他微环境中生物活性分子的检测中。
[Abstract]:The field of nanoscale materials is a core field in nanoscience. The development of its technology leads to the development of new materials and the flourishing development of biomedicine. By connecting biological molecules to nanomaterials, nanotechnology can combine the excellent characteristics of nanomaterials and the recognition ability of biomolecules. A variety of new functional materials have been constructed, which have important applications in biosensing, drug delivery and targeting therapy, material science and DNA nanotechnology. One of the key issues in this field is how to effectively connect biomolecules (such as DNA, protein, etc.) with nanomaterials, and remains well maintained in this process. The activity of biomolecules can form an efficient molecular recognition interface. It is of great significance to study the reactive activity of biomolecules on the nano interface for better performance of the properties of biological nanomaterials. The research work of this paper mainly includes the systematic study of the reactive activity of DNA on the nano interface and the new DNA functionalized nanostructure The main content of the system is as follows:
(1) based on the new block DNA- nano Au couple preparation method developed before the project group, a series of DNA- nanoscale conjugates with controllable density and configuration are constructed, and on this basis, the thermodynamic and kinetic properties of the molecular recognition of DNA at the nano interface and the biological and molecular activity are deeply discussed and analyzed. The DNA self-assembly method, the configuration and the assembly density of DNA molecules affect the stability of DNA at the nano interface and the reaction activity of the molecular recognition process, and establish a theoretical model. Compared with the traditional sulfhydryl chemical modification method, the diblock oligonucleotide DNA- nanoscale system independently regulates the density of DNA on the surface of gold nanoparticles. As a result, the prepared DNA- nanoscale coupling is very suitable as a model to confirm the physical and chemical properties of the molecular recognition of DNA at the nano interface. It is expected to provide a theoretical basis for the construction of the biological nano system.
(2) in the double block DNA- nanoscale gold system, DNA enzyme (DNAzyme) is used as a functional block. A series of DNAzyme-AuNPs complexes with controlled assembly density and morphology are constructed. The reaction activity of DNAzyme on gold nanoparticles can be studied by changing the length of poly A. We compare the nano gold interface. The response ability of DNAzyme with different assembly density and the ability to respond to lead ions by the self assembled DNAzyme by mercapto, and the catalytic ability of the enzyme, are compared with the homogeneous solution system. This study has carried out a more systematic study on the catalytic process and mechanism of 8-17DNAzyme on the surface of gold nanoparticles, which may provide a biological sensing method. An enzyme catalyzed platform with higher activity and better specificity.
(3) a new method of assembling the nano silver oligonucleotide probe was developed by using the adsorption capacity between the cytosine base and the silver nanoparticles. This method is self-assembled on the nano silver through the sequence rich in cytosine, and the self assembled composite probe can remain stable in the salt and high temperature environment, and through this method, DNA can be maintained by this method. In addition, we can adjust the density of oligodeoxynucleotides on nano silver by changing the number of cytosine bases to improve the stability of the nano silver -DNA composite probe. This assembly method does not need to repair oligonucleotides. It reduces the cost of self-assembly and is easy to operate. The assembled probe has excellent optical properties, and may have great potential in the field of optical and biological sensing.
(4) a new method of preparing nano scale flower like ultragold electrode was proposed. The micrometer carbon fiber was etched to nanoscale by flame etching to nanoscale, electroelectrophoretic coatings were deposited on its surface. After heating and baking, nano flower like ultragold electrode was obtained by electrochemical deposition of nano gold. The results of scanning electron microscopy showed that this method could be used. A nano flower like ultramicro gold electrode was obtained on the surface with a size of about 100 m. The nano flower like ultragold electrode was inserted at the nano scale. A super microelectrode sensor based on the conformational change of nucleic acid was developed by combining the nano flower like ultramicro gold electrode with the aptamer technique and electrochemical detection technology. It should be used for the detection of biomolecules (such as cocaine). This system is expected to be applied to the detection of bioactive molecules in single cells and other microenvironments.
【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】：2015
【分类号】：TB383.1;O629.74

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