基于末端保护和纳米材料的生物传感新方法的研究

发布时间：2018-05-10 15:24

本文选题：荧光生物传感技术 + 末端保护　；参考：《湖南大学》2014年硕士论文

【摘要】：生命体中蛋白质、核酸、酶活性以及生物小分子活动信息的研究对生物医学以及临床诊断和治疗有着非常巨大的意义。如何实时、动态、快速、准确地获取这些生命活动的信息,就成为了分析化学科研工作者面临的重大的挑战。为了克服这个难题,就需要我们发展一些准确性高、灵敏度高以及特异性高的定性或定量的方法,达到获取信息的目的。本论文瞄准上述挑战进行研究,基于末端保护技术和纳米材料发展了一系列分别以小分子、核酸、蛋白质和酶活性为检测对象的高灵敏性高选择性的生物传感技术,并通过对实际样品的分析,初步验证了这些技术的可行性和准确性。第2章中,在细胞表面表达的肿瘤特异性蛋白标记物对于癌症早期检测、诊断和治疗是很有价值的目标物。我们基于吸附到DNA3’端的小分子配体和细胞表面表达的蛋白生物标志物之间的相互作用将寡核苷酸标记到细胞表面,然后EXO I能够将非特异性DNA降解,保持无响应,从而确保高特异性检测。这样,一种直接检测核酸的方法就可以替代对于活细胞表面蛋白生物标记物的复杂的、不灵敏的检测方法。由于DNA的信号转换和放大的多种途径,我们基于血红素DNA酶、DNA模板化的AgNCs以及荧光定量PCR,设计了三种信号输出方法分别用于检测、成像以及定量。最终,小分子连接的可编码DNA将会开启一种使用免洗的无标记方法来检测细胞膜蛋白的局面。创立了一种用于细胞膜蛋白质活体检测的简单但有意义的平台。与此同时,将对目标生物标志物的识别转化为对DNA序列的编码,并且还提供了一种同时检测细胞膜表面的多种生物标志物的方法。比如,可以利用设计不同DNA模板来指导合成AgNCs用于多种蛋白质的直接检测与成像。第3章中,多聚核苷酸激酶磷酸酶(PNKP)对DNA的去磷酸化修饰过程在许多的生理活动中起着非常重要的作用。本章以T4多聚核苷酸激酶磷酸酶(T4PNKP)为模型,利用荧光团标记的DNA探针作为T4PNKP的基质,此探针是自组装的单链DNA分子。3’端修饰了磷酸基团作为目标磷酸酶的基质,一旦加入T4PNKP,T4PNKP会将探针的3’磷酸集团水解为羟基,所产生的3’-OH在DNA聚合酶以及dNTPs的作用下迅速扩展,之前的单链DNA变为双链DNA。由于双链DNA和WS2纳米片之间的结合力很弱,延伸后的双链产物在WS2纳米片同时同时存在时有很强的荧光发射,而我们检测到没有聚合酶延伸的探针具有较弱的荧光信号,并且随着WS2纳米片对单链DNA的吸附,荧光猝灭。WS2的超强荧光淬灭能力也为该方法提供了有较宽的线性范围和低的检测限,其检测下限为0.05U/mL。另外,该方法不仅可以对T4PNKP的抑制剂进行定量表征,还适用于复杂生物环境中目标物的测定。本方法设计简单、操作方便、灵敏度高且选择性好,有望成为PNKP酶检测的一种很好的选择并能应用于DNA损伤修复机理的研究。第4章中,SNP对于高危群体的发现、疾病相关基因的鉴定、药物的设计和测试以及生物学的基础研究是非常重要的。氧化石墨烯对于单链的DNA分子具有较强的吸附功能,对于荧光基团具有广泛的猝灭功能,对于单个碱基的荧光不会猝灭。我们基于T7核酸外切酶、单个荧光基团标记的核酸探针和氧化石墨烯构建一种简便有效的高灵敏检测技术用于SNP分析。该方法操作简单,响应快速,并且有较高的灵敏度,其检测下限为10pM。
[Abstract]:The study of protein, nucleic acid, enzyme activity and biological small molecule activity information in the life body has great significance for biomedicine and clinical diagnosis and treatment. How to obtain the information of these life activities in real time, dynamically, quickly and accurately has become a major challenge for the researchers of analytical science and research. This problem requires us to develop some qualitative or quantitative methods of high accuracy, high sensitivity and high specificity to achieve the purpose of obtaining information. This paper aims to study the challenges mentioned above. Based on the end protection technology and nanomaterials, we have developed a series of detection targets for small molecules, nucleic acids, proteins and enzymes, respectively. Highly sensitive and highly selective Biosensing Technology, and through the analysis of actual samples, preliminarily verified the feasibility and accuracy of these technologies.
In the second chapter, the tumor specific protein markers expressed on the cell surface are valuable targets for early detection, diagnosis and treatment of cancer. We mark the oligonucleotides on the cell surface based on the interaction between the small molecular ligands adsorbed to the DNA3 'end and the protein biomarkers expressed on the cell surface, and then EXO I can be used. It is sufficient to degrade nonspecific DNA and maintain no response to ensure high specificity detection. Thus, a method of direct detection of nucleic acids can replace complex, insensitive detection methods for living cell surface protein biomarkers. We are based on heme DNA enzyme, DNA template based on a variety of pathways of signal conversion and amplification. AgNCs and fluorescent quantitative PCR designed three signal output methods for detection, imaging, and quantification. Finally, the small molecule linked coded DNA will open a unlabelled unlabelled method for detecting cell membrane proteins. A simple but meaningful flat for cell membrane protein activity detection is created. At the same time, the identification of target biomarkers is converted into encoding of DNA sequences, and a method for simultaneous detection of a variety of biomarkers on the surface of the cell membrane is provided. For example, the design of different DNA templates can be used to guide the synthesis of AgNCs for direct detection and imaging of various proteins.
In the third chapter, the dephosphorylation of DNA by polynucleotides kinase phosphatase (PNKP) plays a very important role in many physiological activities. This chapter uses T4 polykinase phosphatase (T4PNKP) as a model and uses a DNA probe labeled as a T4PNKP matrix. This probe is a self-assembled single strand DNA molecule.3 'end repair. With the phosphoric acid group as the substrate of the target phosphatase, once the T4PNKP is added, T4PNKP will hydrolyze the 3 'phosphoric acid group of the probe into hydroxyl group, and the produced 3' -OH expands rapidly under the action of DNA polymerase and dNTPs, and the previous single strand DNA becomes double stranded DNA. due to the weak binding force between the double chain DNA and WS2 nanoscale, and the extended double chain. The product has strong fluorescence emission at the same time that the WS2 nanometers simultaneously exist, and we detected that the probe without the polymerase extension has a weak fluorescence signal. And with the adsorption of WS2 nanoscale to single strand DNA, the fluorescence quenching ability of fluorescence quenching.WS2 also provides a wider linear range and low detection limit for this method. The detection limit is 0.05U/mL.. This method can not only quantify the T4PNKP inhibitors, but also be suitable for the determination of objects in complex biological environment. This method is simple in design, convenient in operation, high in sensitivity and good in selectivity. It is expected to be a good choice for PNKP enzyme detection and can be applied to the mechanism of DNA damage repair. Research.
In the fourth chapter, SNP is very important for the discovery of high risk groups, identification of disease related genes, the design and testing of drugs, and basic biological research. The graphene oxide has a strong adsorption function for single strand DNA molecules, the fluorescence group has extensive quenching function, and the fluorescence of a single base will not be quenched. Based on T7 nucleic acid exonuclease, nucleic acid probe labeled by single fluorescent group and graphene oxide, a simple and effective high sensitive detection technique is constructed for SNP analysis. This method is easy to operate, fast response, and has high sensitivity, and its detection limit is 10pM.

【学位授予单位】：湖南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R914

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