几种DNA生物传感器用于微小RNA和真菌毒素的检测

发布时间：2018-09-14 13:22

【摘要】：生物传感器是由生物、化学、物理、光学、电子等学科彼此交叉成长的新技术。其中,基于光电技术的生物传感器的应用最为广泛,它们具有选择性好、灵敏度高、分析速度快、成本低、能在复杂体系中进行在线连续监测等优点。目前,这些生物传感技术已被引入在医学诊断、食品安全分析等多种领域。本论文将DNA(主要以赭曲霉毒素A和微小RNA为检测对象)和相关纳米技术相结合,构建几种新型的光学、电化学DNA生物传感器,并有望将其应用于农产品或中药中真菌毒素的检测和癌症的早期诊断中。本论文正文共有四章。第一到第三章分别介绍三种光学或电化学DNA生物传感器用于赭曲霉毒素A的检测。第四章介绍一种电化学DNA生物传感器用于微小RNA-21的检测。第一章:本章研制了一种基于限制性内切酶和发卡适体探针信号放大技术的DNA电化学生物传感器,用于检测赭曲霉毒素A(Ochratoxin A,OTA)。在最佳条件下,传感器的线性范围为1.0~20 pg/m L,检测限达到0.4 pg/m L。在一定条件下,该传感器能较好地识别复杂体系中的OTA,有望实现实际样本中OTA的检测。第二章:本章基于稀土铽离子,DNA适体构型转变及磁珠技术,研制了一种荧光信号增强型适体生物传感器,用于检测小麦中的OTA。通过这种策略,在最佳条件下,检测OTA的线性范围为0.1 ng/m L~1 ng/m L,检测限可达20 pg/m L。与目前常用的OTA检测方法相比,该方法具有操作简单、快速、无需繁杂的修饰与标记过程等优点。该生物传感器利用铽离子的荧光性质用于OTA的检测,有望建立一种方法用于食品安全的常规质量控制。第三章:本章研制了一种基于DNA保护的银纳米簇荧光探针技术的荧光核酸适体生物传感器,用于农作物中OTA的高灵敏性和选择性检测。在最佳条件下,该传感器对OTA检测的线性范围在0.01到0.3 ng/m L之间,最低检测限为2 pg/m L。通过靶标链释放和DNA骨架模板合成银纳米簇技术研制的适体生物传感器,具有高灵敏性、高选择性、简单快速等优点,可以用于农产品中OTA的检测。第四章:本章研制了基于2'-O-甲基修饰的脱氧核酶(Deoxyribozymes or Catalytic DNA,DNAzyme)和双链特异性核酸酶(Duplex Specific Nuclease,DSN)辅助目标序列循环技术的一种超敏非标记型电化学生物传感器用于微小RNA(micro RNA-21,mi R-21)的检测。通过这种策略,本章研制的适体传感器可以检测低至8 a M的mi R-21,具有较高的特异性,甚至具有很好的单碱基错配识别能力。同时,该生物传感器还具有组装容易,操作方便,成本低廉等优点,使其在癌症早期诊断具有极大的应用潜力。
[Abstract]:Biosensors are new technologies that intersect biology, chemistry, physics, optics and electronics. Among them, the biosensors based on photoelectric technology are most widely used. They have the advantages of good selectivity, high sensitivity, fast analysis speed, low cost, and can be continuously monitored in complex systems. At present, these biosensors have been introduced into many fields, such as medical diagnosis, food safety analysis and so on. In this paper, several novel optical and electrochemical DNA biosensors were constructed by combining DNA (mainly with ochratoxin A and tiny RNA) and related nanotechnology. It is expected to be applied to the detection of mycotoxins in agricultural products or traditional Chinese medicine and the early diagnosis of cancer. There are four chapters in this thesis. In the first to third chapters, three kinds of optical or electrochemical DNA biosensors are introduced for the detection of ochratoxin A. Chapter 4 introduces an electrochemical DNA biosensor for the detection of micro RNA-21. Chapter 1: in this chapter, we develop a DNA electrochemical biosensor based on restriction endonuclease and hairpin probe amplification, which can be used to detect ochratoxin A (Ochratoxin. Under the optimum conditions, the linear range of the sensor is 1.0 ~ 20 pg/m / L and the detection limit is 0.4 pg/m / L. Under certain conditions, the sensor can identify the OTA, in complex system well. It is expected to realize the detection of OTA in real samples. Chapter 2: in this chapter, a fluorescent signal enhanced aptamer biosensor is developed based on the configuration transformation of rare earth terbium ion DNA aptamer and magnetic bead technique, which can be used to detect OTA. in wheat. Under the optimal conditions, the linear range of OTA is 0.1 ng/m L ~ (-1) ng/m / L, and the detection limit is up to 20 pg/m / L. Compared with the commonly used OTA detection method, this method has the advantages of simple operation, fast operation and no complicated modification and marking process. The biosensor uses the fluorescence properties of terbium ion for the detection of OTA and is expected to establish a method for routine quality control of food safety. Chapter 3: in this chapter, a fluorescent aptamer biosensor based on DNA protection silver nanoclusters fluorescence probe technology is developed, which can be used to detect OTA in crops with high sensitivity and selectivity. Under the optimum conditions, the linear range of the sensor for OTA detection is 0. 01 to 0. 3 ng/m / L, and the minimum detection limit is 2 pg/m / L. The aptamer biosensor developed by the technology of target chain release and DNA skeleton template synthesis has the advantages of high sensitivity, high selectivity and simplicity. It can be used for the detection of OTA in agricultural products. Chapter 4: in this chapter, a novel electrochemical biosensor, which is based on 2-Omethyl-modified deoxyribozyme (Deoxyribozymes or Catalytic DNA,DNAzyme) and double-strand specific nuclease (Duplex Specific Nuclease,DSN), has been developed for the detection of micro RNA (micro RNA-21,mi R-21. Through this strategy, the aptamer sensor developed in this chapter can detect mi R-21 as low as 8um, which has high specificity and even good single-base mismatch recognition ability. At the same time, the biosensor has the advantages of easy assembly, easy operation and low cost, so it has great application potential in early diagnosis of cancer.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：Q819;R446

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