几种新型纳米传感器的构建及其在生物分析中的应用

发布时间：2018-12-07 19:57

【摘要】：随着信息技术与生物工程技术的不断发展,生物传感器展现出了越来越大的应用潜力。当今各发达国家都把生物传感器列为21世纪的关键技术,并给予了高度的重视。相比于传统实验室复杂费时的检测方法,新型生物传感器具有高选择性,高灵敏度以及快速、微型化、低成本等优点,不仅被广泛用于环境污染物监测、食品安全领域、生物和医学领域,而且在空间生命科学、军事、航天遥感等方面也表现出巨大的应用前景。然而随着检测对象的日益多样化、复杂化,生物传感方法和分析技术也面临着新的需求与挑战。近年来,纳米材料成为了科学研究者的热门研究对象,纳米尺寸物质拥有与块体材料迥然不同的性质,有的甚至发生了质变,正是由于性质的特异性,纳米尺度材料引发了人们的广泛关注。功能化纳米材料表现出许多优点,比如比表面积大,催化活性高,生物相容性良好等等,因此可以作为敏感元件用于生物传感器的构建,这为构建高选择性、高灵敏、快速高效的生物传感器提供了新的设计思路。在查阅大量相关文献的基础上,围绕功能化纳米材料在生物传感器方面的相关应用,本论文主要研究了几种新型纳米传感器在生物分析中的应用。具体工作如下:第一章:围绕生物传感器和纳米材料对他们的概念、特点、分类及应用意义展开了详细的介绍。第二章:成功开发了一种新型,简单和超灵敏的基于内滤效应(IFE)的荧光分析方法用以检测肿瘤标记物β-葡萄糖醛酸酶(GLU)。发射绿色荧光的氮掺杂的碳量子点(N-CQDs)被用做内滤效应中的荧光团,并且对硝基苯基-β-D-吡喃葡糖醛酸苷(PNPG)用做GLU的底物,GLU催化底物PNPG产生的水解产物对硝基苯酚(PNP)作为内滤效应中的干扰器来猝灭N-CQDs的荧光。这样通过N-CQDs的荧光强度的变化即成功实现了对GLU的活性检测。最后我们还将此方法成功应用于天然产物中的药物筛选。第三章:成功设计了一种比率型荧光探针,其中包含碳量子点(CDs)作为传感信号和罗丹明B(RhB)作为参照信号,用来快速灵敏的检测汞离子(Hg~(2+))和谷胱甘肽(GSH)。此纳米传感器在单一激发波长下会同时显示出CDs和RhB的特征荧光发射峰。此功能化的CDs通过组氨酸和柠檬酸钠的一锅热解法首次在我们的实验室中合成。在Hg~(2+)的存在下,由于Hg~(2+)和CDs表面上的官能团之间的电子转移,导致CDs的荧光发生猝灭。随后,随着加入的GSH越来越多,CDs-Hg~(2+)体系的荧光逐渐得到恢复,这是因为GSH可以竞争性的从CDs表面夺取Hg~(2+),在整个过程中,RhB的荧光强度一直保持不变,通过检测两个荧光峰值的比值变化即可实现对GSH的比率型检测,这可以有效地排除许多背景干扰。最后该方法成功应用于小鼠的氧化应激模型研究。第四章:本实验基于农药残留对于碱性磷酸酶(ALP)的抑制性,将碱性磷酸酶同Ag0-Ag_3PO_4纳米酶结合实现的双重信号放大策略成功应用在了蔬菜中的农药残留的超灵敏比色检测。在实验中,Ag+可以被ALP催化底物产生的抗坏血酸(AA)还原为Ag纳米颗粒,然后Ag纳米颗粒沉积在菱形十二面体Ag_3PO_4的表面上形成Ag0-Ag_3PO_4复合纳米酶,大大提高了光催化活性,从而将TMB直接氧化为oxTMB。通过测定ox TMB的吸光度大小,就可以转换成ALP的活性大小,从而实现对农药残留的定量检测。
[Abstract]:With the development of information technology and bioengineering technology, the biosensor has shown increasing application potential. In today's developed countries, the biosensor is listed as the key technology of the 21st century, and it has given a high degree of attention. Compared with the traditional laboratory complex and time-consuming detection method, the novel biosensor has the advantages of high selectivity, high sensitivity, high speed, miniaturization, low cost and the like, and is not only widely used in environmental pollutant monitoring, food safety, biological and medical fields, but also has great application prospect in the aspects of space science, military and space remote sensing. However, with the increasing diversification of the detection object, the biological sensing method and the analysis technology are also facing new demands and challenges. In recent years, the nano-scale material has become a hot research object of the scientific researchers. The functionalized nano-material has many advantages, such as large specific surface area, high catalytic activity, good biocompatibility, and the like, and can be used as a sensing element for the construction of a biosensor, The rapid and efficient biosensor provides a new design thought. On the basis of consulting a large number of relevant literature, the paper mainly studies the application of several new kinds of nano-sensors in biological analysis. The specific work is as follows: Chapter 1: The concept, characteristics, classification and application significance of the biological sensor and the nano-material are introduced in detail. In the second chapter, a novel, simple and ultra-sensitive fluorescence analysis method based on the internal filtering effect (IFE) was successfully developed to detect the tumor markers of the glucose-glucuronase (GLU). A nitrogen-doped carbon quantum dot (N-CQDs) that emits green fluorescence is used as a fluorophore in the internal filtering effect, and a p-nitrophenyl-1-D-ethanone-glucuronate (PNPG) is used as a substrate for GLU, The hydrolysis product produced by the GLU catalytic substrate PNPG is used as a disturbance device in the internal filtering effect to destroy the fluorescence of the N-CQDs. Thus, the activity detection of GLU was successfully achieved by the change in the fluorescence intensity of the N-CQDs. Finally, we have successfully applied this method to drug screening in natural products. In the third chapter, a ratio-type fluorescent probe was successfully designed, in which the carbon-content sub-point (CDs) of the bag was used as a reference signal for sensing the mercury ions (Hg ~ (2 +)) and the glutathione (GSH). The nano-sensor will show the characteristic fluorescence emission peak of CDs and RhB at the same time under a single excitation wavelength. This functionalized CDs is first synthesized in our laboratory by a pot-thermal solution of histidine and sodium citrate. In the presence of Hg ~ (2 +), the fluorescence of CDs was destroyed due to the electron transfer between the Hg ~ (2 +) and the functional groups on the surface of the CDs. Then, with the increasing of GSH, the fluorescence of the system of CDs-Hg ~ (2 +) was gradually restored, because the GSH could capture the Hg ~ (2 +) from the surface of the CDs, and the fluorescence intensity of RhB remained unchanged throughout the process. The detection of the ratio of GSH can be achieved by detecting the ratio change of the two fluorescence peaks, which can effectively eliminate many background interference. Finally, the method was successfully applied to the oxidative stress model of mice. In the fourth chapter, based on the inhibition of the pesticide residue on the alkaline phosphatase (ALP), the double signal amplification strategy of the alkaline phosphatase and the Ag0-Ag _ 3PO _ 4 nano-enzyme is successfully applied to the ultra-sensitive colorimetric detection of the pesticide residue in the vegetables. In the experiment, the Ag + can be reduced to Ag nanoparticles by the ascorbic acid (AA) generated by the ALP catalytic substrate, and then the Ag nanoparticles are deposited on the surface of the diamond dodecahedron Ag _ 3PO _ 4 to form the Ag0-Ag _ 3PO _ 4 composite nano-enzyme, so that the photocatalytic activity is greatly improved, and the TMB is directly oxidized into the oxyTMB. By measuring the absorbance of the ox TMB, the activity of the ALP can be converted, and the quantitative detection of the pesticide residue can be realized.
【学位授予单位】：曲阜师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TP212

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