聚亚甲基蓝纳米粒子的合成及其在DNA检测中的应用

发布时间：2018-06-16 15:02

本文选题：聚亚甲基蓝纳米粒子 + 反相微乳液法　；参考：《陕西师范大学》2015年硕士论文

【摘要】：随着化学传感器和生物传感器发展,对目标分子进行灵敏度高、选择性好、快速、准确的检测是其研究的主要方向。在最近几年,纳米材料的应用,使化学传感器和生物传感器的性能得到了很大的提高。纳米材料因在传感器方面产生的重大影响,其合成方法有了显著的进步。各种大小、形状、表面电荷和物理化学特性等可控的纳米材料已被合成。其中二氧化硅纳米粒子、纳米金、量子点团簇、半导体量子点、上转换发光纳米粒子、聚合物纳米粒子、碳纳米管以及石墨烯在化学和生物传感器的研究中被广泛使用。纳米材料被广泛用于性能优良的电化学DNA生物传感器,主要原因为：一、通过修饰聚合物和生物活性分子使纳米材料达到良好生物相容性；二、其自身具有独特的电化学活性或可掺杂电活性物质。由于纳米粒子在制备电化学DNA生物传感器时,多数是利用其信号放大技术,有的却需要掺杂或包埋一些电活性物质,比如亚甲基蓝、二茂铁和尼罗蓝等,有的则是通过标记电活性物质,来实现DNA的检测。掺杂型纳米粒子中的电活性物质,其电化学活性不能更好地表现出来,而标记型纳米粒子需要对纳米粒子进行官能团化。基于以上问题,本文旨在合成一种新型的导电聚合物纳米粒子,该纳米粒子本身具有良好的电活性,也易于官能团化,即可以用于信号放大,也可以作为电化学标记物。本文主要由综述和研究报告两部分组成,综述部分介绍了纳米材料、导电聚合物纳米材料、DNA电化学生物传感器和纳米材料在DNA电化学生物传感器的应用,分析了本文选题的目的和意义。研究报告也由两部分组成：1.聚亚甲基蓝纳米粒子的合成及免标记法检测DNA的研究首次在室温下成功合成聚亚甲基蓝纳米粒子(PMBNPs),采用反相微乳液法,用过磷酸铵作氧化剂氧化单体亚甲基蓝,使之在油包水体系中合成了聚亚甲基蓝纳米粒子。再利用透射电子显微镜和循环伏安法,对新合成的聚亚甲基蓝纳米粒子进行表征。为了进一步地研究该纳米粒子,构建了一种新型的免标记的DNA电化学传感器。首先将PMBNPs直接固定在电极表面,在PMBNPs表面电镀一层金纳米粒子,通过金硫键连接带巯基的探针DNA。DNA的杂交一方面使溶液中的对离子向电极表面扩散的通道被部分堵塞导致聚亚甲基蓝纳米粒子的氧化还原电流降低；另一方面也严重阻碍了电子转移通道,减少了电子转移的有效面积,使电活性物质的氧化还原电流降低。所以,用电化学法测定目标DNA,实际上是通过检测固定在电极表面的PMBNPs的氧化还原电流,从而对DNA杂交事件进行识别。在最佳条件下,该传感器的氧化电流与目标DNA在0.2fM到1pM浓度范围内有线性关系,检出限是6.67fM。2.基于DNA对聚亚甲基蓝纳米粒子的吸附作用构建电化学免标记DNA传感器采用上述方法合成的PMBNPs,并用循环伏安和Zeta电位进行表征,结果表明聚亚甲基蓝纳米粒子具有良好的电化学活性,且其表面带正电荷。由于ssDNA与dsDNA所带负电荷数量的不同,所以单、双链DNA吸附正电荷的PMBNPs的量不同。本文通过用差分脉冲伏安法,分别测定被单、双链吸附的PMBNPs,实现对DNA杂交事件的识别。此方法把聚亚甲基蓝纳米粒子作为杂交指示剂,可实现电化学信号放大,完成对DNA高灵敏度检测。
[Abstract]:With the development of chemical sensors and biosensors, the main direction of the research is high sensitivity, selectivity, rapid and accurate detection of the target molecules. In recent years, the application of nanomaterials has greatly improved the performance of chemical sensors and biosensors. The synthetic methods have made remarkable progress. Various sizes, shapes, surface charges and physical and chemical properties of nanomaterials have been synthesized. Among them, silica nanoparticles, gold nanoparticles, quantum dots clusters, semiconductor quantum dots, upconversion luminescent nanoparticles, polymer nanoparticles, carbon nanotubes, and graphene are in chemical and chemical properties. Nanomaterials are widely used in the study of biosensors. Nanomaterials are widely used in electrochemical DNA biosensors with excellent performance. The main reasons are: first, the good biocompatibility of nanomaterials is achieved by modifying polymers and bioactive molecules; two, its own unique electrochemical activity or doping electroactive substances. When nano particles are prepared for electrochemical DNA biosensors, most of them use their signal amplification techniques, some of which need to be doped or embedded in some electroactive substances, such as methylene blue, two ferrocene and Nile blue, and some are used to detect DNA by labeling electroactive substances. Based on the above problems, the aim of this paper is to synthesize a new type of conductive polymer nanoparticles, which have good electrical activity and easy to be functionalized, that is, it can be used in signal amplification and can be used as electrochemistry. This article is mainly composed of two parts. This article is mainly composed of two parts: the summary and the research report. It introduces the applications of nanomaterials, conductive polymer nanomaterials, electrochemical biosensors and nanomaterials in DNA electrochemical biosensors. The purpose and meaning of this topic are analyzed. The research report is also composed of two parts: 1. polymethylmethylene The synthesis of blue nanoparticles and the detection of DNA by non labeling method were the first time to successfully synthesize polymethylene blue nanoparticles (PMBNPs) at room temperature. The polymethylene blue was oxidized by the reverse phase microemulsion and ammonium peroxide as oxidizing agent. The polymethylene blue nanoparticles were synthesized in the oil water system, and then the transmission electron microscope was used. Cyclic voltammetry was used to characterize the newly synthesized polymethylmethylene blue nanoparticles. In order to further study the nanoparticles, a new type of DNA electrochemical sensor was constructed. First, PMBNPs was directly immobilized on the surface of the electrode, electroplating a layer of gold nanoparticles on the surface of PMBNPs, and connecting the probe DNA.D with sulfhydryl group through the gold sulfur bond. On the one hand, the crossbreeding of NA leads to a partial blockage of the channel diffusion to the surface of the electrode resulting in the reduction of the redox current of the polymethylene blue nanoparticles; on the other hand, the electron transfer channel is seriously hindered, the effective area of the electron transfer is reduced and the oxidation-reduction current of the electroactive substance is reduced. So, the use of electricity is used. The determination of target DNA by chemical method is actually by detecting the redox current of PMBNPs fixed on the surface of the electrode and identifying the DNA hybridization event. Under the optimum conditions, the oxidation current of the sensor has a linear relationship with the target DNA in the 0.2fM to 1pM concentration range, and the detection limit is 6.67fM.2. based on DNA against polymethylene blue nanoparticles. The adsorption of the electrochemically labeled DNA sensor is constructed with the PMBNPs synthesized by the above method and characterized by cyclic voltammetry and Zeta potential. The results show that the polymethylmethylene blue nanoparticles have good electrochemical activity and have positive charges on the surface. Because the number of negative charges with ssDNA and dsDNA is different, the single, double chain DNA suction is used. The amount of PMBNPs with positive charge is different. In this paper, by using differential pulse voltammetry to determine the PMBNPs of single and double chain adsorption respectively, the identification of DNA hybridization events is realized. This method uses polymethylmethylene blue nanoparticles as a hybrid indicator to achieve electrochemical signal amplification and high sensitivity detection of DNA.
【学位授予单位】：陕西师范大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：TB383.1;O657.1

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