基于GQDs和SGI构建新型荧光传感器的研究

发布时间：2018-05-16 15:02

本文选题：免标记 + 荧光传感器　；参考：《福建医科大学》2015年硕士论文

【摘要】：现代化的检测技术正朝着快速、灵敏、高通量的方向发展,与此同时,荧光检测因其所具有的操作简便、省时、灵敏等特点受到越来越多的关注,新型荧光检测技术有望成为下一代高通量生物分析技术。现在的荧光检测技术主要通过以下两种技术路线:一:荧光染料标记的荧光生物传感器用于成像与检测,常见的荧光染料有FITC及FAM等;二:应用半导体量子点、金属簇等荧光纳米材料发展荧光检测方法用于DNA、蛋白质和其它小分子等物质的检测。现有的荧光生物传感器通常需要标记处理,造成检测方法费时费力、高成本等不足。因此,开发高灵敏、高特异检测目标物的免标记荧光检测新方法具有广阔的应用前景。基于以上考虑,本课题建立了三种新型的免标记荧光传感体系:第一章基于石墨烯量子点的免标记荧光传感器用于多巴胺检测本章节设计了一种基于石墨烯量子点的免标记荧光传感体系检测多巴胺。通过柠檬酸(Ascorbic Acid,CA)热解法制备石墨烯量子点(Graphene Quantum Dots,GQDs)并作为荧光探针,多巴胺(Dopamine,DA)在碱性条件下自聚合形成聚多巴胺(Polymerization of Dopamine,pDA)并吸附在GQDs表面淬灭荧光,荧光淬灭强度可用于多巴胺检测。利用紫外可见光谱(Ultravioletand Visible Spectrophotometry,UV-vis)傅里叶变换红外光谱(Fourier Transform Infrared Spectroscopy,FTIR)、原子力显微镜(Atomic Force Microscope,AFM)、透射电镜(Transmission Electron Microscopy,TEM)、zeta电势、光电子能谱(X-ray Photoelectron Spectroscopy,XPS)、对本实验体系中GQDs、pDA的形貌以及FRET的反应过程进行表征。在优化的条件下测得多巴胺检测体系的线性范围为0.01-60.0μM,检测限(Limit of Detection,LOD)为0.008μM。本方法可实现多巴胺注射液及血清样品中多巴胺的加标检测,实际样品结果表明,本方法具有良好的选择性,且能够准确检测复杂体系下多巴胺的浓度,为临床便捷检测多巴胺提供新的选择。第二章基于SGI和核酸适配体的ATP检测的免标记荧光传感器本章节建立了一种基于双链DNA嵌合荧光染料(SYBR Green I,SGI)和核酸适配体的免标记荧光传感体系用于三磷酸腺苷(Adenosine Triphosphate,ATP)检测。设计核酸适配体序列以形成长链双链DNA,加入ATP,ATP与适配体双链作用形成G四联体使得双链DNA(Double-stranded DNA,dsDNA)解链,释放原本结合在双链的SGI,使SGI的荧光强度减弱,并且降低的荧光强度与ATP浓度呈正相关,以此实现ATP的检测。本章利用电泳成像技术验证该检测机理。在优化的条件下,ATP检测体系的线性范围为10.0-5000.0μM,LOD为6.1μM,并具有良好的特异性。同时,通过与ATP化学发光试剂盒检测肿瘤细胞中ATP结果作相对误差分析,考察本方法用于实际样品检测的准确性。实验结果表明,本方法可准确检测实际样品的ATP浓度,可为检测复杂体系中ATP含量提供新的方法选择。第三章基于SGI和GO的新型免标记荧光传感器检测汞离子本章节设计了基于SGI和GO的检测汞离子的免标记荧光传感体系。设计T碱基错配探针,与Hg以T-Hg-T反应形成发夹结构DNA并结合SGI产生荧光作为信号实现对汞离子的检测。检测体系中加入GO可降低未嵌入双链DNA的SGI所产生的背景信号以提高信噪比。优化反应条件后,SGI信号与汞离子在10.0-2000.0nM范围内线性相关,LOD为2.2 nM。本方法可实现实际水样中汞离子的准确测定,可推广应用于环境监测、农残分析、食品药品工业等领域中汞离子的检测。
[Abstract]:Modern detection technology is developing towards fast, sensitive and high throughput direction. At the same time, fluorescence detection has attracted more and more attention because of its simple operation, time saving and sensitivity. The new fluorescence detection technology is expected to become the next generation high throughput biosegregation technology. The current fluorescence detection technology is mainly through the following two Technology routes: 1: fluorescent dye labeled fluorescent biosensors for imaging and detection, common fluorescent dyes including FITC and FAM; two: fluorescence detection methods used for the development of fluorescent nanomaterials, such as semiconductor quantum dots, metal clusters, etc., for the detection of DNA, protein and other small components. Therefore, the new method of developing high sensitive and highly specific detection target has broad application prospects. Based on the above considerations, three new type of free fluorescent sensing systems are established in this subject: the first chapter is based on the free standard of graphene quantum dots. The fluorescence sensor is used for dopamine detection in this chapter, a free tagged fluorescence sensing system based on graphene quantum dots is designed to detect dopamine. The graphene quantum dots (Graphene Quantum Dots, GQDs) are prepared by the pyrolysis of Ascorbic Acid (CA) and as a fluorescent probe, and the dopamine (Dopamine, DA) is self polymerized under alkaline conditions. Polymerization of Dopamine (pDA) is formed and adsorbed on the surface of GQDs to quenching fluorescence. The intensity of fluorescence quenching can be used for dopamine detection. The ultraviolet spectrum (Ultravioletand Visible Spectrophotometry, UV-vis) Fu Liye transform infrared spectroscopy (Fourier Transform Infrared), atomic force microscope (atomic force microscope) C Force Microscope, AFM), transmission electron microscopy (Transmission Electron Microscopy, TEM), zeta potential, photoelectron spectroscopy (X-ray Photoelectron Spectroscopy), characterization of the morphology and reaction process of this experimental system. The test limit (Limit of Detection, LOD) is 0.008 mu M. this method can realize dopamine addition detection in dopamine injection and serum samples. The actual sample results show that this method has good selectivity and can accurately detect dopamine concentration under complex system, which provides a new choice for convenient detection of dopamine in the bed. The second chapter is based on this method. The SGI and ATP detection of nucleic acid aptamers free labeling fluorescence sensor in this chapter established a free labeling fluorescence sensing system based on double stranded DNA chimeric fluorescent dye (SYBR Green I, SGI) and nucleic acid aptamers for adenosine triphosphate (Adenosine Triphosphate, ATP) detection. P, ATP and aptamer double chain action formed G four coupling to make double stranded DNA (Double-stranded DNA, dsDNA) dissolve chain, release the original binding of SGI in double chain, weaken the fluorescence intensity of SGI, and reduce the fluorescence intensity with ATP concentration, so as to achieve ATP detection. This chapter uses electrophoretic imaging to verify the detection mechanism. The optimization is optimized. Under the condition, the linear range of ATP detection system is 10.0-5000.0 mu M, LOD is 6.1 mu M and has good specificity. At the same time, the accuracy of this method is used to detect the actual sample by relative error analysis with the ATP chemiluminescence kit for detecting the ATP results in tumor cells. The experimental results show that this method can detect the actual sample accurately. The concentration of ATP can provide a new method for detecting the content of ATP in complex systems. In the third chapter, a new free labeling fluorescence sensor based on SGI and GO is used to detect mercury ions in this chapter. A fluorescent sensing system for detecting mercury ions based on SGI and GO is designed. A T base mismatch probe is designed to form a hairpin structure DNA with Hg with T-Hg-T. SGI produces fluorescence as a signal to detect the mercury ion. Adding GO in the detection system can reduce the background signal produced by the SGI of unembedded double strand DNA to improve the signal to noise ratio. After optimizing the reaction conditions, the SGI signal is linearly related to the mercury ion within the 10.0-2000.0nM range, and the LOD is the 2.2 nM. method for the realization of the mercury ion in the actual water sample. It can be widely applied to the detection of Hg in environmental monitoring, pesticide residue analysis, food and pharmaceutical industry, etc.

【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】：R446.6

【参考文献】