基于多巴胺适配体标记的碳点和纳米石墨的荧光生物传感平台的构建及其应用
发布时间：2018-11-26 15:19
【摘要】：多巴胺(Dopamine, DA)作为中枢神经系统一种重要的单胺类神经递质,对心血管系统、中枢神经系统、内分泌系统等具有广泛而重要的影响。体内DA含量缺乏可能会引起多种神经系统性疾病,如阿尔茨海默症、精神分裂症、帕金森综合征等；而体内DA含量过高则会导致机体处于兴奋或焦虑状态。体内DA含量的快速、准确检测在神经系统性疾病的临床诊断以及病理机制研究方面具有十分重要的作用。但由于生物基质的构成复杂,且DA含量非常低,所以构建高灵敏度、高选择性的DA快速分析方法具有十分重要的现实意义。碳点(Carbon dots, CDs)又称荧光碳纳米颗粒,是一种以碳为骨架结构,粒径小于10 nm的类球形荧光纳米颗粒。CDs不仅具有传统半导体量子点的光学性能(如激发谱带宽、发射谱带窄、荧光强度高、光化学稳定性好等),还具有高量子产率,低细胞毒性,高生物相容性,发射谱激发波长依赖性等特性。目前以CDs为荧光探针构建的生物传感平台多基于荧光猝灭原理,但此类探针的最大缺陷在于：在较强的荧光背景下测定荧光猝灭的程度,将会极大地影响探针检测的灵敏度。因此,为了进一步提高检测的灵敏度,越来越多的研究者开始关注增强型即on-off-on型荧光CDs生物传感平台的构建。本论文以生物样品中痕量DA的检测为目的,以DA核酸适配体标记的CDs(Aptamer-CDs)作为待测物的荧光识别基团,同时以纳米石墨(Nano-graphite.NG)作为荧光猝灭剂构建了能够高选择性、高灵敏度地检测生物样品中痕量DA的荧光生物传感平台,并将其成功应用于生物样品中痕量DA的检测。概要如下：1. Aptamer-CDs/NG生物传感平台的构建CDs表面的羧基经活化后与DA aptamer核酸链上修饰的氨基相作用生成酰胺键,成功合成Aptamer-CDs复合物,然后在体系中引入猝灭剂NG,Aptamer-CDs的荧光被猝灭。当体系中没有DA存在的条件下,Aptamer-CDs的荧光被猝灭；当体系中存在DA时,Aptamer-CDs可以与DA特异性结合形成特定的球状链结构,此结构从NG表面脱落,Aptamer-CDs的荧光恢复。根据Aptamer-CDs的荧光恢复程度与DA浓度之间的线性关系实现对样品中DA含量的定量检测。试验优化了Aptamer-CDs复合物合成以及传感平台应用的反应物比例、温度、时间等条件,最后通过傅立叶红外光谱,琼脂糖凝胶电泳以及高分辨透射电镜对所构建的荧光传感平台进行了结构和形貌表征,证明Aptamer-CDs/NG生物传感平台的成功构建。2. Aptamer-CDs/NG生物传感平台的应用基于Aptamer-CDs和NG,我们构建了新型的on-off-on型荧光生物传感平台,并成功实现对尿液中痕量DA含量的高灵敏度、高选择性测定。干扰试验表明,Aptamer-CDs/NG生物传感平台具有良好的选择性。研究结果表明,在最佳试验条件下,当DA浓度在0.10～5.00 nM范围内,构建的生物传感平台的荧光强度的相对变化值与DA浓度的自然对数值呈良好的线性关系,相关系数为0.9995,检测限为0.055 nM。在该方法建立的基础上,采用标准加入法,测得健康实验室志愿者的尿液(稀释60倍)中DA的含量分别为0.75 nM和0.80 nM,DA的加标回收率均在96.0%～105.2%之间,且相对标准偏差小于18.7%(n=3)。试验结果表明构建的on-off-on型荧光生物传感平台能够满足复杂生物样品中DA的分析测定要求。本论文设计的荧光生物传感平台操作绿色便捷,测定复杂生物样品时仅需进行稀释处理便可实现对样品中所含DA的快速分析测定。
[Abstract]:Dopamine (DA), as an important monoamine neurotransmitter in the central nervous system, has a wide and important influence on the cardiovascular system, the central nervous system and the endocrine system. The lack of in vivo DA content may cause multiple nervous system diseases, such as Alzheimer's disease, schizophrenia, Parkinson's syndrome, etc., while the high in vivo DA content can cause the body to be in an excited or anxious state. The rapid and accurate detection of the content of DA in vivo has a very important role in the clinical diagnosis of nervous system diseases and the research of the pathological mechanism. However, because of the complex composition of the biological matrix, and the content of DA is very low, it is of great practical significance to construct a high-sensitivity and high-selectivity DA rapid analysis method. Carbon dot (CDs), also called fluorescent carbon nanoparticles, is a kind of spherical fluorescent nanoparticles with carbon skeleton structure and particle size less than 10 nm. The CDs not only has the optical properties of the conventional semiconductor quantum dots (such as the excitation spectrum bandwidth, narrow emission spectrum band, high fluorescence intensity, good photochemical stability, etc.), but also has the characteristics of high quantum yield, low cell toxicity, high biocompatibility, emission spectrum excitation wavelength dependence, and the like. The biological sensing platform, which is constructed with CDs as a fluorescent probe, is based on the principle of fluorescence excitation, but the maximum defect of such a probe is that the degree of fluorescence extinction is measured in a stronger fluorescence background, and the sensitivity of the probe detection will be greatly affected. Therefore, in order to further improve the sensitivity of detection, more and more researchers have begun to focus on the construction of the enhancement-on-off-on-type fluorescence CDs biological sensing platform. The purpose of this paper is to detect the trace DA in the biological sample, and the DA nucleic acid aptamer-labeled CDs (Aptamer-CDs) is used as the fluorescent recognition group of the object to be tested, and the nano-graphite (Nano-graphite. NG) is used as the fluorescent quenching agent to construct the high-selectivity. The fluorescence biosensor platform of the trace DA in the biological sample is detected with high sensitivity and is successfully applied to the detection of trace DA in the biological sample. The summary is as follows: 1. Aptamer-CDs/ NG bio-sensing platform constructs the amine bond with the amino phase of the modified amino phase on the DA aptamer nucleic acid chain after activation, and successfully synthesizes the Aptamer-CDs complex, and then introduces the quencher NG in the system, and the fluorescence of the Aptamer-CDs is destroyed. When DA is not present in the system, the fluorescence of Aptamer-CDs is destroyed; when DA is present in the system, Aptamer-CDs can be specifically combined with DA to form a specific spherical chain structure, and the structure is detached from the NG surface, and the fluorescence of Aptamer-CDs is recovered. The quantitative detection of DA content in the sample was achieved according to the linear relationship between the degree of fluorescence recovery and the DA concentration of Aptamer-CDs. The synthesis of Aptamer-CDs and the proportion of reactants, temperature and time of the application of the sensing platform were optimized, and the structure and morphology of the constructed fluorescence sensing platform were characterized by the Fourier infrared spectrum, the agarose gel electrophoresis and the high-resolution transmission electron microscope. The success of the Aptamer-CDs/ NG Biosensing Platform was demonstrated. The application of the Aptamer-CDs/ NG Biosensing Platform is based on Aptamer-CDs and NG, and we construct a new type of on-off-on-type fluorescence bio-sensing platform, and successfully realize high sensitivity and high selectivity for trace DA content in urine. The interference test shows that the Aptamer-CDs/ NG bio-sensing platform has good selectivity. The results show that, under the best test condition, the relative change of the fluorescence intensity of the constructed biosensing platform and the natural logarithm of the DA concentration in the range of 0. 10-5. 00 nM have a good linear relationship with the natural logarithm of the DA concentration, and the correlation coefficient is 0.9995 and the detection limit is 0.055 nM. On the basis of the establishment of this method, the content of DA in the urine (60-fold dilution) of healthy laboratory volunteers was 0. 75 nM and 0. 80 nM, respectively, and the relative standard deviation was less than 18.7% (n = 3). The results show that the on-off-on-type fluorescence biosensor can meet the requirement of the analysis and determination of DA in complex biological samples. The fluorescence bio-sensing platform designed in this paper is simple and convenient to operate, and the rapid analysis and determination of the DA in the sample can be realized only by the dilution treatment when the complex biological sample is determined.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：O657.3;R446.1
本文编号：2358923
[Abstract]:Dopamine (DA), as an important monoamine neurotransmitter in the central nervous system, has a wide and important influence on the cardiovascular system, the central nervous system and the endocrine system. The lack of in vivo DA content may cause multiple nervous system diseases, such as Alzheimer's disease, schizophrenia, Parkinson's syndrome, etc., while the high in vivo DA content can cause the body to be in an excited or anxious state. The rapid and accurate detection of the content of DA in vivo has a very important role in the clinical diagnosis of nervous system diseases and the research of the pathological mechanism. However, because of the complex composition of the biological matrix, and the content of DA is very low, it is of great practical significance to construct a high-sensitivity and high-selectivity DA rapid analysis method. Carbon dot (CDs), also called fluorescent carbon nanoparticles, is a kind of spherical fluorescent nanoparticles with carbon skeleton structure and particle size less than 10 nm. The CDs not only has the optical properties of the conventional semiconductor quantum dots (such as the excitation spectrum bandwidth, narrow emission spectrum band, high fluorescence intensity, good photochemical stability, etc.), but also has the characteristics of high quantum yield, low cell toxicity, high biocompatibility, emission spectrum excitation wavelength dependence, and the like. The biological sensing platform, which is constructed with CDs as a fluorescent probe, is based on the principle of fluorescence excitation, but the maximum defect of such a probe is that the degree of fluorescence extinction is measured in a stronger fluorescence background, and the sensitivity of the probe detection will be greatly affected. Therefore, in order to further improve the sensitivity of detection, more and more researchers have begun to focus on the construction of the enhancement-on-off-on-type fluorescence CDs biological sensing platform. The purpose of this paper is to detect the trace DA in the biological sample, and the DA nucleic acid aptamer-labeled CDs (Aptamer-CDs) is used as the fluorescent recognition group of the object to be tested, and the nano-graphite (Nano-graphite. NG) is used as the fluorescent quenching agent to construct the high-selectivity. The fluorescence biosensor platform of the trace DA in the biological sample is detected with high sensitivity and is successfully applied to the detection of trace DA in the biological sample. The summary is as follows: 1. Aptamer-CDs/ NG bio-sensing platform constructs the amine bond with the amino phase of the modified amino phase on the DA aptamer nucleic acid chain after activation, and successfully synthesizes the Aptamer-CDs complex, and then introduces the quencher NG in the system, and the fluorescence of the Aptamer-CDs is destroyed. When DA is not present in the system, the fluorescence of Aptamer-CDs is destroyed; when DA is present in the system, Aptamer-CDs can be specifically combined with DA to form a specific spherical chain structure, and the structure is detached from the NG surface, and the fluorescence of Aptamer-CDs is recovered. The quantitative detection of DA content in the sample was achieved according to the linear relationship between the degree of fluorescence recovery and the DA concentration of Aptamer-CDs. The synthesis of Aptamer-CDs and the proportion of reactants, temperature and time of the application of the sensing platform were optimized, and the structure and morphology of the constructed fluorescence sensing platform were characterized by the Fourier infrared spectrum, the agarose gel electrophoresis and the high-resolution transmission electron microscope. The success of the Aptamer-CDs/ NG Biosensing Platform was demonstrated. The application of the Aptamer-CDs/ NG Biosensing Platform is based on Aptamer-CDs and NG, and we construct a new type of on-off-on-type fluorescence bio-sensing platform, and successfully realize high sensitivity and high selectivity for trace DA content in urine. The interference test shows that the Aptamer-CDs/ NG bio-sensing platform has good selectivity. The results show that, under the best test condition, the relative change of the fluorescence intensity of the constructed biosensing platform and the natural logarithm of the DA concentration in the range of 0. 10-5. 00 nM have a good linear relationship with the natural logarithm of the DA concentration, and the correlation coefficient is 0.9995 and the detection limit is 0.055 nM. On the basis of the establishment of this method, the content of DA in the urine (60-fold dilution) of healthy laboratory volunteers was 0. 75 nM and 0. 80 nM, respectively, and the relative standard deviation was less than 18.7% (n = 3). The results show that the on-off-on-type fluorescence biosensor can meet the requirement of the analysis and determination of DA in complex biological samples. The fluorescence bio-sensing platform designed in this paper is simple and convenient to operate, and the rapid analysis and determination of the DA in the sample can be realized only by the dilution treatment when the complex biological sample is determined.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：O657.3;R446.1
【参考文献】
相关硕士学位论文 前2条
1 刘威;基于量子点荧光探针的肿瘤标志物GP73和腺苷的分析方法研究[D];南京医科大学;2015年
2 郭艳;碳量子点的结构对荧光发射行为的影响[D];中北大学;2012年
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