新型可控金纳米电极的研制及其用于时空分辨生物信号分子检测

发布时间：2018-05-13 16:33

  本文选题：金纳米颗粒 + 纳米电极　； 参考：《华东师范大学》2015年博士论文

【摘要】：神经细胞之间的信息传递是通过不同神经递质分子的传导来实现的。神经递质传导的紊乱和平衡状态的打破是导致多种中枢神经系统疾病的重要原因之一。因此,能够高灵敏、高时空分辨监测神经递质在神经系统中的动态释放,进而研究中枢神经系统疾病发生前后的神经递质水平的变化,将有助于理解中枢神经系统障碍或疾病的分子机制,为相关疾病的治疗提供方法学指导。 目前,对神经递质进行实时、在体、在线检测的方法主要有微渗析活体取样分析技术和在体快速循环伏安法。微渗析活体采样可以从生物活体内进行动态微量生化取样,有对组织损伤轻、取样少、可与高效液相、毛细管电泳等分离技术有效结合等优点,可应用于实验动物的所有组织和器官,特别是在神经递质的在体检测方面的研究和应用,都得到了很好的结果,为探讨相关神经系统疾病发病的机制提供了有利的分析方法。而在体快速循环伏安法由于微电极尺寸小、时空分辨率高、扩散效率和响应速度快等优点,目前已经被广泛应用于生物活体分析及单细胞分析等领域。 但是,在体快速循环伏安法和微渗析活体取样法只是对扩散至细胞外液中的递质进行研究,还不能直接检测肌体受到刺激后突触间瞬时释放产生的递质水平。由于突触间隙距离很小(小于100nm),若要用电化学方法直接检测间隙内递质分子的释放水平,则需要发展体积小、灵敏度高、时空分辨率高、直径小于100nm的纳米电极。为了制备适于突触间隙分析的纳米电极,本论文设计了简单、绿色环保的方法制备了尺寸可控的金纳米电极。在此基础上,通过在纳米电极组装特异性分子,发展了具有良好选择性、稳定性、灵敏度和生物相容性的功能纳米电极,实现对神经递质的高选择性的直接检出。全文共分为五个部分,具体内容如下： 第一章绪论 本章主要介绍了纳米电极的研究背景、纳米电极的研究意义、纳米电极的分类及制备方法以及纳米电极在生物化学中的应用,着重介绍了纳米电极的制备方法及其表征和纳米电极的应用。 第二章基于混合包裹法和电化学刻蚀可控制备盘状金纳米电极 本工作中,我们先利用电化学刻蚀然后采用混合包裹法制备了一种新型的尺寸可控的盘状金纳米电极,该方法简单、绿色环保。通过TEM和稳态循环伏安法对盘状金纳米电极尖端进行了表征,结果表明：金纳米尖端最小半径为～6nm；电极外部混合绝缘层薄而均匀,厚度在30m左右,经过包裹后的电极尖端整体大小仍能保持在纳米级别；在KCl溶液里通过循环伏安扫描刻蚀裸露的金尖端后得到盘状金纳米电极。电极在K3Fe(CN)6、Fc、K3IrCl6三种标准体系中进行稳态循环伏安扫描,循环伏安曲线具有很好的“S”形貌,说明该方法制备的电极具备了纳米电极的特征；并利用所得的稳态极限电流准确地求出电极的有效半径；电极的尺寸可控且电极的制备重现性好,电极表面的绝缘层在水相和有机相体系中均表现出了良好的稳定性。该部分工作为后续动力学参数的求算和生物中的应用提供了有利的实验工具。 第三章盘状金纳米电极的电化学行为及其在单细胞检测中的应用 本工作中,通过稳态循环伏安法,利用不同有效半径的盘状金纳米电极在K3Fe(CN)6、Fc和K3IrCl6三种标准体系中进行稳态循环伏安扫描,研究盘状金纳米电极表面的动力学行为,通过统计数据求出了三个体系中电极表面的传递系数(a)和异相电子转移速率常数(k°),实验结果与文献报道一致,表明盘状纳米电极表面物质的传递速率快,具有较大的异相电子转移速率常数等优越的电化学性能,有利于电极在生物领域中的应用。电极经nafion修饰后,对多巴胺具有较好的选择性、灵敏度高、低的检测限和宽的线性范围等优点。单细胞实验中,选用PC12(肾上腺嗜铬细胞瘤)细胞为研究对象,nafion修饰的盘状金纳米电极成功地监测到了PC12单细胞中的单个囊泡中的胞吐释放,PC12单细胞中囊泡内的主要成分为多巴胺,与其他文献报道的类似。单细胞膜上并不是所有地方都有囊泡释放,释放点在细胞上成散点分布；活性区域内,在一个释放点释放的囊泡个数也无明显规律；即使是非常相近的活性释放点,释放的囊泡个数也不相同。说明本工作中所制备的纳米电极灵敏度高、检测限低、生物相容性较好,为后续检测神经细胞突触间神经递质分子的释放提供了潜在的分析工具。 第四章纳米毛细管尖端外壁化学沉积金纳米颗粒制备金纳米电极及其对鼠脑中多巴胺的分析检测 本工作中,主要论述在石英毛细管锥形尖端外壁附着一层由金纳米粒子构成的金膜,通过控制电泳时间、电泳电位、电泳次数、金纳米颗粒的沉积时间等来制备一种尺寸可控的金纳米电极,及其经nafion修饰后在活体分析中的应用。首先,通过调节激光拉制仪的五个参数将石英毛细管拉制成纳米尺寸的锥形尖端；然后依次经过羟基化、硅烷偶联剂修饰、附着金纳米颗粒生长因子、在氯金酸和盐酸羟胺混合生长液中附着由金纳米颗粒形成的金膜；最后将附着金纳米颗粒的锥形尖端制备成金电极,经阴极电泳漆绝缘包裹即可以得到金纳米电极。SEM结果表明电极尖端形貌符合半球状金纳米电极的特征。电化学实验结果表明：电极在铁氰化钾和三氯化六氨合钌标准溶液中均呈现良好的“S”图谱,经计算得出该类电极尺寸大小达到了纳米级别。经Nafion修饰的金纳米电极对多巴胺有良好的选择性,线性范围宽,在5.6×10～52×10-8M之间均呈现了良好的线性响应,灵敏度高、检测限低(1×10-8M)。该工作所制备的金纳米电极除尺寸小等优点外,以石英光纤为基底大大地提高了电极的韧性和硬度及电极寿命,成功地被植入已麻醉的SD大鼠大脑纹状体区域并得到了在线的电流-电位曲线,结果表明老鼠纹状体内多巴胺浓度可受外部药物刺激的影响而增大,浓度最高可达49nM。为后续在鼠脑等生物组织中直接检测神经递质分子提供了新的分析方法和工具。 第五章不同形貌的金纳米材料在金纳米电极表面的电化学行为研究 本工作中,我们利用激光拉制法和精细打磨的方法,成功地制备出了尺寸可控、稳定性好的金纳米电极,并以此为基底分别附着上不同形貌的金纳米粒子以考察不同形貌的金纳米粒子对多巴胺的响应。借助光学显微镜、SEM, TEM对电极的表面形貌进行了表征。通过稳态循环伏安法计算电极的有效半径其最小可达6.82nm。在+200mV下,经nafion修饰的电极尖端附着了不同形貌的金纳米颗粒的金纳米电极对多巴胺都有良好的响应,其中锥形金纳米颗粒本身因具有较好的自净能力和很好的稳定性,对多巴胺的响应最好。经校准,对多巴胺的检测限(S/N=3)分别为：锥形金纳米颗粒(AuNP):～5.2×10-9M,棒形金纳米颗粒(AuNR):～1.71×10-8M,球形金纳米颗粒(AuNS):～3.2×10-8M。锥形金纳米颗粒对多巴胺的线性范围为：1×10-8～2.55×10-6M,该类电极具有良好的重现性、稳定性,且电极的利用率很高。以附着有锥形纳米颗粒的金纳米电极为工作电极植入已麻醉的大鼠纹状体内在线研究了脑内多巴胺的释放水平。
[Abstract]:The transmission of information between nerve cells is achieved through the conduction of different neurotransmitters . The disorder of neurotransmitter conduction and the breaking of equilibrium state are one of the important causes of various central nervous system diseases . Therefore , it is possible to monitor the dynamic release of neurotransmitters in the nervous system with high sensitivity and high space - time resolution , and then to study the changes of the neurotransmitter levels before and after the onset of the central nervous system disease , which will help to understand the molecular mechanism of the disorders or diseases of the central nervous system and provide methodological guidance for the treatment of related diseases .

The present invention has the advantages of low micro - electrode size , high space - time resolution , high diffusion efficiency , high response speed and the like , and is widely applied to the fields of living body analysis and single - cell analysis .

In order to prepare the nanoelectrodes suitable for synaptic cleft analysis , the nanoelectrodes with controllable size can be directly detected by electrochemical methods . In order to prepare the nano electrodes suitable for synaptic cleft analysis , the method is simple and environmentally friendly . In order to prepare the nano electrodes suitable for synaptic cleft analysis , the direct detection of high selectivity of neurotransmitters is realized .

Chapter 1 Introduction

This chapter mainly introduces the research background of nano - electrode , the research significance of nano - electrode , the classification of nano - electrode , its preparation method and the application of nano - electrode in biochemistry , focusing on the preparation method and characterization of nano - electrode and the application of nano - electrode .

The second chapter is based on hybrid wrapping method and electrochemical etching to control the disk - shaped gold nano - electrode .

In this work , a novel size - controllable disc - shaped gold electrode with controllable size is prepared by electrochemical etching . The method is simple , green and environment - friendly , and the tip of the disk - shaped gold electrode is characterized by TEM and steady - state cyclic voltammetry . The results show that the minimum radius of the gold nanometer tip is ~ 6 nm ;
the external mixed insulating layer of the electrode is thin and uniform , the thickness is about 30 m , and the overall size of the electrode tip after the wrapping can be kept at the nanometer level ;
The disk - shaped gold nano - electrodes were obtained by cyclic voltammetry scanning in KCl solution . The electrodes were subjected to steady - state cyclic voltammetry scanning in three standard systems of K _ 3Fe ( CN ) 6 , Fc and K _ 3 IrCl6 , and the cyclic voltammogram has a good " S " shape , and the electrode prepared by the method has the characteristics of nano - electrode .
and using the obtained steady - state limit current to accurately calculate the effective radius of the electrode ;
the size of the electrode is controllable and the preparation reproducibility of the electrode is good , and the insulating layer on the surface of the electrode shows good stability in the water phase and the organic phase system .

The Electrochemical Behavior and Its Application in Single Cell Detection

In this work , by steady - state cyclic voltammetry , the dynamic behavior of the surface of the disk - shaped gold nano - electrode was investigated by means of steady - state cyclic voltammetry scanning in three standard systems of K _ 3Fe ( CN ) 6 , Fc and K _ 3 IrCl6 .
There was no obvious regularity in the number of vesicles released at one release point in the active area .
The prepared nano electrode is high in sensitivity , low in detection limit and good in biocompatibility , and provides a potential analysis tool for subsequent detection of the release of nerve cell synaptic neurotransmitter molecules .

Preparation of gold nanoparticles by chemical deposition of gold nanoparticles on the outer wall of nano capillary tip and analysis of dopamine in rat brain

In this work , a gold film composed of gold nanoparticles is attached to the outer wall of the conical tip of the quartz capillary , and the gold nano - electrode with controllable size is prepared by controlling the electrophoresis time , the electrophoresis potential , the number of electrophoresis , the deposition time of the gold nanoparticles and the like .
then the gold film formed by gold nanoparticles is attached in the mixed growth liquid of chloroauric acid and hydroxylamine hydrochloride through hydroxylation , silane coupling agent modification and gold nanoparticle growth factor ;
The results show that the electrode has a good linear response to dopamine in the standard solution of potassium cyanide and ruthenium trichloride . The results show that the electrode has a good selectivity to dopamine and has high sensitivity and low detection limit ( 1 脳 10 - 8M ) . The results show that the concentration of dopamine in the striatum of rat striatum can be increased by the influence of external drug stimulation . The results show that the concentration of dopamine in striatum of rats is up to 49 nM .

Study on the Electrochemical Behavior of Gold Nanomaterials with Different Morphology on the Surface of Gold Nanoelectrodes

In this work , we have successfully prepared gold nano - particles with controllable size and good stability by laser - drawing and fine - polishing . The surface features of the electrodes are characterized by optical microscopy , SEM and TEM . The linear range of the electrodes is 1 脳 10 - 8 锝，

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