【作者】 陈璟；

【导师】 陈杭； 陈星； 张韶岷；

【作者基本信息】 浙江大学 ， 生物医学工程， 2020， 博士

【摘要】 大脑的神经活动是一个电与化学活动相结合的过程,从化学信号(神经递质)的角度去研究神经活动是当前非常重要的一个研究方向。电化学传感方法因为其小型化、易操作、方便快速、可实时在线等的优势,成为了一个越来越受到关注的研究方法。然而,采用电化学传感的方法检测神经递质需要突破两个关键问题。一方面,电化学传感器件(微电极)的尺寸和检测下限难以匹配生理环境,传感器难以兼顾小尺寸、高灵敏度、选择性、稳定性和可重现性的问题;另一方面,缺乏稳定、高精度的便携电化学检测仪器,进一步限制了相应电化学传感器件的实际应用和推广。因此,本论文针对上述两个关键问题,设计和实现了基于电化学传感技术的便携式神经递质浓度检测系统。系统前端以多巴胺和谷氨酸两种代表性的神经递质为主要研究对象,设计了可工作于生物体内复杂环境的高灵敏度、高选择性新型电化学传感器;系统后端针对神经递质检测的快速、高灵敏度、小尺寸和抗干扰的要求,设计了多路可拓展的便携式高精度神经递质浓度检测仪器;两者整合成为一套完整的电化学神经递质浓度检测系统,并应用于实际样品中神经递质的多路浓度同时检测。论文的主要工作内容和创新点如下:1.设计并实现了基于还原型氧化石墨烯与金纳米颗粒复合纳米材料构建的新型铂丝电化学微电极。通过电沉积的方式在铂丝表面形成均匀分布的还原型氧化石墨烯和金纳米颗粒复合膜,构建多巴胺微电极。复合膜高比表面积、高电子传导和良好生物相容的特性有助于对抗多巴胺污垢,解决了当前铂丝电极检测多巴胺时表面聚集和吸附的问题。微电极表现出对多巴胺的高灵敏度和低检测下限(16.57 nM)。同时,电极在复杂环境中能够有效抵抗DA前体和其他单胺类神经递质的干扰。另一方面,电极在重复试验中表现出较高的可重现性(相对标准偏差为3.98%)和稳定性(100次的重复扫描后损耗为3.43%)。通过初步实验验证了电极具备在麻醉大鼠的纹状体内检测多巴胺浓度变化的功能。该电极在灵敏度和选择性等方面具有较高的综合性能,为多巴胺实时动态的检测提供了新方法。2.设计并实现了基于谷氨酸氧化酶的新型谷氨酸电极,电极表面修饰还原型氧化石墨烯、普鲁士蓝、金纳米颗粒以及壳聚糖复合膜。高催化活性的表面使电极表现出对谷氨酸的优越的电催化性能,检测下限达到41.33 nM,并在细胞外间隙的生理浓度范围内表现出浓度-电流的线性依赖关系。电极在100次检测中仅损失3.62%,并在放置14天内保持92.14%以上的初始信号强度。另外,初步实验观察到电极能够在大鼠纹状体内检测到谷氨酸浓度的变化。该电极在尺寸、检测下限、抗干扰性、使用寿命等综合性能上有所提高,为谷氨酸实时动态的检测提供了新方法。3.设计并实现了用于神经递质检测的便携式、高精度、多路可拓展神经递质浓度检测仪器。通过集成微弱信号检测技术和电源抗干扰技术,实现高扰动下的微弱电流信号检测,具有小尺寸、高精度(误差<3%)、高信噪比(77.52 d B)、低检测限(5.35 n A)、宽线性范围且可以无线传输等优点。该仪器能够在标准混合溶液体系中对多巴胺和谷氨酸的浓度实现同步检测,并在大鼠纹状体中检测到多巴胺和谷氨酸的浓度受人为干预产生的变化信号以及动态代谢信号。初步实验验证了系统进行多巴胺和谷氨酸在体检测的可行性。综上所述,本文设计并实现了基于电化学传感技术的便携式神经递质浓度检测系统。该系统包含高灵敏度、高选择性的新型多巴胺和谷氨酸传感器,以及高精度、便携式检测仪器。进行了体内实验的初步验证,结果表明该系统能够在生理环境中检测到大鼠脑内多巴胺和谷氨酸浓度水平的动态变化,有望在今后的在体神经递质浓度实时检测和相关研究中发挥作用。更多还原

【Abstract】 The neural activity of the brain is a process combining electrical and chemical activities.At present,it is a very important research flied to study the brain from the perspective of chemical signals(especially neurotransmitters).The in-situ electrochemical sensing method has attractted more and more attention because it is small,convenient,fast,real-time,online and easy to operate.However,there are two key problems in the detection of neurotransmitters by electrochemical sensing method.On the one hand,the size,lower detection limit,and anti-interference ability of electrochemical sensor devices(microelectrodes)are still far from the requirements of actual detection;on the other hand,the lack of portable and reliable electrochemical detection instrument further restricts the practical application and promotion of the corresponding electrochemical sensor devices.Therefore,aiming at the above two key problems,this theis designs and implements a portable neurotransmitter concentration detection system based on electrochemical sensing technology.The system takes dopamine and glutamate,which are two of the representative neurotransmitters,as the main research objects.Novel dopamine and glutamate electrochemical sensors with high sensitivity and high selectivity that can work in the complex environment of organisms is designed.Moreover,for the requirements of body detection such as fast,high sensitivity,small size and anti-interference,a portable high-precision neurotransmitter detection instrument is designed.Finally,the sensors and the instrument are integrated into the electrochemical neurotransmitter detection system,and applied to the concentration monitoring of specific neurotransmitters in actual samples.The main contents and innovations of the thesis are as follows:1.A novel platinum wire electrochemical microelectrode based on reduced graphene oxide and gold nanoparticle composite nanomaterials is designed and realized.A uniformly distributed composite film of reduced graphene oxide and gold nanoparticles is formed on the surface of the platinum wire by electrodeposition,and the platinum wire microelectrode is constructed.The high specific surface area,high electronic conductivity and good biocompatibility of the composite membrane can resist dopamine fouling and alleviate the problem of surface aggregation and adsorption when detecting dopamine with platinum wire electrodes.The microelectrode shows high sensitivity and low detection limit(16.57 nM)to dopamine.At the same time,the electrode can effectively resist the interference of DA precursors and other monoamine neurotransmitters in a complex environment.Moreover,the electrode shows high reproducibility(relative standard deviation of 3.98%)and stability(3.43% loss after100 repeated scans).In vivo experiments verify that the electrode has the function of detecting changes of dopamine concentration in the striatum of anesthetized rats.The electrode shows great comprehensive performance,such as sensitivity and selectivity,and provides a new method for real-time dynamic monitoring of dopamine.The electrode has the potential to be used to study specific structures or multi-site dopamine detection in organisms.2.A novel microelectrode based on glutamate oxidase is designed and realized by the modification of reduced graphene oxide,Prussian blue,gold nanoparticles and chitosan material.The surface of high catalytic activity makes the electrode show superior electrocatalytic performance to glutamic acid,exhibit superior electrocatalytic performance for glutamate with a detection limit of 41.33 nM,and a linear concentration-current dependence in the physiological concentration range of the extracellular space.The electrode loses only 3.62% of signal intensity in 100 tests,and maintains 92.14% of the initial signal intensity within 14 days.In addition,in vivo experiments verifiy the anti-interference of the electrode in a complex physiological environment,and it is observed that the electrode can detect changes in the concentration of glutamate in the rat striatum.The electrode shows great comprehensive performance in terms of size,lower detection limit,anti-interference,and service life,provides a new method for real-time dynamic monitoring of glutamate,and shows the potential for monitoring multi-site and multi-channel transmitters in samples or organisms for a long time.3.A portable,high-precision,current-type neurotransmitter concentration detection instrument for neurotransmitter detection is designed and realized.By integrating the technology of microsignal detection and anti-jamming of power supply,the detection instrument realizes microcurrent signal detection under high disturbance,showing the advantages of small size,high precision(error<3%),high signal-to-noise ratio(77.52 d B),low detection limit(5.35 n A),wide linear range and wireless transmission.The portable detection instrument can be used to detect the concentration of dopamine and glutamate in the standard mixed solution system synchronously,and detect the change signal and dynamic metabolic signal of the concentration of dopamine and glutamate in the rat striatum,showing the potential of the system for long-term dynamic detection and research of multiple neurotransmitter levels in the biological brain.In summary,a portable neurotransmitter concentration detection system based on electrochemical sensing technology is designed and realized in this thesis.The system includes the dopamine and glutamate sensor with high sensitivity and high selectivity,as well as the high-precision,portable detection instrument.In vivo experiments show that the high-performance neurotransmitter detection system can be used to detect the dynamic changes of dopamine and glutamate concentration in a complex in-vivo environment,which is expected to play a role in the real-time monitoring of in-vivo neurotransmitter concentration and related research in the future.更多还原