New Electroanalytical Methods for Single-Cell Exocytosis

单细胞胞吐作用的新电分析方法

• 批准号: 8650907
• 负责人: Bo Zhang
• 金额: $ 27.82万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-01 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8650907
• 关键词: Address; Affect; Behavioral; Brain; Brain Diseases; Cell membrane; Cell model; Cell physiology; Cells; Central Nervous System Diseases; Characteristics; Chemicals; Communication; Communities; Computer Simulation; Disease; Dopamine; Electrochemistry; Electrodes; Emotional; Event; Exocytosis; Extracellular Space; Geometry; Goals; Heterogeneity; Hormones; Huntington Disease; Image; Immune response; Kinetics; Knowledge; Lead; Location; Mediating; Methods; Microelectrodes; Neuromodulator; Neurons; Neurotransmitters; PC12 Cells; Parkinson Disease; Pheochromocytoma; Physiological Processes; Play; Positioning Attribute; Quartz; Reagent; Relative (related person); Reproducibility; Resolution; Role; Scanning; Single Cell Exocytosis; Techniques; Use of New Techniques; Vesicle; Work; analytical method; base; cellular imaging; design; improved; innovation; nanopore; nervous system disorder; neurotransmission; response; tool; voltage

DESCRIPTION (provided by applicant): We propose to develop and utilize new electroanalytical methods to study single exocytotic events. Exocytosis is of central importance in neuronal transmission, release of hormones and neuromodulators, and immune response and plays an essential role in mediating brain function, emotional and behavioral responses, and many other physiological processes. As such, a detailed understanding of single exocytotic events is needed for better treatments for central nervous system diseases. Electroanalytical methods have played vital roles in this bioanalytical task in the past three decades owning to their unique characteristics, including very high spatial, temporal resolutions, and excellent chemical resolution. However, current electroanalytical methods have significant challenges. For example, the quantal size and release kinetics of the exocytotic event is likely affected by it location relative to the electrode. Array-based electrochemical imaging on single-cells has strong crosstalk. In addition, current methods do not allow for analysis of intracellular vesicles. We propose to address these challenges by developing new electroanalytical techniques and microelectrodes. We emphasize the use of a nanoband electrode integrated with on-chip microelectrodes to increase accuracy and eliminate crosstalk. We also develop new cyclic voltammetric methods to increase temporal resolution and further eliminate crosstalk in voltammetric imaging. Additionally, we use a nanopore electrode to analyze single intracellular vesicles. Building on our strong expertise in single-cell exocytosis, microelectrodes, and nanopores, we propose to accomplish our goal by pursuing three specific aims: Aim 1. To develop and utilize an on-chip microelectrode integrated with a nanoband electrode to analyze single exocytotic events. We will analyze single exocytotic events using an integrated on-chip microelectrode. We will use computer simulation to achieve a deeper understanding of dopamine transport at the electrode/cell interface. We will optimize electrode geometry to improve accuracy and reproducibility in single-cell amperometry. In addition, we will improve their stability and sensitivity through electrode design and chemical functionalization. Aim 2. To eliminated crosstalk and increase temporal resolution in electrochemical imaging utilizing new microelectrode arrays and voltage waveforms. We will image single-cell exocytosis with eliminated crosstalk using amperometry and voltammetry. We will employ new microelectrode arrays and use integrated nanoband electrodes to eliminate crosstalk in amperometry. We will then apply new voltage waveforms in fast-scan CV to improve temporal resolution and eliminate crosstalk in voltammetric imaging. Aim 3. To further develop and utilize a nanopore-based method to simultaneously analyze the sizes and dopamine contents of single intracellular vesicles. A new nanopore electrode has been developed using a quartz nanopore and a microelectrode placed in close proximity to the pore orifice. This microprobe is especially useful for simultaneously determining the sizes and dopamine contents of single intracellular vesicles. We will further develop this new technique and use it to analyze vesicles from model cells. We will use this technique to characterize vesicles from single cells treated by pharmacological reagents. This work will provide new analytical strategies for better understanding single exocytotic events. Our proposed methods have key advantages and can provide new information inaccessible with current techniques. The integration of a nanoband electrode increases the accuracy in determining quantal size and kinetics. New voltage waveforms and microarrays can improve single-cell imaging to better study exocytotic heterogeneity. A nanopore electrode can analyze single intracellular vesicles. We anticipate these new methods and probes will find extensive use in analyzing exocytosis and will be quickly adapted by other groups in the community and become their everyday tools.

描述（由申请人提供）：我们建议开发和利用新的电分析方法来研究单个胞吐事件。胞吐作用对于神经元传递、激素和神经调节剂的释放以及免疫反应至关重要，并且在调节大脑功能、情绪和行为反应以及许多其他生理过程中发挥着重要作用。因此，需要详细了解单个胞吐事件，以便更好地治疗中枢神经系统疾病。过去三十年来，电分析方法凭借其独特的特性，包括极高的空间、时间分辨率和出色的化学分辨率，在生物分析任务中发挥了至关重要的作用。然而，当前的电分析方法面临重大挑战。例如，胞吐事件的量子大小和释放动力学可能受到其相对于电极的位置的影响。基于阵列的单细胞电化学成像具有很强的串扰。此外，目前的方法不允许分析细胞内囊泡。我们建议通过开发新的电分析技术和微电极来应对这些挑战。我们强调使用与片上微电极集成的纳米带电极来提高精度并消除串扰。我们还开发了新的循环伏安法，以提高时间分辨率并进一步消除伏安成像中的串扰。此外，我们使用纳米孔电极来分析单个细胞内囊泡。基于我们在单细胞胞吐作用、微电极和纳米孔方面的强大专业知识，我们建议通过追求三个具体目标来实现我们的目标： 目标 1. 开发和利用与纳米带电极集成的片上微电极来分析单个胞吐事件。我们将使用集成的片上微电极分析单个胞吐事件。我们将使用计算机模拟来更深入地了解电极/细胞界面上的多巴胺传输。我们将优化电极几何形状，以提高单细胞电流分析法的准确性和重现性。此外，我们将通过电极设计和化学功能化来提高它们的稳定性和灵敏度。 目标 2. 至 利用新的微电极阵列和电压波形消除了电化学成像中的串扰并提高了时间分辨率。我们将使用电流分析法和伏安法对消除串扰的单细胞胞吐作用进行成像。我们将采用新的微电极阵列并使用集成纳米带电极来消除电流分析法中的串扰。然后，我们将在快速扫描 CV 中应用新的电压波形，以提高时间分辨率并消除伏安成像中的串扰。 目标 3. 进一步开发和利用基于纳米孔的方法来同时分析单个细胞内囊泡的大小和多巴胺含量。使用石英纳米孔和靠近孔口放置的微电极开发了一种新的纳米孔电极。该微探针对于同时测定单个细胞内囊泡的大小和多巴胺含量特别有用。我们将进一步开发这项新技术，并用它来分析模型细胞的囊泡。我们将使用这种技术来表征经药理学试剂处理的单细胞的囊泡。 这项工作将为更好地理解单个胞吐事件提供新的分析策略。我们提出的方法具有关键优势，可以提供当前技术无法访问的新信息。纳米带电极的集成提高了确定量子大小和动力学的准确性。新的电压波形和微阵列可以改善单细胞成像，以更好地研究胞吐异质性。纳米孔电极可以分析单个细胞内囊泡。我们预计这些新方法和探针将广泛用于分析胞吐作用，并将迅速被社区中的其他群体采用并成为他们的日常工具。