New Electroanalytical Methods for Single-Cell Exocytosis

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外事件。胞吐在神经元传递、激素和神经调节剂的释放、免疫反应中起着至关重要的作用，在调节脑功能、情绪和行为反应以及许多其他生理过程中起着重要作用。因此，对单个胞外事件的详细了解对于更好地治疗中枢神经系统疾病是必要的。在过去的三十年中，电分析方法由于其独特的特性，包括非常高的空间、时间分辨率和优异的化学分辨率，在生物分析任务中发挥了至关重要的作用。然而，目前的电分析方法面临着重大挑战。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性。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解决这些挑战。我们强调使用集成了片上微电极的纳米带电极来提高精度和消除串扰。我们还开发了新的循环伏安方法，以提高时间分辨率，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外分泌、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：开发和利用集成了纳米带电极的片上微电极来分析单个胞外事件。我们将使用集成的芯片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/细胞界面的多巴胺转运。我们将优化电极几何形状，以提高单细胞安培测量的准确性和可重复性。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。来

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.