Spatially and Temporarily Resolved Precision Delivery for Quantitative Biological Studies

项目摘要 细胞环境的时空异质性在生物学过程中有着深远的意义 与人类健康/疾病有关。多年来，许多单细胞分析工具已经开发出来， 细胞之间的异质性，例如，化学物质和离子的空间分布。然而，有一个缺失的部分 在单细胞分析是定量揭示空间和时间异质性细胞的能力， 对化学刺激的反应。这是具有挑战性的，因为控制化学品的精确浓度， 一个特定的位置取决于复杂的细胞内物质运输动力学之间的相互作用， 环境和分子的反应性。事实上，一些生理上重要的分子，包括 活性氧物质（ROS）、活性氮物质（RNS）是高活性的并且具有短的寿命。一 精确传递分子的工具，包括这些反应性分子，是定量研究它们的必要条件。 在单细胞水平上发挥作用。 我们的研究实验室将专注于开发纳米级精确输送工具，以定量控制 递送生物学感兴趣的分子，包括那些高度反应性的分子。该战略基于 能够原位产生感兴趣分子的官能化纳米移液管电极 电化学的空间和时间控制。这将通过一氧化氮（NO）的递送来证明， 一种反应性分子，其瞬时浓度在神经元传递、免疫反应和 血液凝固空间和时间分辨的递送通过结合电化学 化学输送系统与纳米电化学成像技术。这种交付方式可以是 扩展到其他活性分子，包括H2S，CO和ROS。此外，我们将开发一种精确的 交付工具称为数字交付，在那里我们将精确控制生物分子或其他非生物分子的数量。 被递送的生物实体，包括蛋白质和纳米颗粒，通过在递送过程中计数它们的数量， 以电阻脉冲方式输送。最后，我们将定量绘制空间分辨的摄取率， 分子被递送。 最终，在我们拟议的研究中开发的精确输送方法将使定量 研究了许多与反应分子有关的基本生物学和生理学问题， 单细胞水平。例如，这些工具可用于揭示 通过精确传递神经递质或其囊泡的神经元反应。该模式还可以应用于 以在单细胞水平上定量调节或刺激炎症反应。

Project Summary Spatial and temporal heterogeneity in the cellular environment has profound implications in biological processes related to human health/disease. Many single-cell analytical tools have been developed over the years to reveal the heterogeneity among cells, e.g., the spatial distribution of chemicals and ions. However, one missing piece in the single-cell analysis is the ability to reveal quantitatively the spatial and temporal heterogeneity cellular response to chemical stimulus. This is challenging because controlling the exact concentration of chemicals at a specific location depends on the interplay between dynamics of mass transport in the complex cellular environment and the reactivity of the molecules. Indeed, some physiologically important molecules, including reactive oxygen species (ROS), reactive nitrogen species (RNS), are highly reactive and have short lifetimes. A tool for precision delivery of molecules, including these reactive ones, are necessary to quantitatively study their effect at the single-cell level. Our research lab will focus on developing nanoscale precision delivery tools to quantitatively control the delivery of molecules of biological interest, including those highly reactive ones. The strategy is based on a functionalized nanopipette electrode that is capable of in situ generation of the molecule of interest electrochemically with spatial and temporal control. This will be demonstrated by the delivery of nitric oxide (NO), a reactive molecule whose transient concentration is important in neuron transmission, immune response, and blood coagulation. Spatial and temporally resolved delivery is achieved by combining the electrochemical chemical delivery system with nanoscale electrochemical imaging techniques. This delivery modality can be extended to other reactive molecules, including H2S, CO, and ROS. In addition, we will develop a precision delivery tool called digital delivery, where we will precisely control the number of biomolecules or other non- biological entities being delivered, including proteins and nanoparticles, by counting their number during the delivery in a resistive pulse fashion. Lastly, we will quantitatively map the spatially resolved rate of uptake of the molecules being delivered. Ultimately, the precision delivery methods developed in our proposed research will enable quantitative investigation of many fundamental biological and physiological questions related to the reactive molecules at the single-cell level. For example, the tools can be used to reveal the spatial and temporal heterogeneity in the neuron response by precision delivery of neuron transmitters or their vesicles. The modality can also be applied to quantitatively modulate or stimulate the inflammatory response at the single-cell level.