Spatially and Temporarily Resolved Precision Delivery for Quantitative Biological Studies

项目摘要 细胞环境中的时空异质性在生物过程中有着深远的意义。 与人类健康/疾病有关的。多年来，许多单细胞分析工具已经开发出来，以揭示 细胞间的异质性，例如，化学物质和离子的空间分布。然而，有一件事遗漏了 在单细胞分析中，能够定量地揭示细胞的空间和时间异质性 对化学刺激的反应。这是具有挑战性的，因为将化学品的确切浓度控制在 一个特定的位置取决于复杂细胞中质量传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(ROS)，活性氮(RNS)，活性高，寿命短。一个 精确传递分子的工具，包括这些反应性分子，对于定量研究它们的 在单细胞水平上的效果。 我们的研究实验室将专注于开发纳米级的精密输送工具，以定量控制 输送具有生物学意义的分子，包括那些高活性的分子。这一战略是基于 能够原位生成目标分子的功能化纳米管电极 具有空间和时间控制的电化学。这将通过一氧化氮(NO)的传递来证明， 一种反应性分子，其瞬时浓度在神经元传递、免疫反应和 血液凝结。通过结合电化学方法实现空间和时间分辨递送 具有纳米级电化学成像技术的化学输送系统。这种交付方式可以是 延伸到其他活性分子，包括H_2S、CO和ROS。此外，我们将开发一种精确度 传递工具称为数字传递，我们将精确控制生物分子或其他非生物分子的数量 正在交付的生物实体，包括蛋白质和纳米颗粒，通过计算它们在 以阻性脉冲方式输送。最后，我们将定量绘制空间分辨率的摄取率 正在传递的分子。 最终，在我们拟议的研究中开发的精确交付方法将使量化 许多与反应分子有关的基本生物学和生理学问题的研究 单细胞水平。例如，这些工具可以用来揭示 通过精确传递神经递质或其囊泡而产生的神经元反应。情态也可以应用于 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.