Spatially and Temporarily Resolved Precision Delivery for Quantitative Biological Studies - 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.
