7. Project Summary Heavy metals comprise a major class of chemical exposure agents and have a significant impact on public health causing morbidity and mortality following environmental, occupational, and/or ambient exposures.1 Inhalation is one of the most common routes for heavy metal exposures, and is known to cause respiratory inflammation, cancers, metal fume fever, asthma, and reduced physical performance.2-6 Importantly, it is known that aberrant activity of Akt kinase and/or epidermal growth factor receptor kinase (EGFR) occurs within respiratory epithelial tissue following exposures to heavy metals.6-9 However, due to the inherent cellular heterogeneity of cells within the respiratory epithelium and the dynamic responses of single cells to chemical stimuli;10 the effects of heavy metal exposures on the signaling dynamics on single cells across different cell types within the respiratory epithelium is unknown.11 Therefore, a single cell analysis technology that can obtain multiplexed measurements of Akt & EGFR activity in ultra-small samples of primary respiratory epithelial cells, would improve our understanding of the biochemical mechanisms that underlie heavy metal exposures. Importantly, such a technology can potentially enable clinicians to identify early warning signs of heavy metal induced toxicity and/or disease induction in individuals from very small, heterogeneous primary samples. I aim to improve biochemical investigations of the respiratory epithelium, by employing sensor based chemical cytometry. Sensor based chemical cytometry is a single cell analysis method in which biomolecular sensors are used to measure signaling dynamics in small populations of single cells.12-16 Here, I propose the development of a novel set of fluorescent enzyme sensors to obtain multiplexed measurements of Akt & EGFR within single cells using capillary electrophoresis employing fluorescence detection (CE-F). I also aim to improve the design and functionality of the proposed enzyme sensors; by installing photoactivatable moieties on the phosphorylation sites, I expect to improve membrane permeability of the sensors, and gain control over the kinase reaction start time within cells.16 Additionally, I plan to control the kinase reaction stop time in cells by developing a novel chemo-selective reagent which halts intracellular reactions, and facilitates reporter recovery for analysis via CE-F. Studies made possible using these novel enzyme sensors will bolster our understanding of the biochemical mechanisms that govern the induction of disease and/or resilience from ultra-small populations of primary respiratory epithelial cells. Additionally, the knowledge gained from this proposal would improve our understanding of the biochemical mechanisms that underlie heavy metal exposures, while identifying novel strategies to develop cell permeable sensors to achieve temporally controlled reactions within single cells.