Iron Probe Project

Below is a video of my research during my time in graduate school. The video was sponsored by the University of New Hampshire College of Engineering and Physical Sciences. 

The focus of my previous work was on Fe(III) recognition-based ratiometric fluorescent probes. These probes are designed based on peptides with relevant chelators and fluorophores attached. The iron probes are optimized to target the mitochondria to monitor ferroptosis, a form of iron-dependent ROS production leading to cell death. Iron probes are notoriously difficult to synthesize and test due to iron's redox activity & selective coordination chemistry. The project includes optimization of the chelator design to be a tripod; therefore 1 chelator will bind to 1 iron atom. Additionally, the selectivity of the chelator is tested to ensure the probe will not bind to other metals, such as Zn(II) found in cells. The selectivity testing is conducted with Jobs plots, fluorescence studies and titration experiments.

The biological goal of the project is to localize to the mitochondria over other organelles in the cell. To target the mitochondria, a mitochondria localizing fluorophore is utilized. This process is mimicking results our lab found with the thiazole orange fluorophore which allowed us to target the probe to the nucleus over other organelles. 

Finally, since this work is focused on designing a ratiometric fluorophore, the probe design therefore incorporates two fluorophores. One is positioned near the chelator on the N terminus (red in emission wavelength) and another is reactive to ROS species and is positioned near the C terminus of the peptide (green in emission). These fluorophores are separated by amino acids which are strategically positioned to prevent the quenching effect of the chelator on the farther fluorophore. 

The probe is designed to show a stepwise color change: it initially emits red due to the fluorophore near the chelator. Upon mitochondrial localization and ROS interaction, the green fluorophore is deprotected and begins to emit, resulting in a yellow signal from the combined red and green emissions. Finally, interaction with labile iron quenches the red emission, leaving only green. This sequential red–yellow–green shift forms the basis of the 'stop-light' design.