Grant: Discovery Grants Program - Individual

This Discovery grant application describes a program of research that will develop and advance electrochemical methods to quantify molecule transport across biological membranes of fungi, bacteria, and tissue cells. The transport of molecules across cell membranes is one of the most fundamental cell characteristics, regulating cell function and survival. Cell permeability and molecule exchange with the cell environment governs physiological processes, a cell's response to stimuli, and often disease development and progression. The developed methods will provide an understanding of intracellular and membrane--mediated mechanisms that play a role in biochemical cell processes, and cellular features, such as photosensitivity or pesticide resistance. Molecules expelled from living cells diffuse away quickly. Therefore, a microscopic tool able to quantify species locally in proximity to the cell membrane is needed. The long--term objective of this research is to develop and advance electroanalytical methods to detect, identify, and quantify molecule transport across membranes of living cells. The short--term objectives focus on the local quantitative measurement of cell metabolites, pesticides (objective 1), and reactive oxygen and nitrogen species (objective 2) in and outside of cells. The main electroanalytical methods in this research program are scanning electrochemical microscopy (SECM), scanning photoelectrochemical microscopy (SPECM), and scanning gel electrochemical microscopy (SGECM), which employ micro-- and nanoelectrodes that will be positioned in close proximity to the membrane of living cells. Electroactive species released through membranes will be electrochemically converted at the electrode, resulting in a current signal which is proportional to the number of molecules in the solution. Photoactive molecules will be detected by SPECM, combined with micro--optical--ring electrodes (MOREs). These probes can be scanned across samples, allowing microscopic photoelectrochemical imaging. Within an SPECM, MOREs can serve for three types of microscopic analysis: 1. Localized excitation through the optical fiber and concurrent measurement of the electrochemical response, 2. Global excitation through an external light source with a localized spectroscopy probe through the optical fiber and concurrent measurement of the local electrochemical response, and 3. The stimulation of the sample by electrochemistry and concurrent spectroscopic measurement. The proposed research will unravel intracellular mechanisms through the detection of cell metabolites and highly reactive species in living cells, and will investigate under which conditions we can control the cellular response to external stimuli, such as pesticides. Over the course of the five-year funding period, this program aims to train and support three graduate students, one postdoc, and four undergraduate researchers per year on average.