A detailed understanding of human disease at the single cell level constitutes an important frontier of biomedical science. Cell-to-cell heterogeneities exist within tissues, and understanding the nature of these differences in terms of metabolite profile has vast implications regardless the type of disease. Beautiful experiments using fluorescent sensors have been developed to quantify small molecule metabolites within cells; however, the act of shining light on a cell has been shown to have deleterious effects. Further, these sensors are not easily generalizable. We endeavor to make the most accurate measurements of cellular metabolites with minimal perturbation to cellular homeostasis using nanoelectrochemical measurements. These measurements have the potential to open the door to unrealized sensitivity in sub-cellular metabolite quantification. Electrochemistry at nanoelectrodes has been used to interrogate cellular processes and quantify reactive species within cells. However, the time it takes for a nanoelectrode to deliver enough charge to convert a cell's contents during amperometric and voltammetric experiments is on the order of 100 ms. Therefore, novel techniques must be developed to minimize the perturbation to cellular homeostasis to ensure accurate measurements of natural cellular processes. Our group has recently investigated open circuit potentiometry, which was chosen because the measurement is carried out with negligible current. We have discovered that this technique is independent of electrode size. This finding indicates the sensitivity of the measurement will not change with time appreciably, and longitudinal experiments within single cells can be carried out. We propose to develop metabolite-specific nanoelectrodes that operate under open circuit conditions. We will draw from our experience fabricating nanoelectrodes and studying oxygen content within single cells and experience developing sensors to create a generalized platform for single cell metabolomics measurements. Specificity is gained via oxidoreductase enzymes that are trapped on top of an electrode surface by a hydrogel. Metabolite concentration is obtained by the enzymatic turnover rate, which is dependent on substrate concentration. Methodology developed through this grant period has the potential to forge a foundation for generalized metabolomics studies with nanoelectrode sensors, where the library of metabolite of interest depends only on the availability of an associated oxidoreductase enzyme.
A detailed understanding of human disease at the single cell level represents a challenging frontier, and very few measurement techniques are available to accurately quantify metabolites involved in all areas of human health. This MIRA application draws from our experience with nanoelectrochemistry, sensor development, and single cell analysis to innovate a generalizable platform to study metabolite concentrations in cellular compartments with minimal perturbation to cellular homeostasis.
