Dimeric Pdot Metabolite Sensors

About This Grant

Project Summary Quantitation of intracellular metabolite pools is essential for a complete understanding of cell properties, phenotype, and responses in research, biomarker discovery, and screening potential disease therapeutics. Chromatographic separation followed by mass spectrometric detection is the state of the art for high multiplex, selective, and sensitive metabolite quantitation. However, this approach involves attempting to rapidly quench cell metabolism and extract metabolites without impacting metabolite levels. Furthermore, this approach requires multiple types of chromatography for different types of metabolites, and cannot easily provide spatiotemporal information. Spatiotemporal measurements of intracellular metabolites have been achieved by developing genetically encoded metabolite sensors, but these sensors are typically limited to one metabolite, are often not quantitative, and cannot be quickly incorporated into cell samples. We will develop high multiplex metabolite sensors that overcome these limitations to enable new types of observations, experiments, and spatiotemporal metabolomics studies. The sensors will employ Pdots—semiconducting polymers collapsed into nanoparticles—which exhibit high brightness, photostability, and a large color panel for spectral multiplexing. For Pdot sensing, we initially developed ratiometric Pdot sensors sensitive to general physiological conditions (pH, temperature, O2, NADH), then leveraged the O2- and NADH-sensitive Pdots to create two generalizable Pdot metabolite sensor platforms for quantitatively imaging any metabolite that can be oxidized or reduced. These platforms involve attaching a metabolite-specific O2- or NADH-dependent enzyme to an O2- or NADH-sensitive Pdot. However, these enzyme-coated Pdot metabolite sensors require a separate Pdot without an enzyme coating to correct for endogenous O2 or NADH levels. Since O2 and NADH levels can vary spatially within a cell, it is critical that the two Pdots be close together. To achieve this requirement, here we will explore using Pdot dimers consisting of one enzyme-coated Pdot and one uncoated Pdot. We will assess the feasibility of using the Pdot dimer sensors for high-multiplex intracellular metabolite measurements via the following two Aims: (1) Demonstrate accurate multiplex intracellular metabolite measurements in live human cells. (2) Explore two approaches to rapid, scalable cytoplasmic delivery of Pdot sensors.