Single-Particle Analysis of Virus Capsids, Bacteria, and Extracellular Vesicles - Project Summary We are developing micro- and nanofluidic devices to probe virus capsids, bacteria, and extracellular vesicles at the single-particle level with improved spatial and temporal resolution. Single-particle (or digital) measurements provide not only improved sensitivity but also insight into population heterogeneity. Information content is further enhanced by performing these assays in a high-throughput, multiplexed format, where individual events are tracked, but population statistics are also obtained. We are targeting rare or infrequent events, which can significantly impact the overall function or fate of a system, because these events are often obscured when measurements are made on bulk samples. In the first project, we are studying virus capsid assembly and disassembly. To monitor reactions with capsids, we are designing in-plane nanofluidic devices with multiple nanopores in series for resistive-pulse sensing, which is a label-free, nondestructive sizing technique. Resistive-pulse sensing detects events in real time and has sufficient sensitivity to monitor assembly at biologically relevant concentrations and over a range of reaction conditions. With these nanofluidic devices, we are evaluating how assembly effectors and chaotropes impact the assembly process and produce a variety of particle morphologies, including kinetically trapped intermediates and aberrant structures. In the second project, we are tracking development and aging of bacteria with microfluidic devices that have integrated nanochannel arrays to physically trap bacteria. The nanochannels confine growth of bacteria in one dimension, and when coupled with fluorescence microscopy, image analysis is reduced from a three-dimensional to one-dimensional problem and greatly simplified. Growth and division rates, subcellular functions, epigenetic effects, and antibiotic response are easily tracked for extended periods of time and across multiple generations. In the third project, we are profiling N- and O-glycans derived from serum, urine, and ascites fluid, and their extracellular vesicles. For thorough characterization of these glycans, we are combining chemical labeling strategies to neutralize and differentiate sialyl linkage isomers with analysis by microfluidic capillary electrophoresis and capillary electrophoresis-mass spectrometry. Differences in glycan sizes, degrees of fucosylation and sialylation, and ratios of sialyl linkage isomers are quantified in these samples. We are using single-particle techniques to characterize the physical and chemical properties of extracellular vesicles to correlate these properties with their glycan profiles.
