Project Summary / Abstract Small membrane-bound nanostructures are ubiquitous in biology. Occupying the subcellular size regime, biological nanostructures include organelles, secretory vesicles, extracellular vesicles, such as exosomes, microvesicles, apoptotic bodies, and outer membrane vesicles (OMV) produced by Gram-negative bacteria. Additionally, sub-micron structures such as synaptosomes, which are isolated presynaptic terminals of neurons, can be derived from homogenized tissues. All of these structures, even when isolated from a single source, can display an extreme amount of heterogeneity in their chemical, physical, and physiological properties. Biological heterogeneity has long been analyzed and accounted for by making measurements on single cells. Indeed, the broad field of single cell analysis has used traditional analytical techniques, including separations, electrochemistry, and mass spectrometry to reveal properties of single cells or subcellular structures that are hidden from traditional bulk ensemble assays. Bulk ensemble assays are also blind to the asynchronous events that are revealed by single cell, particle, or molecule studies. However, measurements on single cells, particle, or molecules are intrinsically low-throughput unless some sort of multiplexing strategy is employed. Imaging is a common multiplexing approach, however it too can be relatively low-throughput unless steps are taken to pack as many single objects as possible in a field of view. Therefore new strategies are required to make high- throughput measurements on single biological nanostructures to reveal heterogeneities in chemical and physiological properties. Here we propose a high-throughput microfluidic nanoarray approach that facilitates single entity measurements on hundreds to tens of thousands of individual biological nanostructures simultaneously. Our platform relies on ultrahigh density patterning of nanodots of molecules that are used to specifically capture single objects of interest. The nanodot capture arrays are then integrated into multichannel microfluidic devices, and individual liposomes, OMVs, or synaptosomes are captured by the nanodots. Our microfluidic designs allow spatial selectivity in delivery of different reagents or gradients of reagents to different zones of the arrays. This approach is applicable to virtually any membrane-bound nanoscale biological nanostructure. To demonstrate the versatility of this platform, it will be used for a number of different assays on liposomes, OMVs, and synaptosomes. Since these assays are conducted on large groups of individual structures, they can illuminate hidden distributions and heterogeneity of chemical and physiological properties, including toxin content on OMV surfaces or correlation between toxin content and OMV size. In synaptosomes we will examine the heterogeneities in intrasynaptosomal Ca2+ dynamics, neurotransmitter uptake and release, and membrane cycling by endocytosis/exocytosis.