Abstract Secretory proteins are fundamental to local and systemic cellular signaling in biological systems. The regulation and dysregulation of the networks involving these proteins plays a critical role in health, aging, and development of disease. As such, study of these proteins and their networks is critical to our understanding of biology. Not surprisingly, this particular set of proteins constitutes a large proportion of the targets of pharmaceutical drugs, and also represents attractive targets for diagnostics and future precision medicine efforts. Unfortunately, many of these secreted proteins are difficult to detect. There are existing technologies for studying these secreted proteins but they either underperform, are extremely expensive and/or complex given their specific-utility. containing multiple cell types. Although the need to study secreted proteins is ubiquitous, there is not a technology that is widely available (inexpensive) and capable of providing information on multiple secreted proteins simultaneously at single-cell resolution, and we aim to address this need in the current project. At the core of our technologies is the Plasmonic Fluor (PF), our proprietary, ultrabright fluorescent nanoconstruct designed to enhance the performance of fluorescence-based immunoassays. We have already built a high-sensitivity, multiplexed immunoassay platform leveraging this technology, which is currently being tested with external collaborators. This platform includes an inexpensive, high performance, single-laser fluorescence microplate/slide reader optimized for use with our PFs. We will extend this existing platform to accommodate PF- enhanced cellular secretion assays we call PFluorospot. The upgraded platform that will result from the proposed effort will be a truly versatile and powerful tool for the measurement of proteins. In our preliminary work, we have successfully manufactured at commercial scale and with high reproducibility streptavidin-conjugated PFs that are spectrally equivalent to FITC, Cy3, and Cy5 (PF490, PF550, and PF650, respectively). We have also created three PFs with entirely unique excitation/emission spectra: two which have excitation spectra like FITC and emit like Cy3 and Cy5 (PF490_550 and PF490_650, respectively), and one which has an excitation spectrum like Cy3 but emits like Cy5 (PF550_650). These unique PFs were synthesized at a prototype scale which we aim to scale up in this effort because they will allow 3 lasers to be used to interrogate 6 separate targets. In a demonstration of applicability to cellular secretion assays, streptavidin-PF550 and PF650 were conjugated to antibodies at a small scale and used to perform dual-color PFluorospot. These initial PFluorospot experiments show that additional information can be obtained in these assays relative to ELISPOT/FluoroSpot, and are simpler and significantly faster to perform. It was possible to visualize individual cells for adherent cells and better...