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 localize secreting cells. This is particularly important for understanding heterogeneity, spatial effects in co-culture, and polyfunctionality, and we propose to add a 4th laser to our reader to accommodate cell markers. Additionally, a single-color PFluorospot assay was read with our existing reader and demonstrated high sensitivity, high resolution, and short read times. We believe our Pfluorospot platform and reader will become the go-to secretomics platform, and will empower many more biological researchers to measure important, secreted proteins.
Public Health Relevance Statement: Project Narrative There is a significant need in biomedical research for a widely available and inexpensive tool which can be used to simultaneously measure multiple secreted proteins in biological samples with high sensitivity and at single-cell resolution. These proteins are critical to health and disease. We have developed an ultrabright fluorescent nanoconstruct we call a Plasmonic Fluor and an inexpensive reader that will enable researchers to measure these secreted proteins more quickly, at lower cost, and with higher resolution and sensitivity.
Project Terms: Aging; Antibodies; Biological Assay; Assay; Bioassay; Biologic Assays; Biology; Biomedical Research; Cells; Cell Body; Color; Communication; Disease; Disorder; Pharmaceutical Preparations; Drugs; Medication; Pharmaceutic Preparations; drug/agent; Fluorescence; Future; Health; Heterogeneity; Immunoassay; Lasers; Laser Electromagnetic; Laser Radiation; Phenotype; Play; Proteins; Reading; Investigators; Researchers; Research Personnel; social role; Role; Cell Communication and Signaling; Cell Signaling; Intracellular Communication and Signaling; Signal Transduction Systems; Signaling; biological signal transduction; Signal Transduction; Software; Computer software; Staining method; Stains; Technology; Testing; Time; Work; Streptavidin; Strepavidin; Measures; Fluorescein-5-isothiocyanate; 5-Isothiocyanatofluorescein; FITC; Set protein; HLA-DR Associated Protein II; IGAAD; Inhibitor of GZMA-Activated DNase; Phosphatase 2A Inhibitor I2PP2A; SET Translocation Inhibitor-2 of Protein Phosphatase-2A; Template Activating Factor I Beta; base; Procedures; Biological; biologic; Individual; Measurement; Cy5; cyanine dye 5; Co-culture; Cocultivation; Coculture; Coculture Techniques; tool; Diagnostic; Complex; Slide; cell type; Pattern; Techniques; System; Performance; secretory protein; antibody conjugate; PBMC; Peripheral Blood Mononuclear Cell; ELISPOT; enzyme linked immunospot assay; novel; Human Cell Line; Regulation; Sampling; Pharmaceutical Agent; Pharmaceuticals; Pharmacological Substance; Pharmacologic Substance; Cellular Secretion; Cell secretion; Address; Data; Detection; Reader; Reproducibility; Resolution; Development; developmental; cost; design; designing; scale up; prototype; biological systems; plasmonics; precision medicine; precision-based medicine; optical spectra; emission spectra; emission spectrum; optical spectrum; exosome; experimental study; experiment; experimental research; automated analysis
