A High Sensitivity Multiplexed Immunoassay for Aging-Related Inflammation Biomarker Quantification

Abstract Age is the number one risk factor for the major chronic diseases in the developed world. Currently, 81% of adults in the United States are afflicted with one or more chronic diseases. As the average age of the population of the United States continues to grow, we will be faced with a significant disease burden if the percentage of adults with chronic disease remains the same. It is now well established that inflammation, aging, and the development of age-related diseases are linked by a complicated network of underlying biological mechanisms, but these mechanisms and the links between them are not well understood. A major reason for this is the lack of a tool to measure the all of the relevant biomarkers simultaneously over a large range of concentrations. Specifically, there is major need in aging research and clinical diagnostics for a tool which can measure multiple inflammation-related biomarkers quantitatively, reliably, and simultaneously over a large range of concentrations. There is a technology that would be ideal for this application if it were simply more sensitive. The Luminex xMAP system is well-validated and allows measurement of up to 500 biomarkers simultaneously, but these assays often fail to provide a complete characterization of a given sample due to sensitivity limitations of the system. It is simply not possible to use the Luminex technology to detect low abundance biomarkers. Our product, a nanoconstruct which we call a “Plasmonic Fluor”, can be used to enhance Luminex assays to significantly improve the sensitivity limitations of the current assays. The sensitivity limit of the current Luminex assays is determined, in part, by the brightness of the reporter fluorophore used in detecting biomarkers. The currently used fluorophore is R-phycoerythrin (PE), a fluorescent protein that is one of the brightest options available. We have preliminary data suggesting our Plasmonic Fluors are at least 50X brighter than PE, and can easily be substituted to significantly improve Luminex assays without requiring changes to the assay workflow or readout hardware. In this project, we aim to: 1) optimize our Plasmonic Fluor for enhancing Luminex assays; and 2) demonstrate that Plasmonic Fluor-enhanced Luminex is capable of measuring biomarkers at concentrations that are orders of magnitude lower than the current Luminex system. We believe the Plasmonic Fluor will completely replace PE as the standard reporter fluorophore, and Plasmonic Fluor-enhanced Luminex assays will become the de facto standard tool for elucidating the mechanistic linkages between inflammation, aging, and disease.Project Narrative Inflammation is a known characteristic of both aging and development of age-related diseases, but researchers are currently limited in studying and better understanding these processes by the lack of a high-sensitivity, practical, reliable, and cost-effective tool with which to measure many inflammation- and aging-related biomarkers simultaneously. Here we propose to optimize and validate a product we call “Plasmonic Fluor” that can easily be integrated into an existing assay (Luminex) which is already widely used to measure multiple biomarkers simultaneously, but which currently lacks the sensitivity needed to be most useful in the study of inflammation and aging. By switching a single reagent currently used in Luminex assays with our Plasmonic Fluors, we can improve the sensitivity of these assays by orders of magnitude without requiring any changes to the assay workflow or hardware.