Ultrabright Fluorescent Nanoconstruct Enabling Widely Accessible, High Performance Multiplexed Protein Assays

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Abstract Detection and quantification of multiple proteins in biological fluids and tissues is of fundamental importance in biomedical research and clinical diagnostics because it is impossible to understand complex, non-linear, biochemical systems without being able to accurately interrogate the components. The need to interrogate multiple proteins simultaneously is ubiquitous across all domains of biomedical research, and it is a major barrier to fully understanding health, ageing, disease, and response to therapeutic interventions. Though the need may be ubiquitous, there is not a widely accessible solution for researchers to make multiplexed protein measurement with high sensitivity and a large dynamic range. Antibody microarrays have a straightforward, ELISA-like workflow and can be used to measure up to thousands of proteins simultaneously. Unfortunately, they have rather poor sensitivity, and require a specialized and expensive (>$75k) reader to be used. These are significant barriers to adoption for the majority of biomedical researchers. We have developed an ultrabright fluorescent nanoconstruct we call the Plasmonic Fluor, which is >5,000X brighter than the standard fluorescent reporter used in microarrays today. Simply substituting the Plasmonic Fluor for the existing reporter significantly increases the sensitivity of antibody microarrays without requiring any change to the workflow. Importantly, the Plasmonic Fluor is so bright that it allows fluorescent microarrays to be read using more widely available and versatile readers such as a fluorescent Western blot reader. Additionally, it relaxes the requirements on the reader instrumentation significantly, which has enabled us to create an inexpensive reader that any research lab can afford. To allow even easier adoption of the Plasmonic Fluor enhanced microarrays, we have also created software for microarray data analysis. In this project, we aim to: increase the manufacturing scale of our Plasmonic Fluor to an early commercial scale; more extensively validate enhancement of a variety of popular antibody microarrays; finalize the design of our inexpensive reader; and more fully develop our user- friendly microarray analysis software. We believe the Plasmonic Fluor will become the standard fluorescent reporter molecule for all microarrays, and Plasmonic Fluor-enhanced microarrays will become a widely used and powerful tool for elucidating the role of protein networks in health and disease.