STEADI:System for Translational Exosome Analysis and Diagnostics through Integrating electroporation and single-molecule detection - 1 Project Summary/Abstract (30 lines) 2 Extracellular vesicles (EVs) are nanoscale balloon-like particles found in the body that play crucial roles in cell communication 3 and can be used as diagnostic markers for complex diseases. However, the lack of reliable methods to purify, analyze, and 4 manipulate EVs has hindered their widespread use in research and clinical settings. To overcome these limitations, we propose 5 the development of a groundbreaking system called STEADI (System for Translational Exosome Analysis and Diagnostics 6 through Integrating electroporation and single-molecule detection) to enable comprehensive analysis of EVs. 7 STEADI integrates a microfluidic electroporator with a high-throughput size-based particle sorting method and a highly sensitive 8 optical molecular detection technique. By utilizing microbeads of different sizes associated with specific EV surface proteins, 9 STEADI allows precise sorting of EVs based on their surface profiles after efficient loading of molecules into the EVs using 10 electroporation. The loaded and sorted EVs are then released from the carrier microbeads and subjected to detailed analysis 11 using a highly sensitive single-molecule detection technique, enabling accurate quantification and characterization of the diverse 12 contents inside the EVs. The primary objective of STEADI is to provide a user-friendly platform for comprehensive exosome 13 analysis and effective preparation of EVs for research and clinical applications. By combining microfluidic electroporation with 14 a multi-laser cylindrical illumination confocal spectroscopy (CICS) system, STEADI allows for the rapid production of precisely 15 loaded EVs and quantitative profiling of their internal contents. 16 STEADI offers several advantages over existing methods. Firstly, it eliminates laborious pre- and post-purification steps of EVs 17 for molecular loading, molecular detection, and downstream assays, thereby streamlining the process. Secondly, the system 18 enables clean and efficient loading of molecules into EVs, preserving their molecular concentrations and membrane integrity 19 and facilitating the detection of lowly expressed molecules that would otherwise be undetectable. Thirdly, the integration of 20 CICS with microfluidic devices provides enhanced single-molecule detection capabilities, allowing for detailed characterization 21 of exosomal contents with single-EV and single-molecular resolution. 22 The STEADI system has broad applications in biological and clinical research. It facilitates the study of exosome biology and 23 their roles in various physiological and pathological processes. Additionally, it opens doors to exploring EVs' diagnostic and 24 therapeutic potential. Accurately quantifying the molecular properties of rare biomolecules within EVs is crucial for advancing 25 our understanding of disease mechanisms and developing targeted therapies. 26 In conclusion, the STEADI system stands as a remarkable leap forward in EV research, elevating its potential for seamless 27 translation into clinical applications. By providing a comprehensive and user-friendly platform that combines microfluidic 28 electroporation with CICS, it offers new avenues for studying the biology of EVs and their applications in research and medicine. 29 This innovative system has the potential to enhance our understanding of exosome functions and contribute to the development 30 of novel diagnostic and therapeutic strategies across various fields.
