Machine Learning-Assisted Integrated Optofluidic Nanoplasmonic Biosensing for Precision Immune Profiling and Monitoring Abstract: The intricate and dynamic nature of the immune system demands a comprehensive understanding of its functional behavior for effective prediction and treatment of immune-related diseases. Cytokines, vital for intercellular signaling, offer invaluable insights into a range of diseases, including infections, cancer, autoimmune disorders, and allergy transplantation. Prompt and precise multiparametric cytokine detection at the point of care is essential for comprehending patients' dynamic immune responses. In our previous MIRA project, we developed integrated optofluidic nanoplasmonic biosensing platforms for high-throughput, sensitive, and multiplex cytokine detection from whole blood to single-cell levels. Building on our past accomplishments, we propose to develop high performance integrated optofluidic nanoplasmonic biosensing technologies for immune analysis and incorporate machine learning techniques to enable precision Immune profiling and monitoring for better patient care. The primary objectives of this renewal application are to: 1) advance the next generation of serum immunoassays by integrating the power of machine learning (ML) to engineer state-of-art plasmonic nanomaterials and capture probes with significantly enhanced sensing performance for rapid, reliable and effective diagnosis at point-of-care; 2) Develop ML-enhanced micropillar-based in situ immunoassays for real- time immune monitoring of on-chip in vitro models for high-resolution, high-throughput immune profiling towards personalized immunomodulatory therapies; 3) Establish an innovative integrated method by embedding nanoplasmon rulers within a hydrogel matrix to map the 3D spatiotemporal cytokine secretion profiles of individual immune cells encapsulated in the hydrogel, providing new insights into immune cell behavior and communication in a 3D physiologically relevant context. Our vision is to bridge the gap in our fundamental understanding of the immune system and enhance the diagnostic and predictive power for immune system diseases. The advanced integrated optofluidic nanoplasmonic biosensing platforms, empowered by machine learning, will gear the biologists and clinicians with the ability to rapidly and precisely determine patients' immune statuses. This transformative achievement holds enormous potential for both fundamental research and clinical applications, ultimately leading to improved patient outcomes and more effective therapies for immune-related diseases.