A Pre-Equilibrium Single-Molecule Digital Counting Biosensor for Near-Patient Precision Medicine of Life-Threatening Illnesses

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Precision medicine is the emerging approach to treat human diseases by targeting therapy towards the underlying basis of the disease for individual patients. While testing and therapeutic plans for cancers and other chronic diseases are formulated over days to weeks, a potentially life-saving treatment for patients suffering an immediate acute attack must be delivered in minutes to hours, requiring fast diagnostic technologies near the patient. This research aims to meet this need by developing an innovative diagnostic biosensor platform that overcomes the time-consuming requirements of conventional biomarker analysis technologies, with the potential of saving patient lives undergoing crises such as organ failure. This proposed platform will simultaneously achieve higher speed, higher sensitivity, and lower instrumentation cost, while permitting concurrent analyses of multiple blood biomarker proteins at the patient's bedside. The technology developed in this research may enable future biomarker-guided precision treatment of life-threatening illnesses. Additionally, this program will allow the research team to participate in conferences organized by Science-Technology-Engineering-Mathematics (STEM) societies, such as the Society of Hispanic Professional Engineers (SHPE), Advancing Hispanics, Chicanos, and Native Americans in Science (SACNAS) Society, and Society of Women Engineers (SWE), to stimulate the interests of underrepresented minority students in engineering and nanotechnology-based biological sensor device development. The objective of this study is to develop a point-of-care (POC) digital immunoassay biosensor platform to address urgent needs for unconventional diagnostic approaches to precision medicine of severe life-threatening illnesses. The proposed platform is built upon innovative immunoassay technology, which involves (i) confining antibody-conjugated magnetic beads into an array of femtoliter-sized microwells on a microfluidic chip, each forming a fluorescent 'pixel' activated when the target analyte is bound, and (ii) counting the number of fluorescence signal-activated pixels from a 'snapshot' image of the entire microwell array taken for pre-equilibrated analyte binding events. With its high performance and robustness, our new microfluidic digital biosensing technology may enable near-real-time, near-the-patient concurrent quantification of circulating blood cytokine biomarkers. What makes our approach innovative and unique is the unprecedented ability to simultaneously achieve speed ( 10 minutes in total with 30seconds of incubation), sensitivity leading to limit of detection (LOD) of 0.1 - 1 pg/mL, which achieves clinical thresholds, a large linear dynamic range, and multiplexity (up to 24 samples) all together in the same platform. This research will involve the development of a multiphysics model to provide a theoretical foundation and optimization guideline for the proposed biosensor, construction of a self-contained lab-on-a-disk system for portable, semi-automated microarray biosensing of cytokine biomarkers, and validation of the system performance under an intensive care unit setting. The receptor types used in this technology may be further extended to employ a wide variety of antibodies, peptides, and oligonucleotides. This could allow an expansion of this technology for other biological assays including receptor-ligand assays, enzyme assays, and DNA assays.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.