Abstract

Serial measurement of a large panel of protein biomarkers near the bedside could provide a promising pathway to transform the critical care of acutely ill patients. However, attaining the combination of high sensitivity and multiplexity with a short assay turnaround poses a formidable technological challenge. Here, we developed a rapid, accurate, and highly multiplexed microfluidic digital immunoassay by incorporating machine learning-based autonomous image analysis. The assay achieved 14-plexed biomarker detection at concentrations < 10pg/mL with a sample volume < 10 L, including all processes from sampling to analyzed data delivery within 30 min, while only requiring a 5-min assay incubation. The assay procedure applied both a spatial-spectral microfluidic encoding scheme and an image data analysis algorithm based on machine learning with a convolutional neural network (CNN) for pre-equilibrated single-molecule protein digital counting. This unique approach remarkably reduced errors facing the high-capacity multiplexing of digital immunoassay at low protein concentrations. Longitudinal data obtained for a panel of 14 serum cytokines in human patients receiving chimeric antigen receptor-T (CAR-T) cell therapy manifested the powerful biomarker profiling capability and great potential of the assay for its translation to near-real-time bedside immune status monitoring.