Fluorescence-Coded, Magnetic Bead-Enhanced Digital Proximity Extension Assay for Ultrasensitive, Multiplexed, and Accessible Protein Detection

Detecting protein biomarkers with high sensitivity is essential for early disease diagnosis, treatment optimization, and basic biomedical research. Conventional enzyme-linked immunosorbent assays (ELISA) often lack the sensitivity required for low-abundance protein detection, whereas digital ELISA offers ultrahigh sensitivity but faces limitations in multiplexing and accessibility due to reliance on specialized instrumentation. To address these challenges, we developed FluoMag-dPEA (Fluorescence-coded, Magnetic bead-enhanced digital Proximity Extension Assay), a streamlined platform that integrates magnetic bead-enhanced proximity extension assay with digital PCR (dPCR) for highly sensitive and multiplexed protein detection. FluoMag-dPEA platform employs a ratiometric fluorescence coding scheme to achieve scalable multiplexing that can be decoded with any standard two-color dPCR readouts. Target proteins are converted into fluorescence-coded DNA templates on magnetic beads, which are then released for bead-free digital analysis, ensuring broad compatibility with common dPCR systems. The approach achieves attomolar sensitivity and enables precise, simultaneous quantification of multiple proteins. We validated FluoMag-dPEA using an eight-plex cytokine assay to profile secretions from peripheral blood mononuclear cells, demonstrating strong concordance with the benchmark Luminex multiplex protein assay in measuring secreted cytokines while achieving superior sensitivity at lower concentrations, reliably detecting proteins at single-cell equivalents. By offering ultrahigh sensitivity, scalable multiplexing, and accessibility, FluoMag-dPEA represents a powerful tool for protein biomarker detection and holds significant promise for diverse biomedical and clinical applications by many users.