A Multiplexed Digital Platform Enables Detection of Attomolar Protein Levels with Minimal Cross-Reactivity

Protein-based biomarkers are essential for disease diagnostics, yet their low abundance in biofluids often presents significant detection challenges for traditional enzyme-linked immunosorbent assay (ELISA) techniques. While various ultrasensitive methods such as digital ELISA have improved sensitivity, multiplex assays still suffer from considerable cross-reactivities that can compromise result accuracies. To address this challenge, we have developed barcoded Molecular On-bead Signal Amplification for Individual Counting (barcoded MOSAIC), a multiplexed digital ELISA technology that markedly reduces cross-reactivity by pairing barcoded detection antibodies with specific bead types. This approach enables the simultaneous detection of eight analytes from less than 9 μL of blood, with sensitivities ranging from midpicomolar to low-attomolar levels and a collective dynamic range exceeding seven logs across multiple analytes within a single multiplex assay. Additionally, barcoded MOSAIC is compatible with standard immunoassay reagents and workflows, utilizing a rapid, automatable flow cytometric readout for quantification, which makes it a highly accessible benchtop platform that is readily adoptable by both research and clinical laboratories, setting the stage for future translation into point-of-care applications.