A Multiplexed Digital\nPlatform Enables Detection of\nAttomolar Protein Levels with Minimal Cross-Reactivity

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