Expanding High‐Fidelity Multiplexing in Ultrasensitive Single‐Molecule Protein Detection via Proximity Barcoding

ABSTRACT The human proteome presents a vast information reservoir for basic and diagnostics research, yet the low abundances of many proteins in biofluids pose an analytical challenge. While ultrasensitive methods such asdigital enzyme‐linked immunosorbent assay have expanded the window of detectable proteins, multiplexing with high accuracy, sensitivity, and throughput remains limited by cross‐reactivity and signal readout channels. To address this challenge, we introduce PRO‐MOSAIX (PROximity‐barcoded Molecular On‐bead Signal Amplification for Individual MultipleXing), a high‐accuracy multiplex digital immunoassay platform that integrates ultrasensitive single‐molecule protein detection with proximity ligation. PRO‐MOSAIX generates “ON” signals only from matched affinity reagents in proximity, minimizing false positives from cross‐reactive binding. This approach overcomes the multiplexing ceiling imposed by fluorescence spectral overlap by employing a single signal readout channel and DNA barcoding. We further improve multiplexing fidelity by mitigating a secondary source of false positives from DNA‐based signal amplification. As a proof of principle, we establish and validate a 15‐plex PRO‐MOSAIX assay in human plasma, with low femtomolar sensitivities and high measurement accuracies. PRO‐MOSAIX is modular and utilizes common laboratory instrumentation with a high‐throughput flow cytometric readout, providing a broadly accessible tool across research and clinical labs and bridging the gap between analytical sensitivity and high‐order multiplexing.