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

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 as digital 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.