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While ultrasensitive methods such as digital enzyme\u2010linked immunosorbent assay\u00a0have expanded the window of detectable proteins, multiplexing with high accuracy, sensitivity, and throughput remains limited by cross\u2010reactivity and signal readout channels. To address this challenge, we introduce PRO\u2010MOSAIX (PROximity\u2010barcoded Molecular On\u2010bead Signal Amplification for Individual MultipleXing), a high\u2010accuracy multiplex digital immunoassay platform that integrates ultrasensitive single\u2010molecule protein detection with proximity ligation. PRO\u2010MOSAIX generates \u201cON\u201d signals only from matched affinity reagents in proximity, minimizing false positives from cross\u2010reactive 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\u2010based signal amplification. As a proof of principle, we establish and validate a 15\u2010plex PRO\u2010MOSAIX assay in human plasma, with low femtomolar sensitivities and high measurement accuracies. PRO\u2010MOSAIX is modular and utilizes common laboratory instrumentation with a high\u2010throughput flow cytometric readout, providing a broadly accessible tool across research and clinical labs and bridging the gap between analytical sensitivity and high\u2010order multiplexing.<\/jats:p>","DOI":"10.1002\/anie.9011937","type":"journal-article","created":{"date-parts":[[2026,5,28]],"date-time":"2026-05-28T15:11:21Z","timestamp":1779981081000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Expanding High\u2010Fidelity Multiplexing in Ultrasensitive Single\u2010Molecule Protein Detection via Proximity Barcoding"],"prefix":"10.1002","volume":"65","author":[{"given":"Chi\u2010Chia","family":"Wang","sequence":"first","affiliation":[{"name":"Life Sciences Institute University of Michigan  Ann Arbor Michigan USA"},{"name":"Department of Biomedical Engineering University of Michigan  Ann Arbor Michigan 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