One-Step Immunoassay for Biomarker Quantification in Complex Mixtures Based on Phase-Separated Antifouling Coacervates

We develop and present a one-pot sandwich immunoassay (termed oneSTEP) to detect target biomolecules in complex biological fluids based on programmable zwitterionic polymer coacervates. We design these coacervates to selectively recruit target analytes with ultralow nonspecific adsorption. We show that dynamic compartmentalization combined with local target enrichment delivers a rapid and wash-free sandwich immunoassay with high specificity and a high signal-to-noise ratio. The fluorescence-based readout is performed using standard microscopy methods and flow cytometry. We demonstrate the capabilities of the oneSTEP assay by detecting complement component 5 in human serum and the spike protein of severe acute respiratory syndrome coronavirus 2 in artificial saliva with a limit of detection of 300 pM. The results highlight the potential of the oneSTEP sandwich immunoassay as complementary to bead-based approaches in high-throughput screening studies as well as clinical diagnostics.