Direct digital sensing of protein biomarkers in solution

The detection of proteins is of central importance to biomolecular analysis and diagnostics. To date, conventional methods used for protein sensing rely on surface-immobilization of target molecules and multi-step washing protocols. Owing to recent advancements in this field, detection of ultralow analyte concentrations has been achieved, yet fundamental limitations due to the surface-based nature still persist, and limited improvements have been designed to integrate multimodal information beyond concentration measurements. Here we present a protein sensing approach, termed digital immunosensor assay (DigitISA), that realizes a fundamentally new design principle enabling in-solution analyte capture and wash-free removal of unbound probe, allowing protein targets to be detected and their abundance to be quantified a single, solution-phase step. The assay platform is based on microchip electrophoretic separation combined with single molecule detection and enables calibration-free number– concentration quantitation of proteins in complex sensing scenarios. Applying the platform to a range of biomedically relevant targets including amyloid aggregates, exosomes and biomolecular condensates, we demonstrate that the assay provides information beyond monovalent binding interactions, and enables multimodal characterization of immunochemistry/valency, binding stoichiometry, protein biomarker abundance in addition to quantitative information of the probe–analyte interaction and composition. Together, the DigitISA platform constitutes a new experimental paradigm for protein biomarker sensing in a surface-free manner, enabling multimodal in-solution protein quantification, including targets that would otherwise not be addressable by conventional immuno-sensing techniques.