Spherical Nucleic Acid‐Amplified Digital Flow Cytometric Bead Assay for the Ultrasensitive Detection of Protein and Exosome†

Comprehensive Summary Most conventional digital bioassays rely on the use of fully‐sealed microchambers as independent units to compartmentalize the target molecules and the signal generation reaction, which require specialized equipment or proprietary reagents/consumables. Herein, we report a microchamber‐free and spherical nucleic acid (SNA)‐amplified digital flow cytometric bead assay (dFBA) for ultrasensitive protein and exosome analysis with simple workflows, easily accessible instruments/reagents, and high discriminating ability towards the fluorescence‐positive and fluorescence‐negative beads. In this dFBA, microbeads are employed as independent carriers to anchor the single target molecule‐initiated signal amplification reaction, avoiding the use of sealed droplets or microwell microchambers. Meanwhile, antibody‐functionalized SNAs (FSNAs) with a high density of DNA probes act as a bridge for efficiently amplified target‐to‐DNA signal conversion, which allows the use of DNA‐based rolling circle amplification (RCA) as the fluorescence signal amplification technique to quantify non‐nucleic acid targets. Even a single target‐induced on‐bead RCA and fluorescence enriching are sufficient to make the target‐loaded bead bright enough to be clearly discriminated from the negative ones just by use of a most common flow cytometer (FCM). This dFBA has successfully realized the digital analysis of ultralow levels of protein and exosome biomarkers, enlarging the toolbox of digital bioassays for clinical applications.