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.