Multiplexed homogeneous digital immunoassay based on single-particle motion analysis

Homogeneous digital immunoassay is a powerful analytical method for highly sensitive protein biomarker detection with a simple protocol. However, it has not been multiplexed yet. In this study, we developed a multiplexed homogeneous digital immunoassay based on single-particle motion analysis (digital homogeneous non-enzyme-linked immunosorbent assay, digital Ho-Non ELISA). In this assay, multiple target antigen molecules react with the optical subpopulation of magnetic nanobeads labeled with fluorescent dyes and capture antigen-specific antibodies. Then, these beads are magnetically pulled into femtoliter-sized reactors. The surface of these reactors is modified with multiple detection antibodies specific to each antigen by molecular tethers. Each antigen on the particles reacts with the detection antibodies anchored to the surface of the reactors. Magnetic force enhances the efficiency of bead encapsulation in the reactors, and subsequent physical compartmentalization of beads enhances the binding efficiency of the antigen-antibody reaction. The tethered beads show characteristic Brownian motion distinct from free diffusion or non-specific binding of the antigen-free beads. The color of the beads is attributed to target-identification, and the number of tethered beads is attributed to the concentration of the specific target. We measured two biomarkers (PSA and IL6) as model targets by multiplexed digital Ho-Non ELISA. Our method showed higher sensitivity compared to previous digital Ho-Non ELISA and could detect multiple targets simultaneously with the same performance as in single-plex detection. This new strategy has the potential to open a new avenue for next-generation multiplexed immunoassays in in vitro diagnostics. Homogeneous digital immunoassay is a powerful analytical method for highly sensitive biomarker detection with a simple protocol. By using this method, we demonstrated the simultaneous multiple protein detection.