Droplet-free digital immunoassay based on electrochemiluminescence

Here, we report a droplet-free digital immunoassay for goat immunoglobulin G (IgG) based on electrochemiluminescence (ECL) for simplified digital immunoassay. Digital enzyme-linked immunosorbent assay (ELISA) enabled ultrasensitive detection using digital counting immuno-reaction-induced femtoliter-sized droplets. However, the complicated preparation process of femtoliter-sized droplet assay limits the simplicity of digital ELISA. In this study, droplet-free digital ELISA using ECL was developed with digital counting ECL-induced beads instead of droplets along with a labeling reaction of immuno-reacted beads with ECL luminophore of [Ru(bpy)₃]²⁺ in bulk solution. After antigen–antibody reaction on the beads using poly horseradish peroxidase (HRP) as a label, HRP reaction with [Ru(bpy)₃]²⁺-conjugated tyramide was induced to modify the bead with [Ru(bpy)₃]²⁺. The number of beads emitting ECL was counted on an indium tin oxide (ITO) electrode using tri-n-propylamine as a co-reactant. From result of poly HRP assay, our digital assay system required two poly HRP molecules on the bead to confirm ECL. Finally, a digital immunoassay was performed. The limit of detection for goat IgG was 0.7 pg/mL (4.7 fmol/L). Our simplified digital ELISA was less sensitive than conventional digital ELISA by 10-100-fold, however, we successfully performed digital ELISA based on ECL without complicated procedures for droplet array.