Droplet-free digital immunoassay based on electrochemiluminescence

• Published in: Biosensors and bioelectronics. X Vol. 13; p. 100312
• Main Authors: Ito, Kentaro, Inoue, Kumi Y., Ino, Kosuke, Shiku, Hitoshi
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 01.05.2023; Elsevier
• Subjects: Digital immunoassay; Electrochemiluminescence; Electrochemistry; Tyramide signal amplification; Electrochemistry; Electrochemiluminescence; Tyramide signal amplification; Digital immunoassay
• ISSN: 2590-1370; 2590-1370
• DOI: 10.1016/j.biosx.2023.100312

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. [Display omitted] •Digital ELISA was performed using ECL for the first time.•Two poly HRP molecules were detected using our assay system using ECL.•Our system using ECL enabled simple and highly sensitive digital ELISA without droplet.