Electrochemiluminescence Distance and Reactivity of Coreactants Determine the Sensitivity of Bead‐Based Immunoassays

Abstract Herein we report the study of electrochemiluminescence (ECL) generation by tris(2,2′‐bipyridyl)ruthenium (Ru(bpy) 3 2+ ) and five tertiary amine coreactants. The ECL distance and lifetime of coreactant radical cations were measured by ECL self‐interference spectroscopy. And the reactivity of coreactants was quantitatively evaluated in terms of integrated ECL intensity. By statistical analysis of ECL images of single Ru(bpy) 3 2+ ‐labeled microbeads, we propose that ECL distance and reactivity of coreactant codetermine the emission intensity and thus the sensitivity of immunoassay. 2,2‐bis(hydroxymethyl)‐2,2′,2′′‐nitrilotriethanol (BIS‐TRIS) can well balance ECL distance‐reactivity trade‐off and enhance the sensitivity by 236 % compared with tri‐ n ‐propylamine (TPrA) in the bead‐based immunoassay of carcinoembryonic antigen. The study brings an insightful understanding of ECL generation in bead‐based immunoassay and a way of maximizing the analytical sensitivity from the aspect of coreactant.