Transparent Single‐Layer Graphene Electrodes for Deciphering and Enhancing Microbead‐Based Electrochemiluminescence Immunoassay

Abstract Deciphering and enhancing microbead‐based electrochemiluminescence (ECL) immunoassays are imperative for highly sensitive disease‐related biomarker detection. Herein, the fabrication of single‐layer graphene‐coated indium tin oxide (GITO) electrodes with exceptional electrochemical activity are reported, which can accelerate the electrochemical oxidation of tri‐ n ‐propylamine by 9400‐fold and thus significantly enhance the ECL generation from Ru(bpy) 3 2+ ‐labeled microbeads. Furthermore, the high transparency of the GITO electrode enables the precise characterization of ECL generation by spatially mapping the distribution of ECL signals on individual microbeads using ECL self‐interference spectroscopy and inverted ECL microscopy. Additionally, the GITO electrode facilitates the efficient collection of ECL signals from the electrode underneath. Comparative microscopic imaging of single microbeads in both upright and inverted configurations demonstrates that the inverted signal‐collection mode can remarkably reduce the optical loss and enhance the optical signal by a factor of 6, as the optical signal does not need to travel across the microbeads and bulk solution. This enhancement results in a 240% increase in the signal‐to‐noise ratio for bead‐based ECL immunoassays. The study offers valuable insights into ECL generation in bead‐based ECL systems and presents an innovative strategy for enhancing immunoassay sensitivity through the synergistic integration of single‐layer graphene electrodes with an inverted signal‐collection mode.