Redox-Mediated Signal Enhancement of a Spatially Resolved Electrochemiluminescence Immunoassay

Electrochemiluminescence immunoassays (ECLIA) have emerged as powerful tools for sensitive biomarker detection, with increasing interest in their adaptation to miniaturized point-of-care diagnostic platforms. Herein, we report the enhancement of the luminescent signals of a spatially resolved ECLIA (SR-ECLIA) for multiplex biomarker detection on screen-printed carbon electrodes through the incorporation of a water-soluble redox mediator, sulfonated tris(1-phenylpyrazolato)iridium(III) ([Ir(sppz)₃]^3-). Introduction of the nonemissive [Ir(sppz)₃]^3- complex enabled efficient amplification of ECL signals while preserving assay multiplexing capability and compatibility with microarray formats. Application to a duplex SR-ECLIA targeting heart-type fatty acid-binding protein (H-FABP) and glial fibrillary acidic protein (GFAP) demonstrated a marked improvement in assay performance. The redox mediator amplified the absolute ECL immunoassay signals by approximately six-fold and increased the analytical sensitivity (slope of the calibration curve) for both biomarkers five-fold. The enhancement was found to arise from electrocatalysis of coreactant oxidation, which generates more intermediate radicals for the reductive (TPrA^•) excitation of the oxidized luminophore, in conjunction with the previously elucidated redox mediator-enhanced oxidative excitation pathways. Overall, this work highlights the promise of novel Ir(III)-based redox mediators in advancing SR-ECLIA toward highly sensitive, multiplexed, and point-of-care diagnostic platforms.