Circuit Identification in the “Black Box” of Reactive Oxygen Species-Involved Electrochemiluminescence by Time-Synchronized Luminescence-Mass Spectrometry

Reactive oxygen species (ROS)-involved electrochemiluminescence (ECL) systems are increasingly sought after for bioassay and bioimaging. Unraveling the roles of individual ROS at the molecular level is pivotal to rational design and precise modulation of ECL systems, but it remains challenging due to the entangled reaction pathways. Herein, we present a time-synchronized luminescence-mass spectrometry platform (namely, Flex-RT-Triplex) to trace the dynamic fate of intermediates triggered by diverse ROS in correlation with ECL events. By enabling single-variant control over the introduced ROS and applied potential, this platform disentangled the interconnected reaction circuits within the “black box” of ECL networks. Using this platform, four cathodic ECL pathways of lucigenin (Luc) driven by superoxide radicals (O2•–) or hydrogen peroxide (H2O2) were comprehensively validated by tracking the interplay of individual ROS and key intermediates, including Luc radical cation (Luc•+), Luc dioxetane (Luc(OO)), and Luc pseudobase (Luc(OH)+). Moreover, hydroxyl radical (•OH) and H2O2 were found to be contributive to the anodic ECL of rubrene (Rub), contradicting prior speculations. Beyond the understanding and optimization of ROS-involved ECL systems, the proposed methodology may find broader applications in the mechanistic investigation of other ROS-assisted electrochemical transformations, including degradation and synthesis.