Spatial Profiling of Multiple Enzymatic Activities at Single Tissue Sections via Fenton-Promoted Electrochemiluminescence

Profiling multiple enzymatic activities in tissue is crucial for understanding complex metabolic and signaling networks, yet remains a challenge with existing optical microscopies. Here, we developed a Fenton-promoted luminol electrochemiluminescence (ECL) imaging method to achieve the spatial mapping of multiple enzymatic activities within a single tissue section. This method quantitatively visualizes individual enzymatic activity by combining the enzymatic conversion of substrates with the chemical confinement of the locally produced hydrogen peroxide. To achieve high-resolution spatial imaging by limiting the diffusion (∼500 μm) of hydrogen peroxide, iron oxide nanoparticles were coated on the tissue surface to initiate the Fenton process, locally converting hydrogen peroxide into short-lived hydroxyl radicals with a nanometer-scale diffusion range. The Fenton-promoted ECL emission is confined at the enzymatic conversion sites, offering unprecedented spatial visualization of four tumor-associated oxidases within a single tissue section. Colocalization revealed a synergistic effect between lysyl oxidase and quiescin sulfhydryl oxidase on post-translational modifications of tumor extracellular matrix proteins, along with a previously undiscovered interaction with amiloride-sensitive amine oxidase, which could not be distinguished based on expressions or single enzymatic activity alone. This approach offers a novel activity-based protein profiling tool at the tissue level, providing new data for future enzynomic research and multimodal imaging.