Localized Accumulation of Tri-n-Propylamine Prolongs Electrochemiluminescence for Tumor Model Spheroid Analysis

Electrochemiluminescence (ECL) is a technique that couples electrochemical control with photon emission, enabling highly sensitive, label-free imaging without an external light source. The microsecond lifetimes and short diffusion lengths of intermediates generated during the reaction confer excellent spatiotemporal resolution, while the absence of phototoxicity promotes biocompatibility. Previously, ECL microscopy has illuminated systems ranging from single cells to multicellular spheroids. Yet, these works relied on luminol-hydrogen peroxide chemistry, which is limited by weak signals and the sacrificial nature of luminol. Here, we introduce tris(2,2'-bipyridyl)ruthenium(II)/tri-n-propylamine ([Ru(bpy)₃]^{2 +}/TPrA) chemistry as a powerful alternative for spheroid imaging. Pre-incubation of cellular spheroids within TPrA, followed by interrogation in [Ru(bpy)₃]^{2 +} produces markedly sharper spatial resolution and enables continuous imaging for over 3 h, exploiting the recyclability of the luminophore. Furthermore, this strategy reveals the first sustained afterglow chemiluminescence in a biological system, persisting for minutes after the applied potential has ended. Strikingly, we demonstrate that this methodology can elucidate differences in the ECL emission intensity in spheroids from cancerous and non-cancerous cell lines, likely due to differences in TPrA accumulation. These advances establish [Ru(bpy)₃]^{2 +}/TPrA-based ECL as a transformative approach for long-term, high-resolution imaging of three-dimensional cellular architectures with application towards cancerous versus non-cancerous tumor model differentiation.