Electrochemiluminescence microscopy of diffusive biocompounds as co-reactants in cell spheroids with [Ru(bpy)3]2+

In vitro assays using three-dimensional cultured cells, such as spheroids, are widely used in drug screening as an alternative to animal experiments. Biocompounds in the spheroids must be evaluated to ensure their successful application. In this study, electrochemiluminescence (ECL) microscopy was used to detect them. To the best of our knowledge, this is the first report to visualize diffusive biocompounds from spheroids with a simple signal-on ECL device using [Ru(bpy) 3 ] 2+ . This study presents the ECL microscopy of diffusive biocompounds as co-reactants inside cell spheroids using [Ru(bpy) 3 ] 2+ . MCF-7 cell spheroids, a breast cancer cell line, were used in a proof-of-concept study. The spheroids were placed on an electrode for imaging. A potential of 1.2 V was then applied for 15 s to induce electroporation of the cells, allowing the diffusion of biocompounds from the spheroids. Simultaneously, [Ru(bpy) 3 ] 2+ was oxidized to induce ECL emission. We assume that NADH and reduced glutathione (GSH) are candidate diffusive biocompounds, and their intracellular amounts were discussed. The discussion suggests that GSH is expected to be the main biocompound. Thus, the ECL approach using [Ru(bpy) 3 ] 2+ holds potential for spheroid analysis, and the proposed imaging system could be applied for drug screening using various in vitro models, including organoids. • We present novel electrochemiluminescence microscopy for cell analysis. • Electroporation induces extracellular diffusion of biocompounds in spheroids. • This method can visualize diffusive biocompounds in spheroids using [Ru(bpy) 3 ] 2+ . • The primary biocompound is expected to be reduced glutathione.