Thermally annealed large-scale gold nanostructure platform for long-term and label-free electrochemical monitoring of cellular metabolism

In recent developments, an electrochemical method has emerged as a promising tool that enables real-time and non-destructive monitoring of cell viability and functionality in vitro. However, the restricted cell cultivation area and poor adhesion properties of the gold nanostructure film impede its potential as a new analytical tool for cellular research. In this study, we present an advanced platform designed for prolonged cell growth, differentiation, and drug screening, while providing electrochemical detection capability. The thermal annealing process, implemented after gold nanostructure deposition, was found to induce structural reformation on the indium tin oxide (ITO) film through Ostwald ripening and coalescence, leading to increased surface coverage and reduced height. The resulting optimized platform, referred to as the thermally annealed large-scale gold nanostructure (TLGN), offers an expanded cell culture area (about 227 mm²) with sustained resistance to gold film detachment, a critical challenge observed in previously reported electrodes, lasting up to 4 weeks under exposure to cell culture medium. Using TLGN, redox signals were successfully detected from a substantial number of human cervical cancer cells (470,000 cells) at days in vitro 5, a cell count that is 10.35 times higher compared to our previous gold nanostructure electrode. The cells were continuously monitored for up to 4 days under treatment with the anticancer drug (CB-839). Furthermore, the TLGN platform demonstrated its effectiveness in electrochemically monitoring the osteogenesis of human mesenchymal stem cells over a differentiation period of 28 days, based on observed changes in cellular metabolism. In summary, the TLGN platform proves invaluable not only for assessing drug accumulation and potential cytotoxicity at the cellular level but also for real-time monitoring of mitochondrial dynamics during cellular differentiation.