Toxicity analysis of graphene nanoflakes by cell-based electrochemical sensing using an electrode modified with nanocomposite of graphene and Nafion

To evaluate the cytotoxicity of nanomaterials, cell-based electrochemical sensing of extracellular H₂O₂ with a nanocomposite- modified indium tin oxide (ITO) electrode was carried out. The ITO working electrode was modified with a nanocomposite film of Nafion and chemically-driven graphene (G) using the electrospray method. The cyclic voltammetric measurements of H₂O₂ molecules using the nanocomposite-modified electrode showed a high sensitivity (82.6 μA mM ^-1 cm^-2) due to the enhancement of the electrochemical signal by the nanocomposite. The mini-cell culture system integrated with the nanocomposite electrochemical transducer was used to carry out a toxicity analysis of G nanoflakes under size-, concentration- and time-dependent influences. Quantitative sensing of extracellular H₂O₂ released from HeLa cells treated with different sizes and concentrations of G nanoflakes was performed by cyclic voltammetry, and optical bioassays were also used to complement the electrochemical measurements. The H₂O ₂ concentrations after treatment by nanoflakes with different concentrations continuously increased up to 1 M, 20-fold higher than the H ₂O₂ concentration of about 50 mM for untreated cells after 24 h. The results also showed that the increased tendency in measured H ₂O₂ concentration as the concentration of the G nanoflakes increased was consistent with the results of the toxicity analysis data obtained by optical bioassays. Also, we confirmed increased cytotoxicity for smaller G size at the same concentration. These toxicity analyses by cell-based electrochemical sensors are of great interest for the toxicity assessment of nanomaterials having various biomedical and environmental applications.