Laser-assembled conductive 3D nanozyme film-based nitrocellulose sensor for real-time detection of H₂O₂ released from cancer cells

In this work, a nanostructured conductive film possessing nanozyme features was straightforwardly produced via laser-assembling and integrated into complete nitrocellulose sensors; the cellulosic substrate allows to host live cells, while the nanostructured film nanozyme activity ensures the enzyme-free real-time detection of hydrogen peroxide (H₂O₂) released by the sames. In detail, a highly exfoliated reduced graphene oxide 3D film decorated with naked platinum nanocubes was produced using a CO₂-laser plotter via the simultaneous reduction and patterning of graphene oxide and platinum cations; the nanostructured film was integrated into a nitrocellulose substrate and the complete sensor was manufactured using an affordable semi-automatic printing approach. The linear range for the direct H₂O₂ determination was 0.5-80 μM (R² = 0.9943), with a limit of detection of 0.2 μM. Live cell measurements were achieved by placing the sensor in the culture medium, ensuring their adhesion on the sensors' surface; two cell lines were used as non-tumorigenic (Vero cells) and tumorigenic (SKBR3 cells) models, respectively. Real-time detection of H₂O₂ released by cells upon stimulation with phorbol ester was carried out; the nitrocellulose sensor returned on-site and real-time quantitative information on the H₂O₂ released proving useful sensitivity and selectivity, allowing to distinguish tumorigenic cells. The proposed strategy allows low-cost in-series semi-automatic production of paper-based point-of-care devices using simple benchtop instrumentation, paving the way for the easy and affordable monitoring of the cytopathology state of cancer cells.