Long-Term Stabilization of Hydrogen Peroxide on Paper-Based Analytical Devices Incorporating Titanium Dioxide Nanoparticles

Paper-based analytical devices (PADs) have attracted attention because of their portability and low environmental impact. To facilitate point-of-care applications, reagents on PADs must remain stable during transportation and storage. Therefore, stabilizing reagents on paper substrates is critical in PAD fabrication, especially for volatile and unstable reagents that are essential to the detection of target molecules. Hydrogen peroxide (H₂O₂) is an essential cofactor in detection reactions that involve peroxidase, which is used as a biomarker for various critical diseases. However, because of its high volatility and reactivity, H₂O₂ cannot be retained on PADs for extended periods. In this study, we used titanium dioxide (TiO₂) nanoparticles (NPs) to stabilize H₂O₂ on PADs. H₂O₂ is known to be adsorbed onto TiO₂ NPs and then released into aqueous solution. In this study, we developed PADs containing TiO₂ NPs for peroxidase detection. The H₂O₂ on PADs containing TiO₂ NPs remained stable for at least 30 days at 25 °C in air, whereas it degraded and/or evaporated within 1 day on PADs without TiO₂ NPs. At 4 °C in a sealed pouch with silica gel, H₂O₂ was stable for more than 60 days. Furthermore, the PADs accurately determined peroxidase activity in fetal bovine serum even after being stored for 30 days at 25 °C, with no statistically significant difference compared with a commercial peroxidase detection kit. These ready-to-use PADs eliminate the need for external H₂O₂ addition just before analysis and enable facile on-site preparation. In the future, this approach of incorporating TiO₂ NPs into PADs may be extended to a variety of H₂O₂-dependent analyses beyond peroxidase detection.