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 (H 2 O 2 ) 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, H 2 O 2 cannot be retained on PADs for extended periods. In this study, we used titanium dioxide (TiO 2 ) nanoparticles (NPs) to stabilize H 2 O 2 on PADs. H 2 O 2 is known to be adsorbed onto TiO 2 NPs and then released into aqueous solution. In this study, we developed PADs containing TiO 2 NPs for peroxidase detection. The H 2 O 2 on PADs containing TiO 2 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 TiO 2 NPs. At 4 °C in a sealed pouch with silica gel, H 2 O 2 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 H 2 O 2 addition just before analysis and enable facile on-site preparation. In the future, this approach of incorporating TiO 2 NPs into PADs may be extended to a variety of H 2 O 2 -dependent analyses beyond peroxidase detection.