Pre-loaded H₂O₂ breaks the low-pH dependency of plasma-activated water for bacteria inactivation via catalase suppression and oxidative attack

Plasma-activated water (PAW) shows promise as an alternative antimicrobial agent for disinfection and infection control due to its broad-spectrum bactericidal activity and high biosafety. However, the strong acidity required for its efficacy limits its applicability in pH-sensitive scenarios. This Letter proposes that pre-loading 0.1% H₂O₂ prior to plasma activation elevates the bactericidal pH threshold of PAW to milder acidic conditions, achieving >99.999% inactivation of S. aureus at pH = 4.0, with a 5-log improvement compared to conventional PAW. Furthermore, PAW pre-loaded with H₂O₂ contains much higher concentration of ONOO⁻/ONOOH and O₂⁻/HO₂ compared to post-loaded or non-supplemented groups. Mechanistic investigation reveals that reactive species in PAW damage bacterial membrane at low pH (2.0-3.0), which significantly inactivates bacteria, but cannot disrupt membrane at higher pH (4.0) even with pre-loading H₂O₂. In contrast, by observing the bubble generation and intracellular ROS levels, it is discovered that reactive species can inhibit catalase to reduce the decomposition of loaded H₂O₂, thereby achieving bacterial inactivation through H₂O₂-dominated oxidative attack. This Letter proposes a strategy to optimize the adaptability of PAW as an antimicrobial agent for pH-sensitive medical scenarios, offering insights into the roles of acid environments and H₂O₂ in bacterial inactivation.