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.