Extracellular hydrogen peroxide measurements using a flow injection system in combination with microdialysis probes -...

There is a strong need for techniques that can quantify the important reactive oxygen species hydrogen peroxide (H₂O₂) in complex media and in vivo. We combined chemiluminescence-based H₂O₂ measurements on a commercially available flow injection analysis (FIA) system with sampling of the analyte using microdialysis probes (MDPs), typically used for measurements in tissue. This allows minimally invasive, quantitative measurements of extracellular H₂O₂ concentration and dynamics utilizing the chemiluminescent reaction of H₂O₂ with acridinium ester. By coupling MDPs to the FIA system, measurements are no longer limited to filtered, liquid samples with low viscosity, as sampling via a MDP is based on a dynamic exchange through a permeable membrane with a specific cut-off. This allows continuous monitoring of dynamic changes in H₂O₂ concentrations, alleviates potential pH effects on the measurements, and allows for flexible application in different media and systems. We give a detailed description of the novel experimental setup and its measuring characteristics along with examples of application in different media and organisms to highlight its broad applicability, but also to discuss current limitations and challenges. The combined FIA-MDP approach for H₂O₂ quantification was used in different biological systems ranging from marine biology, using the model organism Exaiptasia pallida (light stress induced H₂O₂ release up to ~ 2.7 µM), over biomedical applications quantifying enzyme dynamics (glucose oxidase in a glucose solution producing up to ~ 60 µM H₂O₂ and the subsequent addition of catalase to monitor the H₂O₂ degradation process) and the ability of bacteria to modify their direct environment by regulating H₂O₂ concentrations in their surrounding media. This was shown by the bacteria Pseudomonas aeruginosa degrading ~ 18 µM background H₂O₂ in LB-broth. We also discuss advantages and current limitations of the FIA-MDP system, including a discussion of potential cross-sensitivity and interfering chemical species.