Marine Biological Section, Department of Biology, University of Copenhagen, Denmark.
Department of Clinical Microbiology, Rigshospitalet, Denmark; Department of Immunology and Microbiology, Costerton Biofilm Center, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark.
Free Radic Biol Med. 2018 Nov 20;128:111-123. doi: 10.1016/j.freeradbiomed.2018.05.089. Epub 2018 May 31.
There is a strong need for techniques that can quantify the important reactive oxygen species hydrogen peroxide (HO) in complex media and in vivo. We combined chemiluminescence-based HO 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 HO concentration and dynamics utilizing the chemiluminescent reaction of HO 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 HO 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 HO quantification was used in different biological systems ranging from marine biology, using the model organism Exaiptasia pallida (light stress induced HO release up to ~ 2.7 µM), over biomedical applications quantifying enzyme dynamics (glucose oxidase in a glucose solution producing up to ~ 60 µM HO and the subsequent addition of catalase to monitor the HO degradation process) and the ability of bacteria to modify their direct environment by regulating HO concentrations in their surrounding media. This was shown by the bacteria Pseudomonas aeruginosa degrading ~ 18 µM background HO 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.
目前非常需要能够量化复杂介质和体内重要活性氧物种过氧化氢 (HO) 的技术。我们将基于商业上可用的流动注射分析 (FIA) 系统的化学发光 HO 测量技术与微透析探针 (MDP) 结合使用，通常用于组织内测量。这使得可以利用 HO 与吖啶酯的化学发光反应，对细胞外 HO 浓度和动力学进行微创、定量测量。通过将 MDP 与 FIA 系统耦合，测量不再仅限于低粘度的过滤液体样品，因为通过 MDP 进行采样是基于通过具有特定截止值的可渗透膜进行动态交换。这允许连续监测 HO 浓度的动态变化，减轻测量中潜在的 pH 影响，并允许在不同的介质和系统中灵活应用。我们详细描述了新的实验装置及其测量特性，并结合在不同介质和生物体中的应用示例，突出其广泛的适用性，但也讨论了当前的限制和挑战。用于 HO 量化的 FIA-MDP 联合方法已应用于不同的生物系统，从海洋生物学（使用模型生物 Exaiptasia pallida（光应激诱导 HO 释放高达 ~2.7µM））到生物医学应用（在葡萄糖溶液中定量测定酶动力学（葡萄糖氧化酶产生高达 ~60µM 的 HO，随后添加过氧化氢酶以监测 HO 降解过程））和细菌通过调节周围介质中的 HO 浓度来改变其直接环境的能力。这是通过细菌铜绿假单胞菌在 LB 肉汤中降解 ~18µM 背景 HO 来证明的。我们还讨论了 FIA-MDP 系统的优点和当前的局限性，包括对潜在交叉敏感性和干扰化学物质的讨论。
