Microdialysis Sampling Combined with Electron Spin Resonance for Superoxide Radical Detection in Microliter Samples

Abstract

Quantitation of superoxide radical (O2.-) production at the site of radical generation remains challenging. Microdialysis sampling is an advantageous tool for sampling from localized environments. It is difficult to combine electron spin resonance (ESR) spin traps with microdialysis because O2.- adducts with common nitrone spin traps have shorter half-lives than typical microdialysis collection times. Furthermore, typical dialysate samples (5-15 microL) suffer significant sensitivity loss when diluted for detection in a conventional ESR flat cell (200 microL). To overcome these difficulties, a cyclic hydroxylamine, 1-hydroxy-4-phosphonooxy-2,2,6,6-tetramethylpiperidine (PP-H), which produces a stable nitroxide radical (PP.) product upon reaction with O2.- was employed. Capillary cells (1.4 microL effective volume) coupled with a loop-gap resonator were utilized to measure PP. in microliter microdialysis samples (LOD 0.36 pmol). A xanthine/xanthine oxidase (X/XO) model system provided sustained O2.- production. When PP-H was included in the X/XO medium external to the microdialysis probe, a relative recovery of 22.1 +/- 1.1 and 57.2 +/- 5.7% for PP. was achieved at perfusion fluid flow rates of 0.5 and 1.0 microL/min, respectively. The respiratory burst in interferon-gamma and zymosan-stimulated RAW 264.7 macrophages was also investigated.

Engineered Superoxide Dismutase Monomers for Superoxide Biosensor Applications

“…A major challenge of radical analysis is the short half-life of superoxide (in the ms to s range) due to its spontaneous dismutation to oxygen and hydrogen peroxide. 10 Chromatography, EPR spin trapping, 11,12 and several dyes 13,14 have been used for the quantification of the radical, along with indirect approaches such as the study of radicalmediated damage of biomolecules. 15,16 Protein electrochemistry has been shown to allow real-time, on-line quantification of radical concentrations.…”

Microdialysis of Inflammatory Mediators in the Skin: A Review

“…The analytical bioassays applied in microdialysis experiments have mainly included enzyme-linked immunosorbent assays, radioimmunoassays, spectrophotometric assays, or high-performance liquid chromatography. other approaches for identifying the mediators in microdialysis samples are to use microsphere-based protein micro assays (5,6), electron-spin resonance (11), immunoaffinity capillary electrophoresis, capillary electrochromatography (12), or mass spectrometry-based proteomics (13)(14)(15). Taken together, it is valuable to emphasise that appropriate development and application of an analytical method that can allow for detection of mediators within small aliquots and low concentration is a critical step in microdialysis, which requires time and step optimisation to achieve a proper set up prior to application.…”

Microdialysis sampling techniques applied to studies of the foreign body reaction

“…Given the concerns regarding radical trapping with agents such as hydroxybenzoic acids, which can undergo metabolism or other oxidative chemical transitions, the use of spin trapping agents that can be confirmed for appropriate radical trapped identity has promise in microdialysis sampling (Chen et al, 2004). Using appropriate hardware technology to significantly reduce the volume necessary, dialysates can be monitored for trapped agents using electron paramagnetic resonance (EPR) techniques.…”