Antioxidant activity assays on-line with liquid chromatography

Screening for antioxidants requires simple in vitro model systems to investigate antioxidant activity. High resolution screening (HRS), combining a separation technique like HPLC with fast post-column (bio)chemical detection can rapidly pinpoint active compounds in complex mixtures. In this paper both electrochemical and chemistry-based assays are reviewed and discussed. The focus is on the mechanisms involved and differences between the assays, rather than on the matrix or analytes. With 45 applications high resolution antioxidant screening has now become an almost routine tool for the rapid identification of antioxidants in plant extracts, foods and beverages. The methods based on true reactive oxygen species (ROS) provide the most realistic measure of antioxidant activity. Unfortunately these methods are difficult to set up and control and have not been applied since they were reported. The methods based on electrochemical detection are more practical, but have still received only limited attention for practical screening purposes. The methods based on a single relatively stable reagent such as DPPH and ABTS(+) have become most popular, because of their simple set-up and ease of control. The methods have been combined with on-line DAD, MS and NMR detection for rapid identification of active constituents.     

Bi-Enzyme Alcohol Biosensors Based on Genetically Engineered Alcohol Oxidase and Different Peroxidases

We report on the development of a bi-layer bi-enzyme biosensor architecture using different peroxidases and alcohol oxidase from Hansenula polymorpha C-105 as biological recognition elements. The sensor architecture comprises a first layer containing either horseradish peroxidase or royal palm tree peroxidase crosslinked with an Osmium complex-modified redox hydrogel. On top, a second layer was formed by electrochemically induced precipitation of a cathodic electrodeposition paint simultaneously entrapping alcohol oxidase isolated from a genetically modified strain of Hansenula polymorpha C-105. The sensor architecture was optimized with respect to effective electron transfer and stability of the enzyme. The main characteristics of the biosensors are an apparent maximal current I_max^app of 572–940 nA, an apparent Michaelis constant K_M^app of 9.5 mM, a sensitivity of 60–98 nA mM⁻¹ and an improved operational stability represented by a deactivation constant of 1.5–2.0 × 10⁻⁴min⁻¹.