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The in vitro superoxide scavenging activity (as determined by electrochemical measurement) and the in vivo antioxidant potential (as determined by a mouse model of carbon tetrachloride (CCl4) hepatotoxicity) of methanolic extracts prepared from 10 Chinese tonifying herbs were compared. Electrochemical measurement using a cytochrome c (Cyt. c) sensor showed that all of the tested herbal extracts exhibited a medium superoxide scavenging activity of different potency, as indicated by their IC50 values. The in vivo measurement demonstrated that 80% of the herbal extracts displayed in vivo antioxidant potential, as assessed by the percentage of protection of the activity of plasma alanine aminotransferases and the hepatic glutathione regeneration capacity under CCl4-intoxicated condition. Although the in vitro antioxidant activity did not correlate quantitatively with the in vivo antioxidant potential, for 8 out of 10 samples a similar tendency was found. The rapid amperometric assessment of antioxidant potential by Cyt. c sensor may offer a convenient and direct method for screening as well as the quality control of herbal products. Copyright (C) 2004 John Wiley Sons, Ltd
A novel multilayer cytochrome c electrode for the quantification of superoxide radical concentrations is introduced. The electrode consists of alternating layers of cytochrome c and poly(aniline(sulfonic acid)) on a gold wire electrode. The formation of multilayer structures was proven by SPR experiments. Assemblies with 2-15 protein layers showed electrochemical communication with the gold electrode. For every additional layer, a substantial increase in electrochemically active cytochrome c (cyt. c) was found. For electrodes of more than 10 layers, the increase was more than 1 order of magnitude as compared to monolayer electrode systems. Thermodynamic and kinetic parameters of the electrodes were characterized. The mechanism of electron transfer within the multilayer assembly was studied, with results suggesting a protein-protein electron-transfer model. Electrodes of 2-15 layers were applied to the in vitro quantification of enzymatically generated superoxide, showing superior sensitivity as compared to a monolayer-based sensor. An electrode with 6 cyt. c/PASA layers showed the highest sensitivity of the systems studied, giving an increase in sensitivity of half an order of magnitude versus the that of the monolayer electrode. The stability of the system was optimized using thermal treatment, resulting in no loss in sensor signal or protein loading after 10 successive measurements or 2 days of storage
Because of its high reaction rate and specificity, the enzyme superoxide dismutase (SOD) offers great potential for the sensitive quantification of superoxide radicals in electrochemical biosensors. In this work, monomeric mutants of human Cu,Zn-SOD were engineered to contain one or two additional cysteine residues, which could be used to bind the protein to gold surfaces, thus making the use of promotor molecules unnecessary. Six mutants were successfully designed, expressed, and purified. All mutants bound directly to unmodified gold surfaces via the sulfur of the cysteine residues and showed a quasireversible, direct electron transfer to the electrode. Thermodynamic and kinetic parameters of the electron transfer were characterized and showed only slight variations between the individual mutants. For one of the mutants, the interaction with the superoxide radical was studied in more detail. For both partial reactions of the dismutation, an interaction between protein and radical could be shown. In an amperometric biosensorial approach, the SOD-mutant electrode was successfully applied for the detection of superoxide radicals. In the oxidation region, the electrode surpassed the sensitivity of the commonly used cytochrome c electrodes by similar to 1 order of magnitude while not being limited by interferences, but the electrode did not fully reach the sensitivity of dimeric Cu,Zn-SOD immobilized on MPA-modified gold
This study introduces a thermally responsive, polymer-based electrode system. The key component is a surface-attached, temperature-responsive poly(oligoethylene glycol) methacrylate (poly(OEGMA)) type polymer bearing photoreactive benzophenone and carboxy groups containing side chains. The responsive behavior of the polymer in aqueous media has been investigated by turbidimetry measurements. Polymer films are formed on gold substrates by means of the photoreactive 2(dicyclohexylphosphino)benzophenone (DPBP) through photocrosslinking. The electrochemical behavior of the resulting polymer-substrate interface has been investigated in buffered [Fe(CN)6](3-)/[Fe (CN)6](4-)solutions at room temperature and under temperature variation by cyclic voltammetry (CV). The CV experiments show that with increasing temperature structural changes of the polymer layer occur, which alter the output of the electrochemical measurement. Repeated heating/cooling cycles analyzed by CV measurements and pH changes analyzed by quartz crystal microbalance with dissipation monitoring (QCM-D) reveal the reversible nature of the restructuring process. The immobilized films are further modified by covalent coupling of two small biomolecules - a hydrophobic peptide and a more hydrophilic one. These attached components influence the hydrophobicity of the layer in a different way the resulting change of the temperature-caused behavior has been studied by CV indicating a different state of the polymer after coupling of the hydrophobic peptide.