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- Shleev, Sergey, et al.
(författare)
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Electrochemical characterization and application of azurin-modified gold electrodes for detection of superoxide
- 2006
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Ingår i: Biosensors & bioelectronics. - : Elsevier BV. - 0956-5663 .- 1873-4235. ; 22:2, s. 213-219
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Tidskriftsartikel (refereegranskat)abstract
- A novel biosensor for superoxide radical (O2{radical dot}-) detection based on Pseudomonas aeruginosa azurin immobilized on gold electrode was designed. The rate constant of azurin reduction by O2{radical dot}- was found to be 105 M-1 s-1 in solution and five times lower, i.e., 0.2 × 105 M-1 s-1, for azurin coupled to gold by 3,3′-dithiobis(sulfosuccinimidylpropionate) (DTSSP). The electron transfer rate between the protein and the electrode ranged from 2 to 6 s-1. The sensitivity of this biosensor to O2{radical dot}- was 6.8 × 102 A m-2 M-1. The response to the interference substances, such as uric acid, H2O2, and dimethylsulfoxide was negligible below 10 μM. The electrode was applied in three O2{radical dot}- generating systems: (i) xanthine oxidase (XOD), (ii) potassium superoxide (KO2), and (iii) stimulated neutrophil granulocytes. The latter was compared with luminol-amplified chemiluminescence. The biosensor responded to O2{radical dot}- in all three environments, and the signals were antagonized by superoxide dismutase. © 2006 Elsevier B.V. All rights reserved.
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| 2. |
- Shleev, Sergey, et al.
(författare)
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Simultaneous use of electrochemistry and chemiluminescence to detect reactive oxygen species produced by human neutrophils
- 2008
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Ingår i: Cell Biology International. - : Wiley. - 1065-6995 .- 1095-8355. ; 32:12, s. 1486-1496
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Tidskriftsartikel (refereegranskat)abstract
- A novel approach for the simultaneous optical and electrochemical detection of biologically produced reactive oxygen species has been developed and applied. The set-up consists of a luminol-dependent chemiluminescence assay combined with two amperometric biosensors sensitive to superoxide anion radicals (O-2(center dot-))and hydrogen peroxide (H2O2), respectively. The method permits direct, real-time in vitro determination of both extra-and intracellular O-2(center dot-) and H2O2 produced by human neutrophil granulocytes. The rate of O-2(center dot-) production by stimulated neutrophils was calculated to about 10(-17) mol s(-1) per single cell. With inhibited NADPH oxidase, a distinct extracellular release of H2O2 instead of O-2(center dot-) was obtained from stimulated neutrophils with the rate of about 3 . 10(-18) mol s(-1) per single cell. When the H2O2 release was discontinued, fast H2O2 utilisation was observed. Direct interaction with and possibly attachment of neutrophils to redox protein-modified gold electrodes, resulted in a spontaneous respiratory burst in the population of cells closely associated to the electrode surface. Hence, further stimulation of human neutrophils with a potent receptor agonist (fMLF) did not significantly increase the O-2(center dot-) sensitive amperometric response. By contrast, the H2O2 sensitive biosensor, based on an HRP-modified graphite electrode, was able to reflect the bulk concentration of H2O2, produced by stimulated neutrophils and would be very useful in modestly equipped biomedical research laboratories. In summary, the system would also be appropriate for assessment of several other metabolites in different cell types, and tissues of varying complexity, with only minor electrode modifications.
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