Electrochemical characterization and application of azurin-modified gold electrodes for detection of superoxide

Abstract

The interaction of superoxide radicals with Pseudomonas aeruginosa azurin in solution and in adsorbed form was characterized using spectrophotometric and electrochemical techniques. It was determined that the rate constant for the reduction of azurin by superoxide ion in solution was about 10 5 M -1 s -1 , being 2-3 times lower than the constant for cytochrome c. To prepare superoxide sensitive electrode azurin was immobilised on the surface of gold electrodes. Cyclic voltammetry measurements revealed that the best electron transfer between the protein and the electrode is realised at short-chain thiol, viz. 3,3'-dithiobis(sulfosuccinimidylpropionate,)-(DTSSP-) modified gold. The formal potential of the redox protein on the DTSSP-modified electrode was found to be 380 mV vs. NHE being 100 mV more positive if compared to the standard redox potential of the T1 site of azurin fromPseudomonas aeruginosa. The heterogeneous electron transfer rate constant was determined to be in the range 2-6 s −1 for the azurin adsorbed on the DTSSP-modified gold electrodes.Based on the electrochemical measurements the sensitivity of the biosensor towards superoxide was calculated to be 6. biosensor displayed a response to chemically and enzymatically generated superoxide, and adequate sensitivity to assess the biological response of stimulated human neutrophils.

Utilization of heat-dried Pseudomonas aeruginosa biomass for voltammetric determination of Pb(II)

“…Some investigators have used P. aeruginosa up to now in biosensor constructions [38][39][40][41], and metal biosorption studies [42]. However, heat-dried P. aeruginosa cells have not been studied yet with aim and method mentioned above, thus the present investigation has revealed a simple usage format for P. aeruginosa biomass.…”

Simultaneous use of electrochemistry and chemiluminescence to detect reactive oxygen species produced by human neutrophils

“…•-sensitive gold electrodes Three types of O 2 •-sensitive biosensors were prepared using two different redox proteins, azurin (Shleev et al, 2006) and cyt c (Ge and Lisdat, 2002;Manning et al, 1998) Prior to bio-modification gold electrodes were rinsed thoroughly with Millipore water. The first type of biosensor, based on azurin adsorbed on the DTSSP-modified gold electrode (azurin-DTSSP electrode), was prepared in accordance with our previous studies (Shleev et al, 2006).…”

“…The calibration of the electrodes was performed using chemically or enzymatically produced superoxide as described elsewhere (Ge and Lisdat, 2002;Shleev et al, 2006;Tammeveski et al, 1998). Basic electrochemical characteristics of the biosensors used in the present study are listed in Table 1.…”

“…real-time detection with high sensitivity and selectivity. Although several electrochemical biosensors for the separate measurement of O 2 À and H 2 O 2 have been presented (Alpeeva et al, 2005;Campanella et al, 2000Campanella et al, , 1997Emreguel, 2005;Ferapontova et al, 2001;Ge and Lisdat, 2002;Lindgren et al, 2000;Lisdat et al, 1999;McNeil et al, 1995;McNeil and Manning, 2002;Scheller et al, 1999;Shleev et al, 2006), only a limited number of studies describe their simultaneous use (Krylov et al, 2006;Lvovich and Scheeline, 1997;Shipovskov et al, 2004). Furthermore, to our best knowledge simultaneous optical (using LumH 2 ) and electrochemical (using biosensors) monitoring of O 2 À and H 2 O 2 production by human neutrophil granulocytes has not been performed.…”

Low-interferences Determination of the Antioxidant Capacity in Fruits Juices Based on Xanthine Oxidase and Mediated Amperometric Measurements in the Reduction Mode

“…The electrochemical methods developed for the detection of O2 • -are based on different redox proteins (i.e. cytochrome c at +150 mV, 21 azurin at +500 mV, 23 or hemin at +150 mV), 24 triple potential-step chronoamperometry with platinized carbon microelectrodes for discrimination between different radicals 25 or superoxide dismutase that can be used either for the direct transfer of electrons 26 or to catalyze the O2 • -dismutation to hydrogen peroxide, which is subsequently quantified.…”