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Publication 569
J. Am.
Chem. Soc., 125 (30), 9192 -9203, 2003
DOI: 10.1021/ja0354263 S0002-7863(03)05426-X |
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Quantitative Analysis of Catalysis and Inhibition at Horseradish
Peroxidase Monolayers Immobilized on an Electrode Surface
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Benoit
Limoges, Jean-Michel Savéant and Dounia Yazidi
Contribution from the Laboratoire d'Electrochimie
Moléculaire, Université Paris 7, Denis
Diderot, associé au CNRS (UMR 7591), 2 place
Jussieu, case 7107, 75251, Paris Cedex 05, France
Out of several tries, biotinylation of the electrode surface by means
of a sacrificial biotinylated immunoglobulin, followed by the anchoring
of an avidin-enzyme conjugate appears as the best procedure for depositing
a horseradish peroxidase (HRP) monolayer onto an electrode surface, allowing
a high-yield immobilization of the enzyme within a stable and highly
catalytic coating. Cyclic voltammetry is an efficient means for analyzing
the catalytic reduction of H2O2 at such HRP monolayer
electrodes in the presence of [OsIII(bpy)2pyCl]2+ (with
bpy = bipyridine and py = pyridine) as a one-electron reversible cosubstrate.
The odd shapes of current-potential responses, unusual bell-shaped variation
of the peak or plateau current with the substrate concentration, hysteresis
and trace crossing phenomena, and dependence or lack of dependence with
the scan rate, can all be explained and quantitatively analyzed in the
framework of the same catalysis/inhibition mechanism as previously demonstrated
for homogeneous systems, taking substrate and cosubstrate mass transport
of into account. According to H2O2 concentration,
limiting-behavior analyses based on the dominant factors or complete
numerical simulation were used in the treatment of experimental data.
The kinetic characteristics derived from these quantitative treatments
implemented by the determination of the amount of enzyme deposited by
the newly developed droplet depletion method allowed a comparison with
homogeneous characteristics to be drawn. It shows that HRP remains nearly
fully active once anchored on the electrode surface through the avidin-biotin
linkage. On the basis of this full mechanistic and kinetic characterization,
the analytical performances in H2O2 detection and
amperometric immunosensor applications are finally discussed. |
