Quantitative Analysis of Catalysis and Inhibition at Horseradish Peroxidase Monolayers Immobilized on an Electrode Surface

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Abstract

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 H(2)O(2) at such HRP monolayer electrodes in the presence of [Os(III)(bpy)(2)pyCl](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 H(2)O(2) 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 H(2)O(2) detection and amperometric immunosensor applications are finally discussed.

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