Benoit Limoges

Determination of horseradish peroxidase and a peroxidase-like iron porphyrin at a Nafion-modified electrode

A catalytic coupling reaction between 4-amino antipyrine and a N,N-disubstituted aniline derivative has been exploited in the indirect electrochemical detection of horseradish peroxidase (HRP) and of a biomimetic catalyst, the iron(III) sulfonated tetraphenyl porphyrin. In the presence of hydrogen peroxide and one of the two catalysts a cationic electroactive quinone-iminium dye P+ was formed and detected by linear scan voltammetry using a screen-printed electrode coated with a Nafion film. Detection limits of 10(-12) M for HRP and 4 x 10(-10) M for the iron porphyrin have been achieved. In conclusion the iron porphyrin is considered to be a promising alternative to the HRP label in enzyme immunoassays with electrochemical detection.

Avidin–Biotin Assembling of Horseradish Peroxidase Multi-Monomolecular Layers on Electrodes

The strong avidin–biotin affinity is used to stack up successive monomolecular layers of horseradish peroxidase on carbon electrodes. After a biotinylated immunoglobulin is adsorbed on the electrode surface, alternate deposition of neutravidin and biotinylated HRP allows the assemblage of up to 16 successive active HRP layers. The film build-up is followed by cyclic voltammetry using an osmium complex as soluble mediator and H2O2 as substrate. The variation of the resulting catalytic responses with H2O2 concentration exhibit characteristics qualitatively consistent with the catalysis-inhibition reaction scheme previously established for monomolecular layers. In most cases the catalytic activity increases steadily with the number of monomolecular layers, leading to a significant increase of the analytical sensitivity of the derivatized electrode.