Publication June 2008, 15

CHEMILUMINESCENT DETECTION SYSTEMS OF HORSERADISH PEROXIDASE EMPLOYING NUCLEOPHILIC ACYLATION CATALYST
 
Abstract
The light output of the peroxidase-catalyzed luminol chemiluminescent oxidation reaction can be greatly increased by incorporating different enhancers. Such an increase is attributed to the preferential oxidation of the enhancer by peroxidase intermediates and the rapid formation of enhancer radicals that, in turn, quickly oxidize luminol to its radical anion. These enhancers, which include substituted phenols, substituted boronic acids, indophenols, and N-alkyl phenothiazines, behave as electron transfer mediators. A further, very significant increase in light output was also observed by the addition of nucleophilic acylation catalyst to the enhancer/luminol/oxidant substrate. The effect of the new component is general and applicable to many of the known enhancers but is much more remarkable in association with phenothiazine enhancers (up to 10-fold light output). The addition of a nucleophilic acylation catalyst to these substrates lowered the limit of detection for horseradish peroxidase from 50 to 8 amol. Similar improvements were observed in ‘‘sandwich” enzyme-linked immunosorbent assays and Western blot assays.

Figure: Comparison of working solution pH optimal value for a MORP-catalyzed system and a noncatalyzed system, both having the working solution based on luminol (5 mM)/SPTZ (1.5 mM)/perborate (4 mM)/HRP (227 pM): (a) d, NoMORP (no catalyst added); (b) s, MORP (1.5 mM). Each point is calculated integrating the signal during the first 10-min run of a kinetic measurement.

 
Link: http://www.sciencedirect.com/science/article/pii/S000326970800153X
Publication Date: June 2008, 15