Chemiluminescent detection systems of horseradish peroxidase employing nucleophilic acylation catalysts.
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.
Marzocchi
Ettore
E
Cyanagen SRL, Via Stradelli Guelfi 40/c, 40138 Bologna, Italy.
Grilli
Stefano
S
Della Ciana
Leopoldo
L
Prodi
Luca
L
Mirasoli
Mara
M
Roda
Aldo
A
D015761:4-Aminopyridine / Q000737:chemistry; D000215:Acylation; D000818:Animals; D016227:Benzoquinones / Q000737:chemistry; D002384:Catalysis; D004527:Egg Proteins / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D006735:Horseradish Peroxidase / Q000032:analysis* / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D049449:Luminescence; D008163:Luminescent Measurements / Q000379:methods*; D008165:Luminol / Q000737:chemistry; D046208:Microchip Analytical Procedures; D010084:Oxidation-Reduction; D012680:Sensitivity and Specificity
D016227:Benzoquinones; D004527:Egg Proteins; C004532:quinone; D008165:Luminol; D006861:Hydrogen Peroxide; D015761:4-Aminopyridine; D006735:Horseradish Peroxidase