Comparison of 5-hydroxy-2, 3-dihydrophthalazine-1, 4-dione and luminol as co-substrates for detection of horseradish peroxidase in enhanced chemiluminescent reactions.
The utility of 5-hydroxy-2, 3-dihydrophthalazine-1, 4-dione (HDP) as a co-substrate for the chemiluminescent detection of horseradish peroxidase was assessed. Several substituted aryl boronic acid derivatives (4-phenyl, 4-iodo) acted as potent enhancers of the peroxidase catalyzed reaction. Addition of chelating agents (EDTA) and surfactants (Tween-20 and [poly (vinylbenzyl)tributylphosphonium chloride-poly (vinylbenzyl) trioctylphosphonium chloride copolymer]) modulated background light emission and the intensity and duration of the signal from both HDP and luminol. However, HDP was found to be inferior to luminol in the peroxidase assay. Comparative studies revealed that at 500 amol of peroxidase the S/B was ten-fold higher using a commercial luminol-based signal reagent as compared with an HDP-EDTA-Tween-20 reagent (S/B t = 0 min 21.8 vs 1.7, S/B t = 10 min 17.8 vs 2.0).
Kricka
L J
LJ
Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia 19104, USA.
Ji
X
X
Thorpe
G H
GH
Edwards
B
B
Voyta
J
J
Bronstein
I
I
D001897:Boronic Acids; D004492:Edetic Acid; D006735:Horseradish Peroxidase / Q000032:analysis* / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D007462:Iodobenzenes; D008163:Luminescent Measurements; D008165:Luminol / Q000031:analogs & derivatives* / Q000378:metabolism; D010783:Photometry; D011136:Polysorbates; D013379:Substrate Specificity
C099770:5-hydroxy-2,3-dihydrophthalazine-1,4-dione; D001897:Boronic Acids; D007462:Iodobenzenes; D011136:Polysorbates; C050181:4-iodophenol; D008165:Luminol; D004492:Edetic Acid; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase