Xiaoying Ji

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).

Synergistic enhancement of the horseradish peroxidase-catalyzed oxidation of luminol by 4-substituted phenylboronic acids.

Combinations of 4-substituted phenylboronic acids [phenyl, iodo, bromo, and trans-4-(3-propenoic acid) substituents] have been discovered to have synergistic effects in the horseradish peroxidase (HRP) catalyzed chemiluminescent oxidation of luminol. Three types of effect have been observed: 1. synergistic reduction in the background light emission of a luminol-peroxide assay reagent to a value lower than the background obtained with either enhancer individually; 2. increase in signal to background ratio (S B ) in the presence of HRP to a value higher than the S B obtained with either enhancer individually (synergy) or to a value higher than the combined S B obtained with each enhancer (synergistic enhancement); and 3. for some combinations of enhancers, an increase in signal in the presence of HRP to a value higher than the signal obtained with either enhancer individually (synergy), or to a value higher than the combined signal obtained with each enhancer (synergistic enhancement). The magnitude of the effect was moderate but the synergistic decreases in background and increases in signal produced increases in S B up to four-fold. Examples of synergistic pairs of enhancers included 4-biphenyl and 4-bromophenylboronic acid; 4-biphenyl and 4-iodophenylboronic acid; and trans-4-(3-propenoic acid) and 4-iodophenylboronic acid. Generally, synergy was obtained at several concentrations of all of the combinations of enhancers tested, and at different time points in the reaction due to the different light emission kinetics of the enhanced reactions. The mechanism of this synergistic effect has not been elucidated but may involve the enhancers acting at different points in the complex chemiluminescent peroxidase catalyzed oxidation reaction.

EFFECT OF POLYMERS ON ENHANCED CHEMILUMINESCENT ASSAYS FOR PEROXIDASE AND PEROXIDASE LABELS

Hydroxypropyl methylcellulose, hydroxyethyl cellulose, and hydroxybutyl methylcellulose stabilized light emission in a boronic acid-enhanced chemiluminescent assay for horseradish peroxidase. The stabilization of light emission was concentration-dependent and more effective with substituted boronic acid enhancers (e.g. 4-iodophenylboronic acid) than with substituted phenol enhancers (e.g. 4-iodophenol). Hydroxybutyl methylcellulose improved the linearity of the dose-response curve in a peroxidase-based antioxidant assay and stabilized light emission post-consumption of the antioxidant (Trolox). This polymer had no effect on the signal from a peroxidase label immobilized on a membrane (dot blot) or on the inside surface of a microwell in an enzyme immunoassay for thyrotropin.

Effect of Enhancers on the Pyridopyridazine–Peroxide–HRP Reaction

4-Substituted phenyl boronic acids (e.g., 4-iodo, 4-bromo, 4-phenyl) are effective enhancers of the horseradish peroxidase (Type VIA) catalysed chemiluminescent oxidation of various pyrido[3,4-d]pyridazine-1,4(2H,3H)dione derivatives. The most effective combination was 4-biphenylboronic acid and 8-amino-5-chloro-7-phenylpyrido[3,4-d]- pyridazine-1,4(2H,3H)dione. Generally, the intensity of light emission in the presence of peroxidase was higher with the pyridopyridazines than with sodium luminol. However, the blank light emission was much lower with sodium luminol than with the pyridopyridazines. A synergistic enhancement phenomenon was demonstrated for the combination of a 4-iodophenol and a 4-biphenylboronic acid enhancer with 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H) dione. The combination of these two enhancers produced a light emission intensity in an assay for 5 fmol of peroxidase that was 25% higher than expected from the sum of the individual light intensities.