J.A. González García

Phenol derivatives as enhancers and inhibitors of luminol–H2O2–horseradish peroxidase chemiluminescence

Systematic studies on phenol derivatives facilitates an explanation of the enhancement or inhibition of the luminol‐H2O2‐horseradish peroxidase system chemiluminescence. Factors that govern the enhancement are the one-electron reduction potentials of the phenoxy radicals (PhO ∞ /PhOH) vs. luminol radicals (L ∞ /LH ˇ ) and the reaction rates of the phenol derivatives with the compounds of horseradish peroxidase (HRP-I and HRP-II). Only compounds with radicals with a similar or greater reduction potential than luminol at pH 8.5 (0.8 V) can act as enhancers. Radicals with reduction potentials lower than luminol behave in a different way, because they destroy luminol radicals and inhibit chemiluminescence. The relations between the reduction potential, reaction rates and the Hammett constant of the substituent in a phenol suggest that 4-substituted phenols with Hammett constants (s) for their substituents similar or greater than 0.20 are enhancers of the luminol‐H2O2‐horseradish peroxidase chemiluminescence. In contrast, those phenols substituted in position 4 for substituents with Hammett constants (s) lower than 0.20 are inhibitors of chemiluminescence. On the basis of these studies, the structure of possible new enhancers was predicted.

Enhancer effect of fluorescein on the luminol–H2O2–horseradish peroxidase chemiluminescence: energy transfer process

The chemiluminescence of the luminol-H2O2-horseradish peroxidase system is increased by fluorescein. Fluorescein produces an enhancement of the luminol chemiluminescence similar to that of phenolphthalein, by an energy transfer process from luminol to fluorescein. The maximum intensity and the total chemiluminescence emission (between 380 and 580 nm) of luminol with fluorescein was more than three times greater than without fluorescein; however, the emission duration was shorter. The emission spectra in the presence of fluorescein had two maxima (425 and 535 nm) and the enhancement was dependent on pH and fluorescein concentration. A mechanism is proposed to explain these effects.

Enhanced chemiluminescence kinetic ELISA of dichlorprop methyl ester

A competitive enzyme-linked immunosorbent assay (ELISA) for dichlorprop methyl ester (DIME) with an anti-rabbit secondary antibody conjugated to peroxidase was developed in polystyrene plates. The plate was developed with a luminol-H202-horseredish pemxidese-p-indophenol mixture by using a fibre optic to collect the chemilumincseent emission. The standard curve was produced for 0.05 to 10000 ng ml -i DME. The minimum detectable concentration was 0.11 ng ml -t and the relative standard deviation was 7.26% for a 50 ng ml -t sample of(n = 10). The ELISA peocedure was selective with respect to structurally similar compounds usually found in formulations of pesticides. This method was applied to DME added to wheat grain extracts with recoveries between 97.1 and 103.6% for 5-500 ng ml -t.

P-phenol derivatives as enhancers of the chemiluminescent luminol-horseradish peroxidase-H2O2 reaction: substituent effects

Abstract We studied the substituent effects of ten p-phenol derivatives and aniline on the chemiluminescence from the luminol-horseradish peroxidase-H2O2 system. The enhancer effects of some compounds (phenol, p-cresol, p-coumaric acid, p-hydroxybenzoic acid, p-hydroxymethylbenzoate, p-hydroxybenzaldehyde and aniline) were studied against pH and concentration. These compounds enhanced the chemiluminescent emission of luminol between 2 and 40 times. Phenols with activative substituents (in the para-position) of the benzene ring and phenol, produced a greater increase (at low concentration) in the chemiluminescence than those with deactivating substituents. Phenols with very active or very deactivating substituents of the benzene ring decreased the chemiluminescence of luminol. We found certain relations between the pKa values and the reducing character of these phenol derivatives with their enhancer or inhibition effects on the luminol chemiluminescence.

Chemical indicators as enhancers of the chemiluminescent luminol-H2O2-horseradish peroxidase reaction

The chemiluminescent emission from the luminoI-HaOa-horseradish peroxidase system was enhanced between 2 and 80 times, during the first 5 min, by chemical indicators, such as phenolphthalein, cresolphthalein, phenol red, cresol red, benzidine and o-tolidine. Phenol derivatives showed emission maxima at pH 8.5, while aniline derivatives showed emission maxima at pH 10.5. The enhancers with a methyl group at the ortho or para position to the OH and NH2 groups in the benzene ring showed an inhibitory effect when their concentrations were increased.

Study of the enhanced chemiluminescence from the luminol-horseradish peroxidase-hydrogen peroxide-p-coumaric acid system at very short times: stopped flow selective determination of p-coumaric acid in beers

p-Coumaric acid has a greater enhancing effect on the chemiluminescence of the luminol-H,O,-horseradish peroxidase system, at low concentration, than other phenolic acids studied. We have used this effect to study the variations of the chemiluminescent signal with luminol, hydrogen peroxide, p-coumaric acid, horseradish peroxidase concentrations and pH, using the stopped-flow technique, by monitoring the initial reaction rate. The interference effects of other phenolic acids on the enhanced chemiluminescence with p-coumaric acid (25 nM) were negligible at similar concentrations of phenolic acid. We monitored the chemiluminescence intensity at 10 s for the determination of p-coumaric acid in beers. The detection limit was ca. 0.7 nM and the linear range was O-12.5 nM. The precision of the method, expressed as a relative standard deviation, was 2.5%.

Aniline derivatives as enhancers and inhibitors of the luminol-H2O2-horseradish peroxidase chemiluminescence : Effects of the Hammett constants of the substituents

Abstract The effects of monosubstituted anilines on the luminol-H2O2-horseradish peroxidase chemiluminescence were related with the type of substituent and position. Anilines substituted in meta- and para-positions by substituents with Hammett constants (σ) lesser than −0.27 produced an inhibition of the chemiluminescence. On the contrary, those anilines with substituents with Hammett constants between −0.27 and + 0.18 produced an enhancer effect. Anilines with substituents with Hammett constants greater than + 0.18 produced a slight enhancement or inhibition. An alike evolution was observed in ortho-substituted anilines. These effects were related with the reduction potentials of aniline radicals and the reaction rates of these anilines with the compounds of horseradish peroxidase. The data obtained permitted the prediction of the inhibitor or enhancer character of the aniline derivatives, and even the power of enhancement.

Thin-layer chromatography with chemiluminescent detection of enhancers of the luminol-H2O2-peroxidase system

Abstract p-Iodophenol and p-coumaric acid were separated on cellulose TLC plates using 0.1 M Tris-HCl buffer (pH 8.5) as the mobile phase. The position of the spots in the thin-layer was located with a solution of luminol, H2O2 and horseradish peroxidase. An operational modification was used to keep the plate surface wet, which provided clear advantages for the maintenance of high signals for long periods of time. The chemiluminescent emission was more intense on p-iodophenol and p-coumaric acid than on the background because these compounds are enhancers of the luminol-H2O2-horseradish peroxidase chemiluminescence. The developed plate was scanned by a single optical fibre that collected the emissive signal to the spectrometer. The calibration graph for determining p-iodophenol was linear in the range 154–1320 ng and for p-coumaric acid was linear in the range 82–657 ng. The limits of detection were 38 ng for p-iodophenol and 14 ng for p-coumaric acid.

Relation between the structure of some heterocyclic derivatives and other compounds, and their effects as enhancers or inhibitors of the luminol-H2O2-horseradish peroxidase chemiluminescence

Abstract The enhancement or inhibition produced by 4-hydroxyazobenzene, 4′-hydroxyazobenzene-2-carboxylic acid (HABA), 7-hydroxycoumarin, 7-hydroxy-4-methylcoumarin, 7-hydroxycoumarin-4-acetic acid, 4-hydroxypyridine, 6-hydroxyquinoline, vanillin, p -rosolic acid, phenylacetate, 4-phenylphenolacetate, 5-aminoquinoline and 5-aminoisoquinoline on the luminol-H 2 O 2 -horseradish peroxidase chemiluminescence were studied. These phenomena were compared with the effects produced by some enhancers known with similar structures to the previous compounds, such as, phenol, 4-phenylphenol, 4-hydroxycinnamic acid, 2-naphthol, 4-hydroxybenzaldehyde, phenolphthalein and 1-aminonaphthalene. The enhancer activity was related to particular structures of each compound, reaction rate constants with horseradish peroxidase, pH and enhancer concentration.