Characteristics and applications of the enhanced HRP-catalyzed oxidation of luminol
Gilbert, Brian Douglas
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https://hdl.handle.net/2142/23642
Description
Title
Characteristics and applications of the enhanced HRP-catalyzed oxidation of luminol
Author(s)
Gilbert, Brian Douglas
Issue Date
1989
Department of Study
Chemistry, General
Chemistry, Analytical
Chemistry, Biochemistry
Discipline
Chemistry, General
Chemistry, Analytical
Chemistry, Biochemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, General
Chemistry, Analytical
Chemistry, Biochemistry
Language
eng
Abstract
Chemiluminescence (CL) is the process in which the energy from a chemical reaction is released and thus produces an electronically excited species which, upon decay to the ground state, emits light. One of the most efficient chemiluminescent molecules is luminol. As a result of this, luminol has received much attention in chemical analysis. An analytically useful CL system which has also received much attention in recent years is the enhanced horseradish peroxidase (HRP)-catalyzed luminol reaction. Generally, the enhancers are halogenated phenols or aromatic amines. Unfortunately, only limited work has been done to characterize the action of these enhancers. The work reported to date has been empirical characterization of enhanced CL oxidations with regards to enhancer identity and concentration, pH, CL molecule, oxidants, HRP isoenzyme composition and the CL emission spectrum. This research project studied the p-iodophenol enhanced HRP-catalyzed oxidation of luminol improve the understanding of this reaction. The rate and total yield of photon emission, the rate and extent of luminol oxidation, $\phi\sb{\rm CL}$ (quantum efficiency of chemiluminescence), $\phi\sb{\rm fl}$ (quantum efficiency of fluorescence) and $\phi\sb{\rm ex}$ (quantum efficiency of excitation) were determined for this reaction under a variety of conditions. In addition, compounds with structural similarity to known enhancers were studied via flow injection analysis (FIA) to determine if a correlation between structure and enhancer ability could be made.
It was found that the photon emission, the rate and extent of reaction increased in the presence of p-iodophenol. However, this enhancer had little effect on $\phi\sb{\rm CL}$, $\phi\sb{\rm fl}$ and $\phi\sb{\rm ex}$. Thus, it seems that the major means of enhancement is an acceleration of the luminol reaction. The FIA studies of various phenols and amines showed that most candidate compounds produced suppression of the HRP-catalyzed oxidation of luminol. However, this procedure led to the discovery of a new enhancing compound (4-chloro-3-methyl phenol). This screening also allowed some structural correlations to be made (such as position and type of substitution on the phenolic ring). Optimization of this FIA system with respect to HRP concentration, luminol concentration, H$\sb2$O$\sb2$ concentration and the observation time (i.e., flow rate) allowed detection of p-iodophenol as low as 670 nM.
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