Evaluation of the catalytic decomposition of H2O2 through use of organo-metallic complexes – A potential link to the luminol presumptive blood test

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Abstract

Forensic scientists use several presumptive tests to detect latent blood stains at crime scenes; one of the most recognizable being the luminol reagent. Luminol, under basic conditions, reacts with an oxidizing species which, with the help of a transition metal catalyst facilitates a luminescent response. The typical oxidizing species used in the luminol reaction is hydrogen peroxide (H2O2). While the luminol reaction has been studied since its inception, the mechanistic pathway is still an area of great debate. Previous work suggests that the luminol reaction with latent blood stains possesses a correlation to the Fenton-Decomposition reaction mechanism, which decomposes H2O2 into the strongly oxidizing hydroxyl radical (*OH) species. This work seeks to understand the luminol reaction on a mechanistic level and to determine if a synergy exists between the chemiluminescence observed in the reaction and the production of the hydroxyl radical via Fenton-like processes. Results indicate that organo-metallic complexes produce hydroxyl radicals at different rates and different concentrations. These findings appear to be related to structural differences in the organo-metallic complex, which conform to the 18 electron rule or are one electron rich/deficient. Furthermore, the production of *OH is controlled by the chemical environment which governs complex stability at high pH conditions, reflective of the luminol process. Model hemoglobin systems reveal a strong correlation between the rate of *OH production via the Fenton-like pathway and maximum chemiluminescent intensity.

Introduction

Luminol (3-aminophthalic hydrazide) has been used by forensic science for the resolution of latent blood stains. Tobe et al. evaluated six different presumptive blood tests, revealing that luminol has the greatest sensitivity and specificity for the detection of blood [1]. In its basic form, luminol reacts with an oxidizing species, traditionally H2O2, resulting in the chemiluminescent detection of latent blood stain patterns [2], [3], [4], [5]. The applicability of luminol as an indicator of the post-mortem interval (PMI) of human skeletal remains is currently being studied. The remaining hemoglobin is protected by the bone structure, maintaining stability and denaturing as the bone decomposes over time. This relative stability allows hemoglobin to react with luminol and produce a luminescent response, indicative of the PMI. The accuracy of luminescent responses is limited, thus the luminol reagent is used to determine PMI only in conjunction with other forensic techniques [6], [7], [8], [9].

It is well documented that this chemiluminescence stems from the oxidation of luminol resulting in the formation of the excited state dianion intermediate (3-aminophthalate), which upon return to the ground state emits a broad spectrum (λmax 425 nm) of light [2], [3], [10]. Previous studies have reported that strongly oxidizing radical species, such as the hydroxyl radical (*OH), superoxide anion radical (*O2), and others, are responsible for chemiluminescence via luminol oxidation [11], [12], [13]. Since H2O2 is the oxidizing reagent typically used, one possible oxidizing species responsible for the excitation to the excited state is *OH. Tangent to the field of forensics, several methods of *OH generation, as well as their usefulness in removing potential pollutants have revealed the role of Fenton Decomposition as a key mechanistic pathway [14], [15], [16], [17], [18].

Fenton Decomposition involves a cyclic process in which the transition metal iron is used as a catalyst to produce the *OH species. For example, Fe2+ is oxidized by H2O2 to produce *OH in the following reaction [19], [20]:Fe2+ + H2O2  Fe3+ + *OH + OH

Fe3+ can then react with either H2O2, a hydroperoxyl radical (*OOH), or a superoxide radical anion (O2*) in the system to regenerate Fe2+, giving way to iron's ability to serve as a catalyst in a cyclic oxidation–reduction process. The hydroxyl radical produced via this catalytic cycle acts as a reactive intermediate with the end products of the reactions being water and oxygen gas. These reactions generally take place under acidic conditions due to the solubility limitations resulting in Iron (III) oxyhydroxides at basic pH. The resultant oxyhydroxides significantly reduce the production of *OH from H2O2. However, as some reports have shown, the use of chelating agents (such as EDTA, various amino acids, and porphyrin structures) can allow the production of oxidizing species to occur at basic pH levels while restricting the production of insoluble iron compounds [21], [22]. However, it is not only iron which can act as a metal catalyst. Several transition metals can serve as electron donors/acceptors in oxidation–reduction reactions and possess the ability to form metal-centered complexes with chelating ligands. Thus, different metals and metal–ligand complexes can serve as catalysts to produce *OH through Fenton-like processes [23], [24], [25].

This work explores the catalytic capabilities of several different transition metal–ligand complexes to produce *OH. The overall catalytic production of *OH was monitored using UV–vis spectroscopy and a widely accepted *OH trapping agent, p-nitrosodimethylaniline (PNDA). The overall rate constants and concentrations of *OH in solution were determined for model organo-metallic complexes, as well as hemoglobin. The relationship between the structures of these organo-metallic complexes and their ability to catalyze the decomposition of H2O2 via Fenton-like processes is explored. Furthermore, the chemiluminescence of luminol was explored using each organo-metallic complex, including hemoglobin, to determine if a correlation between *OH production and chemiluminescent intensity exists.

Section snippets

Materials and methods

The following reagents were used: sodium hydroxide (98%, VWR), CuSO4·5H2O (Cynmar), NiSO4·6H2O (Mattheson, Coleman & Bell), FeSO4·7H2O (Merck), Fe2(SO4)3·xH2O assumed to contain 6 moles of water (97%, Sigma Aldrich), molecular biology grade glycine (VWR), molecular biology grade β-alanine (MP Biomedicals, LLC), ethylenediaminetetraacetic acid magnesium disodium salt (Spectrum Chemical), 30% H2O2 (JT Baker), lyophilized powdered hemoglobin (Sigma Aldrich), p-nitrosodimethylaniline (97%, Alfa

Catalytic OH radical production kinetics

Hydroxyl radicals react with the nitroso functional group (single bondNdouble bondO) of the PNDA molecule, converting it into its radical bearing counterpart [26]. As the hydroxyl radicals are trapped, the total conjugation of π electrons decreases due to structural changes in the molecule resulting in decreased absorbance at PNDA's maximum absorbance wavelength (440 nm) [26]. The rate constants for PNDA's reaction with *OH, as well as the concentrations of the *OH produced, can be indirectly determined from the

Conclusions

This work has shown the strongly oxidizing *OH, produced via the Fenton-like Decomposition of H2O2, is the species which initiates the luminol reaction, which requires the same reagents as the Fenton Decomposition pathway: an oxidizing agent (typically H2O2) and a metallic catalyst (present as an ion or as part of an organo-metallic complex). Hemoglobin, a major component of blood, exhibits both the greatest amount of *OH over time and luminescence intensity compared to other organo-metallic

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