A comprehensive experimental study of industrial,domestic and environmental interferences with the forensic luminol test for blood

This paper presents the ?rst comprehensive and quantitative study of substances that interfere with the forensic luminol test for blood. Two hundred and ?fty substances have been selected on the basis of modern lifestyles and of contiguity with crime scenes. The intensity of the chemiluminescence produced by each substance has been measured relative to that of haemoglobin and the peak wavelength shift has also been determined. The following is a short list of nine substances that produce chemiluminescence intensities comparable with that of haemoglobin: turnips, parsnips, horseradishes, commercial bleach (NaClO), copper metal, some furniture polishes, some enamel paints, and some interior fabrics in motor vehicles. Care needs to be taken when the luminol test for blood is used in the presence of these substances. Copyright © 2003 John Wiley & Sons, Ltd.     

Attempted cleaning of bloodstains and its effect on the forensic luminol test.

The forensic luminol test has long been valued for its ability to detect trace amounts of blood that are invisible to the naked eye. This is the first quantitative study to determine the effect on the luminol test when an attempt is made to clean bloodstained tiles with a known interfering catalyst (bleach). Tiles covered with either wet or dry blood were tested, and either water or sodium hypochlorite solution (bleach) was used to clean the tiles. As expected, the chemiluminescence intensity produced when luminol was applied generally decreased with the number of times that a tile was cleaned with water, until the chemiluminescence was neither visible nor detectable. However, when the tiles were cleaned with bleach there was an initial drop in chemiluminescence intensity, followed by a rise to a consistently high value, visibly indistinguishable from that of blood. Examination of bleach drying time suggested that any interfering effect becomes negligible after 8 h.     

The assaying of haemoglobin using luminol chemiluminescence and its application to the dating of human skeletal remains

The luminol chemiluminescence reaction has, for some time, been used as a tool for the detection of haemoglobin at crime scenes. More recently, the luminol test has been suggested as a possible tool for estimating the post‐mortem interval (PMI) of skeletal remains. The preliminary results from the following study indicate that the chemiluminescent luminol test is a relatively easy and economical method for distinguishing between remains of medico‐legal (≤100 years) and historical (>100 years) interest. The femur was the preferred bone for PMI measurements using the luminol test, due to its robustness and relative resistance to diagenesis. Initial results suggest that bone that was historical in nature, produced a demonstrably weaker reaction than that of medico‐legal interest. These results suggest that the luminol test is a promising technique, albeit with some limitations, for the assessment of skeletal material that may be potentially of medico‐legal interest. Copyright © 2009 John Wiley & Sons, Ltd.     

Kinetic characteristics of luminol chemiluminescence caused by chloramine compounds

The decaying part of the kinetic curves of luminol chemiluminescence (0.02 mM) induced by N-chlorphenylalanine is approximated by an exponential dependence, which varies insignificantly as chloramine concentration is changed from 0.2 to 0.7 mM. On the whole, the chemiluminescence of luminol is a result of its oxidation, which occurs in three stages with the formation of two intermediate products. N-Chlorphenylalanine is involved in the process at the initial stage. The reciprocal of the time the luminescence reaches a maximum increases linearly with the growth of N-chlorphenylalanine concentration. According to the calculations using the equations that reflect three stages of luminol conversion in the presence of excess chloramine, the rate constant for the initial stage is about 10(3) l/(mol.min). The rate constant for one stage of the conversion of luminol oxidation product is approximately 0.2 min-1, and the rate constant of the other is severalfold greater. Luminol chemiluminescence induced by low concentrations of N,N-dichlortaurine is more durable. Probably, it is composed of two types of emission one of which slowly decays.     

Determination of bisphenol A using a flow injection inhibitory chemiluminescence method.

A new flow injection chemiluminescence (CL) method has been developed for the determination of bisphenol A (BPA), based on the inhibitory effect of BPA on the chemiluminescence reaction between luminol and potassium hexacyanoferrate. Under optimum conditions, the decrease in CL emission intensity was linear with BPA concentration in the range 8.0 x 10(-7)-1.2 x 10(-5) mol/L, and the detection limit was 3.1 x 10(-7) mol/L. The relative standard deviation (RSD) of 11 replicate measurements was 2.6% for 2.0 x 10(-6) mol/L BPA (n = 11). The sampling frequency was calculated to be ca. 120/h. This method has been successfully used to determine the content of BPA in aqueous solution of polycarbonate materials. A brief discussion on the possible chemiluminescence reaction mechanism is presented.     

Influence of different luminols on the characteristics of the chemiluminescence reaction in human neutrophils

In search for a luminol with very high output of light, 20 different luminol samples were tested for their ability to enhance the chemiluminescence reaction in phorbol myristate acetate activated human neutrophils. We found that the majority of luminols tested (17 samples) gave almost the same light output from neutrophils, and that the major part of the activity was from an intracellular origin. Owing to the fact that three isoluminol samples were unable to monitor respiratory burst activity taking place intracellularly, a very low level of chemiluminescence was obtained with these samples. Their light output was, however, greatly increased when horseradish peroxidase or myeloperoxidase was added, showing that the light-generating reaction with isoluminol as well as with luminol is peroxidase-dependent. The fact that isoluminol could also use myeloperoxidase as amplifying peroxidase, suggests that the lack of measurable intracellular activity in the presence of isoluminol is somehow related to a limited or restricted diffusion of the molecule to intracellular sites. The isoluminol system constitutes a sensitive system for measuring release of oxygen metabolites from phagocytic cells.     

Chemiluminescence in oxidation of luminol by chloramine derivatives of biogenic compounds

Chloramine derivatives of amino acids induce chemiluminescence of a luminol solution. The chemiluminescence is more prolonged than the emission of luminol produced by hypochlorite. Persistent chemiluminescence also appears under the action of hypochlorite on a mixture of luminol and amino acids. It is assumed that the chemiluminescence of luminol in suspensions of stimulated phagocytes may be associated with its oxidation by chloramines.     

Chemiluminescence of enterococci isolates from freshwater

All Enterococcus spp., isolated from environmental water samples (n=81), emitted a high chemiluminescence signal in the presence of luminol (10(-2) M). Kinetic studies of chemiluminescence show a close correlation between chemiluminescence and growth curves during the exponential phase, with a maximum chemiluminescence reached just before bacterial growth entered in the stationary phase. On the other hand, genera closely related to Enterococcus such as Streptococcus or Lactococcus produced a very weak chemiluminescent signal. Chemiluminescence of enterococci could therefore offer a rapid test, in aiding the identification of the genus Enterococcus and in the survey of the microbiological quality of water supplies.     

Measurement of whole blood phagocyte chemiluminescence in the Wistar rat

The factors influencing the rat whole blood chemiluminescence (CL): concentrations of blood, luminol, zymosan or opsonized zymosan, volume of the reaction mixture, storage time of blood samples and the presence of anticoagulants were evaluated. The CL micromethod described provides a fast and sensitive tool for the determination of metabolic activity of phagocytes in the microlitre range of rat whole blood. © 1997 John Wiley & Sons, Ltd.     

Estimation of tyrosine by the attenuation of 4-iodophenol enhanced chemiluminescence

Tyrosine markedly attenuates the chemiluminescence output intensity from the 4-iodophenol enhanced chemiluminescence assay system in a manner consistent with competition between the amino acid and luminol for the 4-iodophenoxy radical. This effect provides the basis for a sensitive assay of tyrosine. Interference by the other amino acids has been assessed; major interference by cysteine can be removed by incubation with iodoacetic acid.     

A chemiluminescence method to detect hydroquinone with water‐soluble sulphonato‐(salen)manganese(III) complex as catalyst

A water‐soluble sulphonato‐(salen)manganese(III) complex with excellent catalytic properties was synthesized and demonstrated to greatly enhance the chemiluminescence signal of the hydrogen peroxide ? luminol reaction. Coupled with flow‐injection technique, a simple and sensitive chemiluminescence method was first developed to detect hydroquinone based on the chemiluminescence system of the hydrogen peroxide–luminol–sulphonato‐(salen)manganese(III) complex. Under optimal conditions, the assay exhibited a wide linear range from 0.1 to 10 ng mL^–1 with a detection limit of 0.05 ng mL^–1 for hydroquinone. The method was applied successfully to detect hydroquinone in tap‐water and mineral‐water, with a sampling frequency of 120 times per hour. The relative standard deviation for determination of hydroquinone was less than 5.6%, and the recoveries ranged from 96.8 to 103.0%. The ultraviolet spectra, chemiluminescence spectra, and the reaction kinetics for the peroxide–luminol–sulphonato‐(salen)manganese(III) complex system were employed to study the possible chemiluminescence mechanism. The proposed chemiluminescence analysis technique is rapid and sensitive, with low cost, and could be easily extended and applied to other compounds. Copyright © 2015 John Wiley & Sons, Ltd.     

Microflow‐injection chemiluminescence of luminol and hypochlorite enhanced by phloxine B

We report for the first time that the sensitivity of the luminol–hypochlorite chemiluminescence (CL) reaction was enhanced approximately 10 times by the addition of phloxine B. The maximum wavelength of CL emission shifted from 431 to 595 nm in the absence and presence, respectively, of phloxine B, suggesting that an efficient chemiluminescence resonance energy transfer occurred between a luminol donor and a phloxine B acceptor in the luminol–hypochlorite–phloxine B system. Based on this observation, a simple, rapid and sensitive microflow injection CL method, using a microchip with spiral channel configurations, was developed for the determination of hypochlorite. Under optimized conditions, a linear calibration curve (R² = 0.9944) over the range 0.1–10.0 µmol/L was obtained, with a detection limit of 0.025 µmol/L (S:N = 3). The relative standard deviation (RSD) was found to be 4.2% (n = 10) for 2.5 µmol/L hypochlorite. The sample consumption was only 2 μL, with a sample throughput of 90/h. The method has been used for determining trace amounts of hypochlorite in water samples with satisfactory results. Copyright © 2012 John Wiley & Sons, Ltd.     

Chemiluminescence of the polymorphonuclear leukocytes-luminol system in the presence of biogenic chloramines

It was demonstrated that N-chlorphenylalanine and other chloramines strengthen sharply chemiluminescence in the polymorphonuclear leukocytes (PML)-luminol system without special activation of cells. The intensity of chemiluminescence is higher than the intensity of luminol solution emission induced by N-chlorphenylalanine. But it was nearly equal to chemiluminescence intensity of a mixture of luminol, N-chlorphenylalanine and 20-30 nM H2O2. The increase in chemiluminescence in the PML-luminol system in the presence of N-chlorphenylalanine is not related to PML activation but is the result of direct oxidation of luminol by N-chlorphenylalanine. Chloramine derivatives of amino acids and taurine at final concentrations of 0.01-0.1 mM do not suppress luminol chemiluminescence in suspension of PML stimulated by phorbol-12-myristate-13-acetate. At the same time, hypochlorite inhibits sharply luminol emission induced by stimulated cells.     

Improvements to enhanced horseradish peroxidase detection sensitivity

At very low horseradish peroxidase (HRP) concentrations, the enhanced chemiluminescence reaction is often characterized by a lag time between initiation of the reaction and beginning of light output. In this study, four treatments of luminol solution were examined in an effort to remove the lag time and to improve chemiluminescence light output. Addition of ammonium persulphate stimulated light output more than tenfold. Ultraviolet irradiation and photoactive dye pretreatment of luminol solution both increased light output fourfold. Luminol purity was the most important factor affecting detection sensitivity. Recrystallization of luminol from base improved the detection limit 13-fold although there was an improvement in the detection limit from 13 attomoles per millilitre to 5 attomoles per millilitre with highly purified luminol when photoactive dye pretreatment was utilized. The results are consistent with a simple interference mechanism whereby enhancer radicals produced by the enzyme are preferentially quenched by contaminants present in the luminol, in the enhancer and in the solvent used to dissolve the enhancer. Consumption of these interferences prior to light emission results in a lag time and a less favourable HRP detection limit.     

Determination of parathion residues in rice samples using a flow injection chemiluminescence method

A sensitive ?ow injection chemiluminescence method is described for the determination of parathion pesticide, based upon its direct chemiluminescence reaction with luminol and hydrogen peroxide in the presence of non‐ionic surfactant polyethylene glycol 400. Under the selected experimental conditions, the concentration of parathion is proportional to the CL intensity in the range 0.02–1.0 mg/L. The detection limit was 0.008 mg/L and the relative standard deviation was 2.8% for 0.2 mg/L parathion solution (n = 11). This method was successfully applied to the determination of parathion residue in rice samples. Copyright © 2003 John Wiley & Sons, Ltd.     

Evaluation of nongermicidal handwashing protocols for removal of transient microbial flora

A method has been described which can compare the efficacy of different nongermicidal handwashing protocols for removal of transient microbial flora without the necessity of establishing or relying on a previously determined baseline for an individual subject. The wash effluent is collected, and colony counts for the effluent reflect the number removed by the wash protocol. A second standard wash in a handwashing machine is performed, and the test criterion is the percent removed in the test wash based on the sum of total CFU recovered from the two washes. The method was used to compare an 8-s cycle for a newly developed handwashing machine with a conventional 15-s Ivory soap wash. When machine pressure was adequate (42 lb/in2), there was no statistically significant difference in the percent removal of transient flora by the two methods (48.8% from the machine versus 45.1% from the Ivory soap wash). At 32 lb/in2, the Ivory soap wash recovered 60.3%, whereas the machine recovered 45.1%.     

Flow‐injection chemiluminescence method for the determination of chloramphenicol based on luminol–sodium periodate order‐transform second‐chemiluminescence reaction

A new chemiluminescence (CL) reaction was observed when chloramphenicol solution was injected into the mixture after the end of the reaction of alkaline luminol and sodium periodate or sodium periodate was injected into the reaction mixture of chloramphenicol and alkaline luminol. This reaction is described as an order‐transform second‐chemiluminescence (OTSCL) reaction. The OTSCL method combined with a flow‐injection technique was applied to the determination of chloramphenicol. The optimum conditions for the order‐transform second‐chemiluminescence emission were investigated. A mechanism for OTSCL has been proposed on the basis of the chemiluminescence kinetic characteristics, the UV‐visible spectra and the chemiluminescent spectra. Under optimal experimental conditions, the CL response is proportional to the concentration of chloramphenicol over the range 5.0 × 10^?7–5.0 × 10^?5 mol/L with a correlation coefficient of 0.9969 and a detection limit of 6.0 × 10^?8 mol/L (3σ). The relative standard deviation (RSD) for 11 repeated determinations of 5.0 × 10^?6 mol/L chloramphenicol is 1.7%. The method has been applied to the determination of chloramphenicol in pharmaceutical samples with satisfactory results. Copyright © 2011 John Wiley & Sons, Ltd.     

Detection of chemiluminescence in lipid peroxidation of biological systems and its application to HPLC

A combined system of chemiluminescence detection and high performance liquid chromatography (CL–HPLC) was developed to determine primary peroxidation products in biological tissues, such as phosphatidylcholine hydroperoxide (PCOOH). The CL–HPLC assay consists of separation of lipid classes with HPLC and detection of hydroperoxide-specific chemiluminescence. Hydroperoxides react with heme compounds to produce oxidants as suggested by our early studies on tissue low-level chemiluminescence in which singlet molecular oxygen is generated as one of the excited species in several biological systems involving free radical events. In the CL–HPLC method, a cytochrome c–luminol mixture was used as a hydroperoxide-specific luminescent reagent, and the quantification of hydroperoxide was performed by detecting chemiluminescence due to the luminol oxidation caused by the oxidant produced during the lipid hydroperoxides with heme. The detection limit of PCOOH was 10 pmole hydroperoxide–O₂. PCOOH in normal human blood was found to be 10–500 pmol/ml plasma and significantly higher levels of PCOOH were observed in some hospitalized patients.     

Evaluation of nongermicidal handwashing protocols for removal of transient microbial flora.

A method has been described which can compare the efficacy of different nongermicidal handwashing protocols for removal of transient microbial flora without the necessity of establishing or relying on a previously determined baseline for an individual subject. The wash effluent is collected, and colony counts for the effluent reflect the number removed by the wash protocol. A second standard wash in a handwashing machine is performed, and the test criterion is the percent removed in the test wash based on the sum of total CFU recovered from the two washes. The method was used to compare an 8-s cycle for a newly developed handwashing machine with a conventional 15-s Ivory soap wash. When machine pressure was adequate (42 lb/in2), there was no statistically significant difference in the percent removal of transient flora by the two methods (48.8% from the machine versus 45.1% from the Ivory soap wash). At 32 lb/in2, the Ivory soap wash recovered 60.3%, whereas the machine recovered 45.1%.     

Chemiluminescence determination of pharmacologically active compounds by capillary electrophoresis.

A simple and rapid capillary electrophoresis with direct chemiluminescence method has been developed for the determination of five natural pharmacologically active compounds including rutin, protocatechuic aldehyde, chlorogenic acid, luteolin and protocatechuic acid. The luminol as a component of the separation electrolyte buffer was introduced at the head of the separation capillary. The separation of five compounds was carried out in a fused-silica capillary with 15.0 mmol/L tetraborate, 1.0 mmol/L SDS and 0.42 mmol/L luminol (pH 8.5). The analytes was determined by enhancing the chemiluminescence of luminol with 0.13 mmol/L K3Fe(CN)6 in 0.05 mol/L NaOH, which was introduced at the post-column stage. The voltage applied was 16 kV. Under the optimum conditions, the analytes were separated within 10 min. The excellent linearity was obtained over two to three orders of magnitude with a detection limit (signal:noise = 3) of 0.012-0.055 micromol/L for all five analytes. The method was successfully used in the analysis of pharmaceutical and biological samples, and the assay results were satisfactory.