Luminol demonstrations

It is assumed that common reaction intermediates are readily available to would-be demonstrators. Luminol, acridine, TMAE, diphenylanthracene, rubrene, fluorescein, rhodamine B are all obtainable from the Aldrich Chemical Company, which has branches in most countries. Violanthrone can be obtained from ICN Pharmaceuticals Inc. K and K Labs Division, 121, Express Street, Plainview, New York 11803, USA. 

Another rather striking example demonstrates that the fluorescence efficiency of the respective dicarboxylates is not the most important factor in determining the chemiluminescence efficiencies of the hydrazides 9.10-diphenylanthracene-2.3-dicarbonic acid 25 has a fluorescence efficiency of about 0.9 (as has the parent compound 9.10-di-phenylanthracene) 94>. The corresponding hydrazide 26, however, gives a quantum yield of 48% that of luminol only (in DMSO/t-BuOK/ O2) 95) although 3-aminophtalic acid has a fluorescence efficiency of about 0.3 only. 

This preliminary work demonstrated well the use of CL as a highly sensitive and selective detection method in CE by its application to the separation of luminol and ABEI using the same experimental conditions cited previously for the CL reaction of luminol in HPLC. Detection limits (S/N = 3) of 100 amol and 400 amol were obtained for the compounds mentioned, respectively, achieving an improvement in sensitivity of 2-3 orders of magnitude with respect to the ones obtained using UV absorption for detection. 

Figure 1 shows the graphs of the PCL that were recorded with riboflavin as the photosensitizer and luminol as the detector for free radicals [21], The course of the PCL reaction has two maxima at approximately 30 s and 3 min after the start of irradiation. It has been demonstrated by analysis of kinetics after addition of the reactants at varying times that the first maximum is riboflavin-dependent. Luminol is needed only for visualization of the superoxide radicals. 

Commercially available horseradish peroxidase (crystalline) will substitute for luriferase in the foregoing reaction. In addition, a compound of known structure. 5-amino-2, 3-dihvdro-l, 4-phthalazinedione (also known as luminol), will substitute for luciferin. The mechanisms appear to be the same regardless of the way in which the crosses are made. Thus, a model bioluminescent system is available and can be used as a sensitivity assay for H2O2 at neutral pH. The identification of luciferase as a peroxidase is of interest since this represents the only demonstration of a bioluminescent system in which the catalytic nature of a luciferase molecule has been defined. 

Fang and co-workers [115] have reported a flow injection chemiluminescence assay for the detection of maleic hydrazide (MH). The imprinted poly(MAA-co-EDMA) polymer selectively retained the herbicide that was further treated with alkaline luminol-potassium periodate to produce a strong CL signal. Upon reaction, the absorbed MH was destroyed and removed by the flowing solution. The polymer was reused up to ten times and the linear response was between 3.5 x 10 and 5.0 x 10 2 mg mL-1 with a detection limit of 6.0 x 10 5 mg mL-1. The CILA flow injection system was used for the analysis of potato and onion samples spiked with 1.0, 2.0, and 4.0 x 10-3 mg mL-1 of MH. Recoveries ranged from 98% to 103% (RSD 2.3%), demonstrating the successful application of the method. 

Kinetic analysis of luminol-dependent chemiluminescence demonstrated that a solution of berbamine (20 pM) was found to inhibit the generation of various types of reactive oxygens by guinea-pig neutrophils. The results of these and other experiments suggest that berbamine inhibits the active oxygen generation via the stabilization of plasma membrane and the inhibition of phospholipid-dependent protein kinase (PKC) and NADPH oxidase activation [178]. 

The present study demonstrates the pH-dependent bimodal chemiluminescence of 6,8-diaryl-imidazopyrazinone la induced by superoxide. Despite its lower sensitivity to superoxide than CLA la showed enough sensitivity to superoxide in comparison with luminol and appears to be capable of superoxide detection. What the advantage of la over CLA is that it exhibits the pH-dependent bimodal chemiluminescence, and, therefore, the direct estimation of the local pH in a cell or a droplet is feasible based on the luminescence ratio, Iank /(la nii+Iaiiiai)- This property of la allows us to apply la for simultaneous detection of the two concurrent events, superoxide evolution and pH alteration, in biological systems. 

Luminol (3-aminophthalhydrazide) is used in a commercially available portable device called the Luminox that measures minute concentrations (parts per billion) of the pollutant nitrogen dioxide in air. Luminol is also used ftequently in laboratory demonstrations of the chemiluminescence phenomenon. Luminol-mediated chemiluminescence is the result of an oxidation reaction. The oxidation proceeds in two steps, which ultimately lead to the production of the aminophthalate anion in an excited state and the elimination of water and molecular nitrogen. The formation of the strong triple bond (N=N) is a major factor in the release of energy in the form of light. 

Curve c depicts opsonification by specific antibody as previously described. Phagocytosis results from contact of the Fc-labeled microbe with the Fc-receptors on the PMN membrane. Immune opsonification by this mechanism was unchanged following heat treatment. Demonstration of the classical complement amplification of specific antibody reaction is presented in curve d. Note the acceleration in rate obtained with addition of complement. Curve a depicts the CL response from PMN, luminol, and . aeruginosa in the absence of antibody and complement. 

Besides fluorescence FIIAs, chemiluminescence FIIAs have also been developed, whereby mainly luminol and acridinium ester were used as chemiluminescence labels. High sensitivity was reported with systems using flow-cells packed with immobilized antibodies or antigens, whereby the luminescence is measured directly at the cell. The assays are carried out by stepwise injection of all components. Detection limits in the attomole range have been reported with such systems. Recently, the stereoselective recognition ability of antibodies raised against one enantiomer has been demonstrated. On this basis an enantioselective chemiluminescence HIA for amino acids, which allows the detection of less than 0.01% of the unwanted enantiomer, has been developed. 

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